{
 "built": "2026-10-28",
 "domains": [
  {
   "domain": "vascular",
   "title": "Blood flow, vessels and blood",
   "overview": "The most consistent vascular findings in ME/CFS concern circulation when upright: cerebral blood flow falls much more than normal on tilt, stroke volume is low, red-cell and blood volume are below predicted in a large minority to about half, and during exercise the heart is under-filled (preload failure). These are reported by several groups, though the cerebral blood flow and invasive exercise data each come mostly from one centre, and a 2026 study found blood volume did not predict who has orthostatic abnormalities. Reduced endothelial function (FMD, PORH) has been found in three independent cohorts, and newer work shows fewer muscle capillaries than bed rest would explain and narrowed retinal vessels in post-COVID ME/CFS. Complement activation is replicated in long COVID but inconsistent in ME/CFS. Microclots remain contested on method, and pooled blood proteomics do not show raised coagulation, platelet or endothelial-activation proteins.",
   "findings": [
    {
     "id": "vascular-low-red-cell-volume",
     "subdomain": "Blood volume",
     "finding": "Red cell volume and total blood volume are below predicted in a large minority to about half of ME/CFS patients measured by isotope or CO-rebreathing methods.",
     "grade": "replicated",
     "plain": "Many patients have less blood than expected for their size, mainly because they have fewer red cells. Less blood means less returns to the heart when upright.",
     "detail": "Hurwitz 2009 (Clin Sci): total blood volume -15%, plasma volume -13%, red cell volume -19% in CFS, and the blood-volume deficit explained 91-94% of group differences in cardiac volume indices. Newton 2016 linked lower plasma volume to smaller cardiac MRI volumes and to fatigue severity but not to illness duration (argues against pure deconditioning). Five independent groups report a red-cell-volume deficit (Streeten 2000, Hurwitz, Newton, van Campen 2018, Yamazaki 2026). Measurement methods differ (I-131 albumin, Cr-51, CO-rebreathing) and predicted values depend on body-size formulas, so prevalence figures are not comparable across studies.",
     "studies": [
      {
       "cite": "Hurwitz 2009, Clin Sci",
       "n": "56 CFS (30 severe, 26 non-severe) vs 90 controls",
       "design": "case-control, isotope blood volume + echo",
       "result": "TBV -15.4%, PV -13.2%, RBCV -19.1%; deficit explained most of the lower stroke index",
       "url": "https://doi.org/10.1042/CS20090055",
       "verified": true
      },
      {
       "cite": "Newton 2016, Open Heart",
       "n": "CFS vs controls (cardiac MRI + red cell mass)",
       "design": "case-control",
       "result": "Lower cardiac volumes tracked plasma and red cell volume; PV correlated with fatigue severity; no link to illness duration",
       "url": "https://doi.org/10.1136/openhrt-2015-000381",
       "verified": true
      },
      {
       "cite": "Yamazaki 2026, Physiol Rep",
       "n": "49 ME/CFS",
       "design": "cross-sectional, blood volume + 10-min lean test",
       "result": "RBC volume deficient in 49%; TBV hypovolaemic 35%, normal 43%, hypervolaemic 22%",
       "url": "https://doi.org/10.14814/phy2.71087",
       "verified": true
      },
      {
       "cite": "Streeten & Bell 2000, Am J Med Sci",
       "n": "15 CFS",
       "design": "case series, Cr-51 red cell mass",
       "result": "Low red cell mass in most patients; orthostatic symptoms",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Streeten+Bell+2000+red+cell+mass+chronic+fatigue",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Yamazaki 2026, Physiol Rep",
       "n": "49 ME/CFS",
       "design": "cross-sectional",
       "result": "Blood-volume category did not predict any lean-test abnormality (POTS, hypocapnia, hypotension); 22% were hypervolaemic",
       "url": "https://doi.org/10.14814/phy2.71087",
       "verified": true
      }
     ],
     "open_questions": [
      "Why does red cell mass fall: reduced marrow output (Solving ME/CFS re-analysis of FinnGen labs found lower reticulocytes and soluble transferrin receptor with normal iron), inactivity, or altered EPO signalling?",
      "Does blood volume change with severity or after crashes?",
      "If low blood volume does not predict orthostatic intolerance (Yamazaki 2026), how much of the symptom burden does it explain?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "pituitary-adh"
     ],
     "test_available": "Haemoglobin mass by CO-rebreathing (some sports-science and research labs); isotope blood volume (Daxor BVA-100, a few US centres). Reticulocytes, ferritin, transferrin saturation are routine."
    },
    {
     "id": "vascular-raas-paradox",
     "subdomain": "Blood volume",
     "finding": "The hormones that should rise to restore blood volume (renin, aldosterone, EPO) are not raised in ME/CFS or POTS despite low volume, and blood proteomics shows no consistent change in them.",
     "grade": "several",
     "plain": "If blood volume is low the body should release hormones to retain salt and water. In many patients these hormones stay normal or low, which suggests the volume set-point itself is off.",
     "detail": "In POTS, low aldosterone and an inappropriately low renin for the plasma-volume deficit (the renin-aldosterone paradox) were described by Raj et al. 2005; adrenal responsiveness to ACTH is intact (Laurin 2023), so the defect sits upstream. Older ME/CFS work reported low renin in tilt-positive patients and normal EPO. A Solving ME/CFS re-analysis of SomaScan and UK Biobank Olink data (about 150 patients plus UKB) found renin, ACE, BNP, EPO and hepcidin flat across cohorts.",
     "studies": [
      {
       "cite": "Raj 2005, Circulation",
       "n": "POTS vs controls",
       "design": "case-control",
       "result": "Plasma volume deficit with low renin and aldosterone for the degree of hypovolaemia",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Raj+renin-aldosterone+paradox+postural+tachycardia+2005",
       "verified": false
      },
      {
       "cite": "Laurin 2023, Auton Neurosci",
       "n": "POTS vs HC",
       "design": "ACTH stimulation",
       "result": "Adrenal aldosterone and cortisol responses to ACTH intact in POTS",
       "url": "https://doi.org/10.1016/j.autneu.2023.103105",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "Hoel, Germain, Walitt SomaScan + UKB Olink",
       "design": "secondary analysis",
       "result": "RAAS/natriuretic, EPO, hepcidin sets null (q>0.7)",
       "url": "/#data",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is the low-renin pattern present in ME/CFS without POTS?",
      "Does ADH/copeptin respond normally to upright posture?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "pituitary-adh",
      "hpa-endocrine"
     ],
     "test_available": "Supine and upright renin/aldosterone; copeptin. Routine labs, but interpretation needs posture and salt-intake control."
    },
    {
     "id": "vascular-small-heart-stroke-volume",
     "subdomain": "Cardiac size and stroke volume",
     "finding": "Left-ventricular volumes and stroke volume are smaller in ME/CFS at rest and fall more than normal on tilt, largely in proportion to low blood volume rather than to weak heart muscle.",
     "grade": "replicated",
     "plain": "The heart is not damaged, but it is under-filled, so each beat pumps less blood, especially when upright.",
     "detail": "Hurwitz 2009: severe CFS had about 10% lower stroke index and end-diastolic volume; adjusting for blood volume removed most of the difference. Hollingsworth 2012 (cardiac MRI) reported markedly lower end-diastolic volume and stroke volume. Miwa reports a 'small heart' (cardiothoracic ratio <0.42) subgroup with orthostatic intolerance. van Campen 2024 (233 POTS, many more normal HR-BP response) showed larger stroke-volume-index falls on tilt in all ME/CFS groups than in controls, with two patterns: venous pooling (HR rise 30-39) and a hyperadrenergic pattern (HR rise >=40) with relatively preserved SVI.",
     "studies": [
      {
       "cite": "van Campen 2024, J Clin Med",
       "n": "233 ME/CFS with POTS + normal-response patients vs HC",
       "design": "retrospective tilt with echocardiographic SVI",
       "result": "SVI fall larger than HC in all groups; two POTS haemodynamic profiles",
       "url": "https://doi.org/10.3390/jcm13247726",
       "verified": true
      },
      {
       "cite": "Hollingsworth 2012, Eur J Clin Invest",
       "n": "CFS vs controls",
       "design": "cardiac MRI",
       "result": "Lower LV end-diastolic volume and stroke volume",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Hollingsworth+cardiac+MRI+chronic+fatigue+syndrome+2012",
       "verified": false
      },
      {
       "cite": "Miwa 2026, Front Med",
       "n": "review of Japanese series",
       "design": "narrative review",
       "result": "Small heart and downregulated renin-aldosterone/ADH systems in OI subgroup",
       "url": "https://doi.org/10.3389/fmed.2026.1744154",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Olimulder 2016, Neth Heart J",
       "n": "ME/CFS vs controls",
       "design": "cardiac MRI",
       "result": "Cardiac dimensions within normal range after indexing in a referral cohort",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Olimulder+chronic+fatigue+syndrome+cardiac+magnetic+resonance",
       "verified": false
      }
     ],
     "open_questions": [
      "Do stroke-volume deficits normalise after volume loading, and does that predict symptomatic benefit?",
      "How much of the van Campen echo data has been reproduced in another centre with blinded readers?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Echocardiogram (routine, but usually reported normal at rest); tilt with stroke-volume monitoring (Finapres/bioimpedance at autonomic units)."
    },
    {
     "id": "vascular-preload-failure-icpet",
     "subdomain": "Exercise haemodynamics",
     "finding": "During upright invasive exercise testing most ME/CFS patients have abnormally low right-atrial filling pressure (preload failure), and a high-flow subgroup shows poor systemic oxygen extraction.",
     "grade": "several",
     "plain": "When patients exercise, too little blood returns to the heart. In some, blood also bypasses the muscles, so oxygen goes unused.",
     "detail": "Joseph/Systrom 2021 (Chest): of 1,516 iCPETs, 160 met ME/CFS criteria with RAP <6.5 mmHg; peak RAP 1.9 vs 8.3 mmHg, peak VO2 80% vs 101% predicted. Low-flow tertile had lower peak cardiac output; high-flow tertile had lower systemic O2 extraction (0.74 vs 0.86-0.88). Selection by low RAP is built into the cohort, so the prevalence of preload failure in ME/CFS generally is not established. Novak 2026 reports preload failure in 92% of the 66 ME/CFS patients who had iCPET (same Harvard group). Long COVID iCPET shows the same pattern (Singh 2022; Joseph 2023 review). Independent replication outside the Systrom network is lacking.",
     "studies": [
      {
       "cite": "Joseph 2021, Chest",
       "n": "160 ME/CFS vs 36 controls",
       "design": "retrospective iCPET + skin biopsy",
       "result": "Peak RAP 1.9 vs 8.3 mmHg; impaired O2 extraction in high-flow tertile; SFN in 31%",
       "url": "https://doi.org/10.1016/j.chest.2021.01.082",
       "verified": true
      },
      {
       "cite": "Novak 2026, PLOS One",
       "n": "170 ME/CFS, 143 long COVID, 73 HC, 290 hEDS",
       "design": "retrospective autonomic + iCPET",
       "result": "Preload failure 92% ME/CFS (of 66 tested), 96% LC (of 25)",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "Joseph 2022, Chest (pyridostigmine RCT)",
       "n": "45 ME/CFS",
       "design": "RCT, single-dose crossover-style iCPET",
       "result": "Pyridostigmine improved peak VO2 vs placebo via higher cardiac output (acute effect)",
       "url": "https://doi.org/10.1016/j.chest.2022.04.146",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What is the preload-failure prevalence in an unselected ME/CFS cohort?",
      "Can non-invasive tests (pulse pressure narrowing on stand, Day 2023; CO-rebreathing cardiac output) stand in for iCPET?",
      "Does preload failure precede or follow illness onset?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre",
      "small-vessel-capillary"
     ],
     "test_available": "Invasive CPET at a few US centres (Brigham, Yale, Mount Sinai); not routine. Two-day CPET is non-invasive but does not measure preload."
    },
    {
     "id": "vascular-oxygen-extraction",
     "subdomain": "Exercise haemodynamics",
     "finding": "A subset of patients with unexplained exertional intolerance cannot extract oxygen normally from blood during exercise, associated with hypocapnia and possible arteriovenous shunting.",
     "grade": "early",
     "plain": "Blood arrives at the muscles but leaves with too much oxygen still in it, so the muscles get less than they need.",
     "detail": "Melamed 2019 (Systrom group) described a poor-extraction phenotype with relative hypocapnia (PaCO2 29.9 vs 37.5 mmHg) and high mixed-venous O2 saturation at peak (44% vs 31%). Proposed causes: microvascular shunting from small-fibre neuropathy, mitochondrial limitation, or a left-shifted oxyhaemoglobin curve from hyperventilation. Muscle-oxygenation work by the Wüst group (NIRS post-occlusion) points in the same direction. All iCPET data come from one network.",
     "studies": [
      {
       "cite": "Melamed 2019, Eur J Appl Physiol",
       "n": "unexplained exertional intolerance cohort",
       "design": "retrospective iCPET",
       "result": "Poor systemic O2 extraction with hypocapnia; high peak SvO2",
       "url": "https://doi.org/10.1007/s00421-019-04222-6",
       "verified": true
      },
      {
       "cite": "Joseph 2021, Chest",
       "n": "160 ME/CFS",
       "design": "iCPET",
       "result": "High-flow tertile extraction 0.74 vs 0.86-0.88",
       "url": "https://doi.org/10.1016/j.chest.2021.01.082",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Which contributes most: shunting, mitochondria, or CO2-related Bohr shift?",
      "Is impaired extraction present at rest or only at peak?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "autonomic-small-fibre",
      "mitochondrial-energy"
     ],
     "test_available": "Research only (iCPET); muscle NIRS in research labs."
    },
    {
     "id": "vascular-cbf-tilt",
     "subdomain": "Cerebral blood flow",
     "finding": "Cerebral blood flow falls about 26% during head-up tilt in ME/CFS versus 7% in controls, including in patients with normal heart rate and blood pressure responses.",
     "grade": "several",
     "plain": "Standing up reduces blood flow to the brain far more than normal, even when pulse and blood pressure look fine, and the drop tracks orthostatic symptoms.",
     "detail": "van Campen 2020 (429 patients, 44 HC, extracranial carotid+vertebral Doppler): 82% of normal-response, 98% of delayed-OH and 100% of POTS patients exceeded the control lower limit (13%). Flow recovery after tilt is incomplete and slowest in severe patients (van Campen 2021). The CO-CBF relation is roughly 1:1 in most patients, unlike the 3:1 seen in health (van Campen 2024, 2025). Transcranial Doppler work from other groups (Medow/Stewart; Novak 2026: reduced orthostatic CBF velocity in 88% ME/CFS) agrees. Caveats: most volume-flow data come from one Dutch clinic, operators are not blinded to diagnosis, controls are few relative to patients, and the 2025 POTS paper found CBF reduction did not differ between its two haemodynamic subgroups.",
     "studies": [
      {
       "cite": "van Campen 2020, Clin Neurophysiol Pract",
       "n": "429 ME/CFS vs 44 HC",
       "design": "case-control, tilt with extracranial Doppler",
       "result": "End-tilt CBF -26% vs -7%; ~90% abnormal",
       "url": "https://doi.org/10.1016/j.cnp.2020.01.003",
       "verified": true
      },
      {
       "cite": "van Campen 2021, Clin Neurophysiol Pract",
       "n": "ME/CFS patients",
       "design": "tilt with post-tilt recovery",
       "result": "CBF reduced after tilt, slowest recovery in most severe",
       "url": "https://doi.org/10.1016/j.cnp.2021.09.001",
       "verified": true
      },
      {
       "cite": "Novak 2026, PLOS One",
       "n": "170 ME/CFS, 143 LC, 73 HC",
       "design": "retrospective TCD tilt",
       "result": "Reduced orthostatic CBFv in 88% ME/CFS, 92% LC",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "van Campen 2025, J Clin Med",
       "n": "ME/CFS with POTS",
       "design": "retrospective tilt CO + CBF",
       "result": "Two-thirds ~1:1 CO-CBF reduction; one-third limited CO fall with high HR (hyperadrenergic)",
       "url": "https://doi.org/10.3390/jcm14113648",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent replication of extracranial volume-flow method with blinded readers",
      "Does CBF reduction predict response to fluids, compression, or vasoconstrictors?",
      "What happens to CBF in sitting (the posture patients actually spend time in)?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre",
      "vascular-endothelial"
     ],
     "test_available": "Extracranial Doppler on tilt (Stichting CardioZorg, Netherlands); transcranial Doppler on tilt at some autonomic labs."
    },
    {
     "id": "vascular-hypocapnia",
     "subdomain": "Breathing and CO2",
     "finding": "Many ME/CFS patients over-breathe when upright (orthostatic hypocapnia), and during exercise hyperventilation and dysfunctional breathing are common.",
     "grade": "replicated",
     "plain": "When standing, some patients breathe off too much CO2. Low CO2 narrows brain arteries, which can add to dizziness and brain fog.",
     "detail": "End-tidal CO2 falls on tilt in several groups (Naschitz 2000, Razumovsky 2003, Natelson 2007, Medow 2014, Novak's 'hypocapnic cerebral hypoperfusion'). Yamazaki 2026: postural orthostatic syndrome of hypocapnia (POSH) in 41% of 49 on a lean test. Mancini 2025: hyperventilation and/or dysfunctional breathing during CPET common, with VE/VCO2 34.7 vs 28.1 in hyperventilators. But van Campen 2023 found CO2 reactivity normal and the PETCO2 fall explained only a modest share of the CBF fall, so hypocapnia is probably a contributor, not the main driver. Whether over-breathing is primary (chemoreflex/autonomic) or compensatory (e.g. to aid venous return via thoracic pump) is unresolved.",
     "studies": [
      {
       "cite": "Medow 2014, J Appl Physiol",
       "n": "CFS with OI vs controls",
       "design": "tilt + n-back",
       "result": "Upright hyperventilation and CBFv fall; phenylephrine restored n-back performance",
       "url": "https://doi.org/10.1152/japplphysiol.00527.2014",
       "verified": true
      },
      {
       "cite": "Yamazaki 2026, Physiol Rep",
       "n": "49 ME/CFS",
       "design": "lean test with capnography",
       "result": "POSH 40.8%, most common orthostatic abnormality",
       "url": "https://doi.org/10.14814/phy2.71087",
       "verified": true
      },
      {
       "cite": "Mancini 2025, Front Med",
       "n": "58 ME/CFS + sedentary controls",
       "design": "CPET breathing analysis",
       "result": "18 hyperventilators; elevated VE/VCO2 or dysfunctional breathing in 83% of them",
       "url": "https://doi.org/10.3389/fmed.2025.1669036",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "van Campen 2023, Physiol Rep",
       "n": "female ME/CFS vs HC",
       "design": "tilt with PETCO2 and CBF",
       "result": "CO2 reactivity normal; PETCO2 explains only a modest part of CBF fall",
       "url": "https://doi.org/10.14814/phy2.15639",
       "verified": true
      }
     ],
     "open_questions": [
      "Is upright hypocapnia a cause, a compensation, or a marker of sympathetic activation?",
      "Does breathing retraining or added CO2 change orthostatic symptoms?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "low-blood-volume-preload"
     ],
     "test_available": "Capnography during a 10-minute stand or tilt (cheap; available in some respiratory/autonomic labs); CPET with breathing-pattern analysis."
    },
    {
     "id": "vascular-vasoconstrictor-phenylephrine",
     "subdomain": "Cerebral blood flow",
     "finding": "Raising blood pressure with phenylephrine during tilt blocked hyperventilation, restored cerebral blood velocity and improved working memory in ME/CFS, whereas CO2 or acetazolamide alone did not.",
     "grade": "early",
     "plain": "In a lab test, a drug that tightens blood vessels improved both brain blood flow and thinking speed when patients were upright.",
     "detail": "Medow 2014 and Medow & Stewart 2024 (same New York group) used acute infusions with n-back testing; numbers are small (single-digit to low teens per condition). These are mechanistic studies, not treatment trials. Compression of legs and abdomen reduced cardiac output fall (-15% vs -27%) and CBF fall (-14% vs -25%) on tilt in van Campen 2021 (n=16).",
     "studies": [
      {
       "cite": "Medow & Stewart 2024, Am J Physiol Regul",
       "n": "ME/CFS with OI vs controls",
       "design": "acute intervention crossover",
       "result": "Only phenylephrine improved upright n-back and phase synchronisation",
       "url": "https://doi.org/10.1152/ajpregu.00071.2024",
       "verified": true
      },
      {
       "cite": "van Campen 2021, Front Neurol (compression)",
       "n": "16 ME/CFS",
       "design": "within-subject tilt",
       "result": "Compression reduced CO and CBF falls",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=van+Campen+compression+stockings+cerebral+blood+flow+ME%2FCFS",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do oral analogues (midodrine) reproduce the cognitive effect in daily life?",
      "Placebo-controlled trials of compression garments in ME/CFS"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Research only; midodrine and compression are used clinically for orthostatic intolerance."
    },
    {
     "id": "vascular-endothelial-fmd-porh",
     "subdomain": "Endothelium",
     "finding": "Large-artery flow-mediated dilation and microvascular post-occlusive reactive hyperaemia are reduced in ME/CFS in two independent Norwegian cohorts and by peripheral arterial tonometry in Berlin.",
     "grade": "replicated",
     "plain": "The lining of blood vessels does not widen properly when it should. This has been found in several groups with different methods.",
     "detail": "Sandvik 2023 (RituxME substudy, 39 patients): FMD 5.1% vs 8.2% (p<0.0001, adjusted for diameter and sex), PORH 1354 vs 2208 PU; rituximab had no effect; PORH but not FMD improved over 18 months and PORH correlated with daily steps. Sørland 2021 (cyclophosphamide cohort, 35 patients): FMD 5.9% vs 7.7%, PORH 1331 vs 1886 PU. Scherbakov 2020 (EndoPAT): endothelial dysfunction (RHI <=1.81) in 51% of 35 vs 20% of 20 controls, linked to severity. Haffke 2022: reduced RHI in 5/14 post-COVID ME/CFS. Caveats: inactivity lowers FMD, controls were mostly not activity-matched, and FMD and PORH did not correlate with each other.",
     "studies": [
      {
       "cite": "Sandvik 2023, PLOS One",
       "n": "39 ME/CFS vs healthy controls",
       "design": "case-control within RCT",
       "result": "FMD 5.1% vs 8.2%; PORH 1354 vs 2208 PU",
       "url": "https://doi.org/10.1371/journal.pone.0280942",
       "verified": true
      },
      {
       "cite": "Sørland 2021, Front Med",
       "n": "35 ME/CFS vs 66 HC",
       "design": "case-control within open-label trial",
       "result": "FMD 5.9% vs 7.7%; PORH lower; no change with clinical response",
       "url": "https://doi.org/10.3389/fmed.2021.642710",
       "verified": true
      },
      {
       "cite": "Scherbakov 2020, ESC Heart Fail",
       "n": "35 ME/CFS vs 20 HC",
       "design": "case-control, EndoPAT",
       "result": "ED in 51% vs 20%; associated with severity",
       "url": "https://doi.org/10.1002/ehf2.12633",
       "verified": true
      },
      {
       "cite": "Haffke 2022, J Transl Med",
       "n": "14 PCS-ME/CFS, 16 PCS, HC",
       "design": "case-control, EndoPAT + biomarkers",
       "result": "ED in 5/14 and 5/16, none of HC; ET-1 raised",
       "url": "https://doi.org/10.1186/s12967-022-03346-2",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is endothelial dysfunction cause, consequence of inactivity, or a marker of an immune/serum factor?",
      "Does it track PEM or orthostatic severity over time?",
      "Activity-matched control studies"
     ],
     "hypotheses": [
      "vascular-endothelial",
      "serum-factor",
      "autoimmunity"
     ],
     "test_available": "FMD ultrasound and EndoPAT in vascular research labs; not standard clinical tests."
    },
    {
     "id": "vascular-serum-endothelial-in-vitro",
     "subdomain": "Endothelium",
     "finding": "Plasma or purified IgG from ME/CFS patients alters cultured endothelial cells: lower nitric oxide production, more mitochondrial fragmentation, and anti-endothelial antibody binding.",
     "grade": "early",
     "plain": "Something in patients' blood seems to make lab-grown vessel-lining cells work less well. This has been seen by three labs using different tests, none yet repeated by another lab using the same test.",
     "detail": "Bertinat 2022: HUVECs exposed to ME/CFS plasma (11 vs 12) produced less NO with higher inhibitory eNOS Thr495 phosphorylation. Flaskamp 2022 (Charité): more anti-endothelial antibody binding in post-COVID ME/CFS, and sera lacked the pro-angiogenic effect seen in non-ME post-COVID sera. Prusty 2026 (BBIH): purified IgG from 39 ME/CFS and 15 post-COVID ME/CFS fragmented endothelial mitochondria versus MS and healthy IgG; single lab, nominal p-values over many readouts (summarised in Solving ME/CFS serum-factors review). No lab has reproduced another's assay.",
     "studies": [
      {
       "cite": "Bertinat 2022, Vascul Pharmacol",
       "n": "11 ME/CFS vs 12 HC plasma",
       "design": "in vitro HUVEC",
       "result": "Lower NO production, higher eNOS Thr495 phosphorylation",
       "url": "https://doi.org/10.1016/j.vph.2022.106953",
       "verified": true
      },
      {
       "cite": "Flaskamp 2022, Cells",
       "n": "PCS-ME/CFS, PCS, HC sera",
       "design": "in vitro HMEC-1",
       "result": "Higher AECA binding in PCS/CFS; no pro-angiogenic effect",
       "url": "https://doi.org/10.3390/cells11152376",
       "verified": true
      },
      {
       "cite": "Liu/Prusty 2026, Brain Behav Immun Health",
       "n": "39 ME, 15 PCS-ME, 20 MS, 41 HC IgG",
       "design": "in vitro",
       "result": "Endothelial mitochondrial fragmentation with ME IgG, not MS or HC IgG",
       "url": "https://doi.org/10.1016/j.bbih.2026.101187",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Blinded multi-lab exchange of the same coded sera",
      "What is the antigen, and is the effect IgG-dependent in every assay?"
     ],
     "hypotheses": [
      "serum-factor",
      "autoimmunity",
      "vascular-endothelial"
     ],
     "test_available": "Research only."
    },
    {
     "id": "vascular-retinal-microvasculature",
     "subdomain": "Microvasculature",
     "finding": "Retinal imaging shows narrower arterioles, lower arteriolar-to-venular ratio, reduced flicker-induced venular dilation and lower capillary vessel density in post-COVID patients, most pronounced in those meeting ME/CFS criteria.",
     "grade": "several",
     "plain": "The tiny vessels at the back of the eye, which can be photographed without needles, look narrower and respond less in post-COVID ME/CFS.",
     "detail": "Wallraven 2026 (BMC Med, 'All Eyes on PCS'): 102 PCS (62 with ME/CFS) vs 102 recovered and 96 never-infected; CRAE 178 vs 186 um, AVR 0.83 vs 0.87, venular flicker dilation 3.7% vs 4.8%; ME/CFS subgroup most affected (AUC 0.79); robust to CV-risk adjustment except AVR attenuated. Schlick 2022 (Erlangen, OCT-A): lower vessel density in superficial and intermediate capillary plexus in PCS, lower still with chronic fatigue. Data are almost entirely post-COVID; pre-2020 ME/CFS cohorts have not been imaged with these methods.",
     "studies": [
      {
       "cite": "Wallraven 2026, BMC Med",
       "n": "102 PCS (62 ME/CFS), 102 recovered, 96 never-infected",
       "design": "prospective case-control, retinal vessel analysis",
       "result": "Narrower arterioles, lower AVR and flicker dilation; ME/CFS most affected; AVR tied to IL-6, ICAM-1, VCAM-1",
       "url": "https://doi.org/10.1186/s12916-026-05144-9",
       "verified": true
      },
      {
       "cite": "Schlick 2022, Int J Mol Sci",
       "n": "PCS vs controls",
       "design": "case-control, OCT-A",
       "result": "Lower retinal capillary vessel density; worse with chronic fatigue",
       "url": "https://doi.org/10.3390/ijms232213683",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is the same seen in pre-pandemic, non-COVID ME/CFS?",
      "Do retinal measures change with PEM or with treatment?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial",
      "innate-hyperactivation"
     ],
     "test_available": "OCT-A at ophthalmology clinics (widely available, though reference values for vessel density vary by device); dynamic vessel analysis research only."
    },
    {
     "id": "vascular-sublingual-capillary",
     "subdomain": "Microvasculature",
     "finding": "Sublingual video-microscopy shows reduced perfused capillary density and glycocalyx changes in long COVID, persisting many months after infection.",
     "grade": "early",
     "plain": "Under the tongue, fewer small vessels carry blood in long COVID patients.",
     "detail": "Osiaevi/Rovas/Kümpers (Münster) used sideStream dark-field microscopy with automated analysis (GlycoCheck) and reported persistent capillary rarefaction in long COVID (Nephrol Dial Transplant 2024; Angiogenesis 2023). No ME/CFS-specific cohort has been published with this method; whether rarefaction correlates with ME/CFS-defined PEM is unknown.",
     "studies": [
      {
       "cite": "Osiaevi 2024, Nephrol Dial Transplant",
       "n": "long COVID vs controls",
       "design": "case-control, sublingual video-microscopy",
       "result": "Capillary rarefaction proposed as possible cause of long COVID",
       "url": "https://doi.org/10.1093/ndt/gfae026",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Apply sublingual microscopy to pre-pandemic ME/CFS",
      "Does capillary density recover over time?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial"
     ],
     "test_available": "Research only (GlycoCheck/SDF microscopy)."
    },
    {
     "id": "vascular-muscle-capillary-basement-membrane",
     "subdomain": "Microvasculature",
     "finding": "Muscle biopsies show fewer capillaries per fibre in ME/CFS and less capillary supply per fibre size in both ME/CFS and long COVID, unlike the pattern after 60 days of bed rest; thickened capillary basement membrane has been reported but is not yet peer-reviewed.",
     "grade": "early",
     "plain": "Muscles of patients have fewer and possibly thicker-walled small blood vessels, and this is different from what happens to healthy people kept in bed.",
     "detail": "Charlton 2026 (Nat Commun, Wüst group): ME/CFS (n=23-24) had lower capillary-to-fibre ratio than controls (p<0.001, n=30) and long COVID; both patient groups had lower capillary density and lower capillary supply for a given fibre area; healthy bed rest (n=17-24) instead raised capillary density because fibres shrank. This argues against inactivity as the full explanation. Slaghekke 2025 (conference poster, electron microscopy, about 11 patients per group) reported basement membrane thickening (p<0.0001) and smaller capillary lumens in ME/CFS and post-COVID; the composite CF x collagen IV measure that correlated with CPET has been questioned. Appelman 2024 found amyloid-containing deposits in long COVID muscle that increased after induced PEM.",
     "studies": [
      {
       "cite": "Charlton 2026, Nat Commun",
       "n": "~25 LC, ~24 ME/CFS, 30 HC, 24 bed rest",
       "design": "case-control + bed-rest comparison, vastus lateralis biopsy",
       "result": "ME/CFS lower capillary-to-fibre ratio and density; patterns differ from bed rest",
       "url": "https://doi.org/10.1038/s41467-026-75725-y",
       "verified": true
      },
      {
       "cite": "Slaghekke 2025, ME/CFS Research Conference poster",
       "n": "~11 per group",
       "design": "electron microscopy",
       "result": "Thicker capillary basement membrane, smaller lumen",
       "url": "https://mecfs-research.org/wp-content/uploads/2025/04/Anouk-Slaghekke_Poster_Conference_2025.pdf",
       "verified": false
      },
      {
       "cite": "Appelman 2024, Nat Commun",
       "n": "25 LC vs 21 recovered",
       "design": "longitudinal case-control, biopsy pre/post exercise",
       "result": "Amyloid-containing deposits and myopathy worse after PEM",
       "url": "https://doi.org/10.1038/s41467-023-44432-3",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent replication of basement membrane thickening with blinded EM",
      "Is thickening caused by repeated ischaemia, immune deposition, or pericyte loss?",
      "Severe and housebound patients have not been biopsied"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial",
      "mitochondrial-energy"
     ],
     "test_available": "Research only (needle biopsy at a research centre)."
    },
    {
     "id": "vascular-pericytes-mural",
     "subdomain": "Microvasculature",
     "finding": "Pericytes and vascular smooth-muscle cells have not been examined directly in ME/CFS tissue, but blood proteins from mural cells and vessel matrix (NOTCH3, ITGAV, TGF-beta set, LUM) are lower across several ME/CFS cohorts and in long COVID.",
     "grade": "early",
     "plain": "Pericytes are the cells wrapped around capillaries that control their width. Nobody has looked at them in ME/CFS tissue yet; indirect blood clues suggest they may be fewer or less active.",
     "detail": "Solving ME/CFS re-analysis of SomaScan (Hoel, Germain, Walitt) and UK Biobank Olink data: ITGAV d -0.34 (UKB q=0.002), NOTCH3 -0.32, THBS1 -0.50, LUM -0.92; vascular smooth-muscle tissue score d -0.48 (p=0.001) but flat in UKB competitive tests. The long COVID UK Explore set also showed lower endothelial/TGF-beta proteins (ANGPT2 -0.84, EDN1 -0.65). Lower circulating NOTCH3 could reflect less shedding rather than fewer cells. The basement-membrane thickening reported in muscle (where pericytes help build the membrane) is a possible link.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "3 SomaScan cohorts + UKB Olink + LC UK",
       "design": "secondary meta-analysis",
       "result": "Lower mural/ECM proteins in 3-4 cohorts and long COVID",
       "url": "/#data",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "UKB",
       "design": "competitive gene-set test",
       "result": "Vascular smooth-muscle set flat in UKB",
       "url": "/#data",
       "verified": false
      }
     ],
     "open_questions": [
      "Stain pericyte markers (PDGFRB, NG2, alpha-SMA) in existing muscle and skin biopsies",
      "Relate NOTCH3/ITGAV levels to orthostatic stroke volume"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial",
      "connective-tissue"
     ],
     "test_available": "Research only."
    },
    {
     "id": "vascular-vascular-proteins-blood",
     "subdomain": "Blood biomarkers",
     "finding": "Proteomic panels do not show a consistent rise in classic endothelial-activation or vasoactive proteins in ME/CFS; most named candidates are null and the endothelial-activation set trends down.",
     "grade": "contested",
     "plain": "Blood tests for vessel damage markers mostly come back normal in ME/CFS when large datasets are pooled, which differs from what small studies suggested.",
     "detail": "Solving ME/CFS re-analysis: VWF, SELP, SELE, VCAM1, ANGPT1/2, VEGFA, EDN1, ADM, ACE, REN, NPPB null; ICAM1 +0.20 (driven by UKB), ACE2 +0.21, NPPC +0.23; endothelial-activation set d -0.30 (q=0.09, down). By contrast, smaller ELISA studies report raised endothelin-1 (Haffke 2022) and Wallraven 2026 found raised IL-6, ICAM-1, VCAM-1 in PCS and ME/CFS. Post-COVID ME/CFS may differ from pre-pandemic cohorts.",
     "studies": [
      {
       "cite": "Haffke 2022, J Transl Med",
       "n": "14 PCS-ME/CFS, 16 PCS, HC",
       "design": "case-control ELISA",
       "result": "ET-1 raised in both patient groups",
       "url": "https://doi.org/10.1186/s12967-022-03346-2",
       "verified": true
      },
      {
       "cite": "Wallraven 2026, BMC Med",
       "n": "102 PCS (62 ME/CFS) vs 198 controls",
       "design": "case-control",
       "result": "IL-6, ICAM-1, VCAM-1 raised",
       "url": "https://doi.org/10.1186/s12916-026-05144-9",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "~150 ME/CFS SomaScan + UKB Olink",
       "design": "secondary analysis",
       "result": "Named endothelial/vasoactive proteins null; activation set lower",
       "url": "/#data",
       "verified": false
      },
      {
       "cite": "Scherbakov 2020, ESC Heart Fail",
       "n": "35 ME/CFS",
       "design": "case-control",
       "result": "sVCAM and sICAM did not differ between patients with and without ED",
       "url": "https://doi.org/10.1002/ehf2.12633",
       "verified": true
      }
     ],
     "open_questions": [
      "Are post-COVID cohorts more inflammatory/endothelial than pre-pandemic ME/CFS?",
      "Do any of these markers change after exertion?"
     ],
     "hypotheses": [
      "vascular-endothelial",
      "innate-hyperactivation"
     ],
     "test_available": "Endothelin-1, ICAM-1 and VCAM-1 are available from some specialist labs but not validated for ME/CFS."
    },
    {
     "id": "vascular-vegf",
     "subdomain": "Blood biomarkers",
     "finding": "VEGF-A has been reported lower, not higher, in ME/CFS plasma, and proteomic re-analysis finds no consistent change; long COVID cohorts more often report raised VEGF.",
     "grade": "contested",
     "plain": "VEGF is a signal that makes new small vessels. Results in ME/CFS are inconsistent, and they differ from long COVID.",
     "detail": "Landi 2016 reported clustered reductions of IL-7, IL-16 and VEGF-A in ME/CFS that were not seen in liver-disease fatigue controls. Large cytokine panels (e.g. Montoya 2017) did not find VEGF among robust differences; the Solving ME/CFS proteome re-analysis found VEGFA null. In long COVID several cohorts report raised VEGF (e.g. Patel 2022). Flaskamp 2022 found post-COVID non-ME sera pro-angiogenic but post-COVID ME/CFS sera not, consistent with a missing compensatory angiogenic response in ME/CFS.",
     "studies": [
      {
       "cite": "Landi 2016, Cytokine",
       "n": "ME/CFS vs controls and liver-disease controls",
       "design": "case-control multiplex",
       "result": "Lower VEGF-A, IL-7, IL-16",
       "url": "https://doi.org/10.1016/j.cyto.2015.11.018",
       "verified": true
      },
      {
       "cite": "Flaskamp 2022, Cells",
       "n": "PCS-ME/CFS vs PCS vs HC sera",
       "design": "in vitro tube formation",
       "result": "PCS sera pro-angiogenic; PCS-ME/CFS sera not",
       "url": "https://doi.org/10.3390/cells11152376",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "SomaScan + Olink cohorts",
       "design": "secondary analysis",
       "result": "VEGFA null",
       "url": "/#data",
       "verified": false
      }
     ],
     "open_questions": [
      "Is there a tissue-level angiogenic deficit (e.g. muscle capillary rarefaction) that blood VEGF misses?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial"
     ],
     "test_available": "Research only."
    },
    {
     "id": "vascular-rbc-deformability",
     "subdomain": "Red blood cells",
     "finding": "Red blood cells from ME/CFS patients were stiffer in one microfluidic study, and real-time deformability cytometry found altered red-cell deformation persisting after COVID-19.",
     "grade": "early",
     "plain": "Red cells need to bend to squeeze through capillaries. In a small study, patients' red cells were stiffer.",
     "detail": "Saha 2019 (Stanford/OMF, custom microfluidic channels, small paired sample, about 12-14 patients) reported slower entry and reduced elongation of ME/CFS red cells. Kubánková 2021 (Biophys J, real-time deformability cytometry) found altered erythrocyte deformation and size heterogeneity persisting months after COVID-19. No independent ME/CFS replication; older reports of abnormal red-cell shape (stomatocytes) are from the 1990s and used subjective scoring.",
     "studies": [
      {
       "cite": "Saha 2019, Clin Hemorheol Microcirc",
       "n": "small ME/CFS vs age-matched HC sample",
       "design": "case-control microfluidics",
       "result": "ME/CFS RBCs significantly stiffer",
       "url": "https://doi.org/10.3233/CH-180469",
       "verified": true
      },
      {
       "cite": "Kubánková 2021, Biophys J",
       "n": "17 COVID-19, 14 recovered, 24 HC",
       "design": "RT-DC",
       "result": "Erythrocyte deformation changes persisted after recovery",
       "url": "https://doi.org/10.1016/j.bpj.2021.05.025",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication with RT-DC or ektacytometry in a larger ME/CFS cohort",
      "Is stiffness linked to oxidative stress or membrane lipids?"
     ],
     "hypotheses": [
      "small-vessel-capillary"
     ],
     "test_available": "Research only (ektacytometry exists in some haematology labs)."
    },
    {
     "id": "vascular-microclots",
     "subdomain": "Coagulation",
     "finding": "Fibrinaloid microclots (thioflavin-T-positive amyloid fibrin deposits) are reported to be increased in platelet-poor plasma in ME/CFS and long COVID, but the method is not standardised and haemostasis experts dispute the inference that they cause symptoms.",
     "grade": "contested",
     "plain": "One research network sees tiny clot-like particles in patients' plasma. Other experts say the method is unreliable and the particles may not exist in circulating blood. Anticoagulant treatment based on them is not supported.",
     "detail": "Nunes 2022 (25 ME/CFS vs 15 HC): microclot image area commonly >10-fold higher, platelet spreading score 2.72 vs 1.00, hypercoagulable TEG; load lower than in long COVID. Kruger 2022 found entrapped inflammatory proteins in long COVID microclots. Dalton 2024 (Sheffield, independent group, semi-automated counting) found higher counts in long COVID as a cohort, with wide overlap and high counts also after recent COVID. Critiques: visual scoring, uncertain blinding, ex vivo artefact possible, no in vivo demonstration; Hunt 2024 (RPTH) concluded the hypothesis is not evidence-based and unlicensed antithrombotics or apheresis are not justified outside trials; Kell 2024 rebutted. Solving ME/CFS proteome re-analysis found a platelet/microclot protein set slightly lower, not higher.",
     "studies": [
      {
       "cite": "Nunes 2022, Pharmaceuticals",
       "n": "25 ME/CFS vs 15 HC",
       "design": "case-control, fluorescence microscopy + TEG",
       "result": "Microclot area >10x; platelet hyperactivation",
       "url": "https://doi.org/10.3390/ph15080931",
       "verified": true
      },
      {
       "cite": "Dalton 2024, medRxiv",
       "n": "long COVID vs controls",
       "design": "case-control, semi-automated counting",
       "result": "Higher counts in LC as a group, broad overlap",
       "url": "https://doi.org/10.1101/2024.04.04.24305318",
       "verified": true
      },
      {
       "cite": "Kruger 2022, Cardiovasc Diabetol",
       "n": "long COVID",
       "design": "proteomics of microclots",
       "result": "Entrapped inflammatory molecules, alpha-2 antiplasmin",
       "url": "https://doi.org/10.1186/s12933-022-01623-4",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Hunt 2024, Res Pract Thromb Haemost",
       "n": "review",
       "design": "critical appraisal",
       "result": "Microclot inferences not evidence-based; antithrombotics not justified",
       "url": "https://doi.org/10.1016/j.rpth.2024.102442",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "SomaScan + Olink cohorts",
       "design": "secondary analysis",
       "result": "Platelet/microclot protein set d -0.33 (down)",
       "url": "/#data",
       "verified": false
      }
     ],
     "open_questions": [
      "Blinded, multi-lab, standardised counting in ME/CFS with disease controls",
      "Do microclots exist in vivo (e.g. capillaroscopy, biopsy)?",
      "Any controlled trial of antithrombotic therapy"
     ],
     "hypotheses": [
      "microclots",
      "complement-thromboinflammation"
     ],
     "test_available": "Not reliably: some private labs offer microclot imaging; no validated clinical test."
    },
    {
     "id": "vascular-platelets",
     "subdomain": "Coagulation",
     "finding": "Platelet activation findings in ME/CFS are inconsistent: one network reports hyperactivated platelets, while an earlier controlled study found no platelet activation or hypercoagulability.",
     "grade": "contested",
     "plain": "Whether platelets are overactive in ME/CFS depends on who measured and how.",
     "detail": "Nunes 2022 reported PAC-1 and CD62P staining and spreading consistent with activation. Kennedy 2006 (Dundee) measured platelet aggregation, P-selectin and coagulation markers and found no evidence of activation or hypercoagulability, explicitly arguing against antiplatelet therapy. In long COVID, Cervia-Hasler 2024 found monocyte-platelet aggregates and platelet-activation markers raised. No ME/CFS-specific flow cytometry replication.",
     "studies": [
      {
       "cite": "Nunes 2022, Pharmaceuticals",
       "n": "25 vs 15",
       "design": "case-control",
       "result": "Platelet hyperactivation by microscopy",
       "url": "https://doi.org/10.3390/ph15080931",
       "verified": true
      },
      {
       "cite": "Cervia-Hasler 2024, Science",
       "n": "113 COVID-19 (40 LC) + 39 controls",
       "design": "longitudinal proteomics",
       "result": "Platelet activation and monocyte-platelet aggregates in active LC",
       "url": "https://doi.org/10.1126/science.adg7942",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Kennedy 2006, Blood Coagul Fibrinolysis",
       "n": "CFS vs controls",
       "design": "case-control",
       "result": "No platelet activation or hypercoagulable state",
       "url": "https://doi.org/10.1097/01.mbc.0000214705.80997.73",
       "verified": true
      }
     ],
     "open_questions": [
      "Standardised whole-blood flow cytometry in ME/CFS with exercise challenge"
     ],
     "hypotheses": [
      "microclots",
      "complement-thromboinflammation"
     ],
     "test_available": "Research only."
    },
    {
     "id": "vascular-coagulation-markers",
     "subdomain": "Coagulation",
     "finding": "Routine coagulation markers (D-dimer, fibrinogen, PT/APTT) are generally normal in ME/CFS; claims of hypercoagulability rest on small 1990s studies and on thromboelastography from one network.",
     "grade": "contested",
     "plain": "Standard clotting blood tests are usually normal in ME/CFS.",
     "detail": "Berg 1999 reported a 'hypercoagulable state' (soluble fibrin monomer, thrombin-antithrombin) in a small sample; Kennedy 2006 found no coagulation differences. Proteomic re-analysis found the coagulation set null. Thromboelastography abnormalities (Nunes 2022; Saleem 2026 review) are not validated as ME/CFS markers.",
     "studies": [
      {
       "cite": "Berg 1999, Blood Coagul Fibrinolysis",
       "n": "small CFS sample",
       "design": "case-control",
       "result": "Reported hypercoagulable markers",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Berg+1999+hypercoagulable+chronic+fatigue+syndrome",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Kennedy 2006, Blood Coagul Fibrinolysis",
       "n": "CFS vs controls",
       "design": "case-control",
       "result": "No coagulation differences",
       "url": "https://doi.org/10.1097/01.mbc.0000214705.80997.73",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "SomaScan + Olink cohorts",
       "design": "secondary analysis",
       "result": "Coagulation set null",
       "url": "/#data",
       "verified": false
      }
     ],
     "open_questions": [
      "Do markers change transiently after exertion?"
     ],
     "hypotheses": [
      "microclots",
      "complement-thromboinflammation"
     ],
     "test_available": "D-dimer, fibrinogen, PT/APTT are routine; expected normal."
    },
    {
     "id": "vascular-complement-long-covid",
     "subdomain": "Complement and thromboinflammation",
     "finding": "Active long COVID shows persistent complement dysregulation (terminal complex, alternative and classical pathway activation) with thromboinflammation markers, replicated across several cohorts.",
     "grade": "replicated",
     "plain": "Complement is part of the innate immune system. In long COVID it stays switched on months after infection, together with signs of platelet and vessel injury.",
     "detail": "Cervia-Hasler 2024 (Science): >6,500 proteins in 268 longitudinal samples; terminal complement dysregulation, classical activation linked to herpesvirus antibody titres, raised haemolysis, tissue-injury and platelet markers. Baillie 2024 (Med, 166 LC vs 79): iC3b, TCC, Ba and C5a predicted LC (AUC 0.785). Further partial replication in Klein 2023 and Barratt-Due 2024; one adjusted null (Farztdinov preprint). Long COVID only; ME/CFS evidence is separate.",
     "studies": [
      {
       "cite": "Cervia-Hasler 2024, Science",
       "n": "113 COVID-19 (40 LC) + 39 controls",
       "design": "prospective longitudinal proteomics",
       "result": "Terminal and alternative/classical complement activation; thromboinflammation",
       "url": "https://doi.org/10.1126/science.adg7942",
       "verified": true
      },
      {
       "cite": "Baillie 2024, Med",
       "n": "166 LC vs 79 recovered",
       "design": "case-control",
       "result": "Complement activation fragments raised; 4-marker AUC 0.785",
       "url": "https://doi.org/10.1016/j.medj.2024.01.011",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Farztdinov 2024, preprint",
       "n": "29 vs 56",
       "design": "case-control",
       "result": "Complement differences not significant after age/BMI adjustment",
       "url": "https://www.medrxiv.org/search/Farztdinov%20complement%20long%20COVID",
       "verified": false
      }
     ],
     "open_questions": [
      "Does complement activation persist in long-duration LC that meets ME/CFS criteria?",
      "Trials of complement inhibitors (none completed in LC or ME/CFS as of late 2026 to our knowledge)"
     ],
     "hypotheses": [
      "complement-thromboinflammation",
      "viral-persistence",
      "herpes-reactivation",
      "innate-hyperactivation"
     ],
     "test_available": "C3, C4, CH50, sC5b-9 available in some immunology labs; not validated for long COVID or ME/CFS."
    },
    {
     "id": "vascular-complement-mecfs",
     "subdomain": "Complement and thromboinflammation",
     "finding": "In ME/CFS, complement findings are heterogeneous: an exercise-induced C4a rise, a genetically defined alternative-pathway subgroup, and proteomic signals that point in different directions across cohorts.",
     "grade": "contested",
     "plain": "Complement may be abnormal in some ME/CFS patients, but studies disagree on which part and in which direction.",
     "detail": "Sorensen 2003/2009 (CDC): C4a rose after exercise in CFS, with lectin-pathway MASP2 transcript differences. Maya 2026 (IJMS, CDC; 50 ME/CFS vs 121 controls from population sample): complement pQTL carriers showed alternative-pathway dysregulation (high C3/low Bb), six pQTLs linked to fatigue phenotypes in UK Biobank; SC5b-9 association possibly genetic. Solving ME/CFS re-analysis: alternative/terminal complement up in Hoel and UKB, null in Germain, down in Walitt (I2 0.7-0.9); CSF complement/coagulation sets lower in ME/CFS with POTS (Bragée 2026, 10 vs 21, exploratory).",
     "studies": [
      {
       "cite": "Sorensen 2009, Mol Med",
       "n": "CFS vs controls, exercise",
       "design": "case-control, exercise challenge",
       "result": "Lectin-pathway changes underlying post-exercise C4a rise",
       "url": "https://doi.org/10.2119/molmed.2008.00098",
       "verified": true
      },
      {
       "cite": "Maya 2026, Int J Mol Sci",
       "n": "50 ME/CFS vs 121 non-fatigued",
       "design": "population-based case-control + pQTL",
       "result": "Alternative-pathway subgroup (high C3/low Bb) linked to pQTLs",
       "url": "https://doi.org/10.3390/ijms27031574",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome), 2026",
       "n": "4 cohorts",
       "design": "secondary meta-analysis",
       "result": "Complement direction inconsistent between cohorts",
       "url": "/#data",
       "verified": false
      }
     ],
     "open_questions": [
      "Measure activation fragments (C5a, sC5b-9, Ba) with careful pre-analytics before and after exertion",
      "Is the long COVID complement signal present in pre-pandemic ME/CFS?"
     ],
     "hypotheses": [
      "complement-thromboinflammation",
      "innate-hyperactivation",
      "genetic-neuronal"
     ],
     "test_available": "Research only for activation products; C3/C4 routine but non-specific."
    },
    {
     "id": "vascular-pots-overlap",
     "subdomain": "POTS overlap",
     "finding": "POTS is present in roughly a fifth of ME/CFS patients on formal testing (prevalence varies 6-43%), but cerebral hypoperfusion and preload failure are found in most patients regardless of whether they meet POTS heart-rate criteria.",
     "grade": "replicated",
     "plain": "POTS is a fast heart rate on standing. Only some ME/CFS patients have it, but most have reduced brain blood flow when upright anyway.",
     "detail": "Novak 2026: POTS 19% ME/CFS and 22% long COVID, but reduced orthostatic CBFv 88-92%. van Campen 2020: CBF abnormal in 82% of patients with normal HR/BP response. van Campen 2024/2025 split ME/CFS-POTS into a venous-pooling profile and a hyperadrenergic profile (HR rise >=40) with different CO-CBF relations, which may have different treatment implications. Yamazaki 2026: hypocapnia more common than POTS on a lean test. Low plasma volume and the renin-aldosterone paradox are well described in POTS itself.",
     "studies": [
      {
       "cite": "Novak 2026, PLOS One",
       "n": "170 ME/CFS, 143 LC",
       "design": "retrospective",
       "result": "POTS 19%/22%; orthostatic CBFv reduction 88%/92%",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "van Campen 2024, J Clin Med",
       "n": "233 ME/CFS-POTS",
       "design": "retrospective tilt",
       "result": "Two haemodynamic POTS profiles",
       "url": "https://doi.org/10.3390/jcm13247726",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Prospective trial stratified by POTS profile (venous pooling vs hyperadrenergic)",
      "Standardised orthostatic testing duration and posture across studies"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Tilt table or 10-minute NASA lean test (widely available); add capnography and stroke-volume monitoring where possible."
    }
   ],
   "hypotheses_notes": [
    {
     "id": "low-blood-volume-preload",
     "for": "Red-cell volume deficits in 5 groups; small, under-filled heart explained by volume; preload failure on iCPET in most tested; CBF falls on tilt in about 90%; compression and vasoconstrictors reduce CBF falls.",
     "against": "Blood-volume category did not predict orthostatic abnormalities (Yamazaki 2026); iCPET cohorts pre-selected for low RAP; one Dutch clinic supplies most CBF data; volume-regulating hormones not consistently abnormal."
    },
    {
     "id": "vascular-endothelial",
     "for": "FMD/PORH/EndoPAT reduced in 3 independent cohorts; patient plasma/IgG impairs endothelial cells in vitro in 3 labs; retinal vessel narrowing in post-COVID ME/CFS.",
     "against": "Inactivity alone lowers FMD; blood endothelial-activation proteins are not raised in pooled proteomics; no in vitro assay replicated across labs."
    },
    {
     "id": "small-vessel-capillary",
     "for": "Lower muscle capillarisation unlike bed rest (Charlton 2026); basement membrane thickening (poster); retinal and sublingual capillary loss in long COVID; poor O2 extraction subgroup.",
     "against": "Mostly long COVID or small samples; basement-membrane data not peer-reviewed; no severe patients biopsied."
    },
    {
     "id": "microclots",
     "for": "Pretorius/Kell network reports raised microclot load in ME/CFS and LC; Dalton 2024 independent count in LC higher on average.",
     "against": "Method unstandardised and visually scored; wide overlap with controls; no in vivo proof; haemostasis critiques (Hunt 2024); platelet/coagulation proteins not raised in pooled proteomics; Kennedy 2006 no platelet activation."
    },
    {
     "id": "complement-thromboinflammation",
     "for": "Replicated complement activation in active long COVID; exercise C4a rise and genetic alternative-pathway subgroup in ME/CFS.",
     "against": "ME/CFS proteomic complement direction inconsistent across cohorts; no complement-inhibitor trial."
    },
    {
     "id": "autonomic-small-fibre",
     "for": "SFN in about a third to half of iCPET/autonomic cohorts; shunting proposed to explain poor extraction; hyperadrenergic POTS profile.",
     "against": "Denervation severity did not correlate with exertional measures (Joseph 2021)."
    },
    {
     "id": "serum-factor",
     "for": "Patient plasma lowers endothelial NO; IgG fragments endothelial mitochondria (MS-controlled).",
     "against": "Single-lab assays, no cross-lab replication."
    }
   ],
   "glossary": [
    {
     "term": "Preload",
     "plain": "How much blood fills the heart before each beat. Low preload means each beat pumps less."
    },
    {
     "term": "Invasive CPET (iCPET)",
     "plain": "An exercise test with catheters in the heart and an artery, measuring filling pressures and oxygen use directly."
    },
    {
     "term": "Flow-mediated dilation (FMD)",
     "plain": "Ultrasound measure of how much an arm artery widens after a cuff is released; a test of the vessel lining."
    },
    {
     "term": "PORH",
     "plain": "Post-occlusive reactive hyperaemia: the skin blood-flow surge after a cuff is released, a test of small vessels."
    },
    {
     "term": "Hypocapnia",
     "plain": "Low carbon dioxide in the blood, usually from over-breathing; it narrows brain arteries."
    },
    {
     "term": "Basement membrane",
     "plain": "A thin protein layer around each capillary; thickening can slow oxygen passing to tissue."
    },
    {
     "term": "Pericyte",
     "plain": "A cell wrapped around capillaries that controls their width and helps keep their walls intact."
    },
    {
     "term": "Complement",
     "plain": "About 30 blood proteins in the innate immune system that tag and punch holes in targets; terminal complex (sC5b-9) marks full activation."
    },
    {
     "term": "Fibrinaloid microclot",
     "plain": "A clot-like particle of misfolded fibrin seen under a microscope with an amyloid dye; its significance is disputed."
    },
    {
     "term": "Red cell volume",
     "plain": "The total amount of red blood cells in the body, measured with tracer methods, not by a standard blood count."
    },
    {
     "term": "OCT-A",
     "plain": "Optical coherence tomography angiography: a non-invasive scan of the tiny vessels in the retina."
    }
   ],
   "key_reviews": [
    {
     "cite": "Joseph 2021, Chest",
     "url": "https://doi.org/10.1016/j.chest.2021.01.082",
     "why": "Defines preload failure and impaired extraction phenotypes in the largest iCPET series."
    },
    {
     "cite": "van Campen 2020, Clin Neurophysiol Pract",
     "url": "https://doi.org/10.1016/j.cnp.2020.01.003",
     "why": "Largest cerebral blood flow on tilt study (429 patients)."
    },
    {
     "cite": "Nunes, Kell, Slaghekke, Wüst 2026, Cell Death Dis",
     "url": "https://doi.org/10.1038/s41419-025-08162-2",
     "why": "Recent synthesis proposing endothelial senescence as a unifying model (hypothesis paper, not evidence)."
    },
    {
     "cite": "Hunt 2024, Res Pract Thromb Haemost",
     "url": "https://doi.org/10.1016/j.rpth.2024.102442",
     "why": "Haemostasis-expert critique of the microclot hypothesis."
    },
    {
     "cite": "Novak 2026, PLOS One",
     "url": "https://doi.org/10.1371/journal.pone.0341278",
     "why": "Shared autonomic/cerebrovascular phenotype of long COVID and ME/CFS in 313 patients."
    },
    {
     "cite": "Westermeier, Pretorius, Untersmayr 2026, Cardiovasc Diabetol",
     "url": "https://doi.org/10.1186/s12933-026-03316-8",
     "why": "Cardiometabolic and vascular perspective on PEM."
    }
   ],
   "gaps": [
    "No blinded, multi-centre replication of the van Campen cerebral blood flow method.",
    "Preload failure prevalence in unselected ME/CFS (iCPET cohorts are pre-selected).",
    "No placebo-controlled trials of volume expansion (saline, desmopressin, fludrocortisone) targeting measured low blood volume in ME/CFS.",
    "Microclots: no standardised blinded multi-lab assay and no in vivo demonstration.",
    "Retinal, sublingual and muscle capillary studies are almost all post-COVID; pre-pandemic ME/CFS is rarely studied.",
    "Severe and very severe patients are almost absent from vascular studies.",
    "Pericyte markers have never been stained in ME/CFS tissue.",
    "Complement activation fragments have not been measured with modern pre-analytics before and after exertion in ME/CFS.",
    "Whether endothelial dysfunction exceeds what activity-matched inactive controls show.",
    "Mechanism of low red-cell mass (marrow output vs destruction vs EPO signalling) is unstudied."
   ]
  },
  {
   "domain": "muscle-exertion",
   "title": "Muscle, exertion and energy",
   "overview": "People with ME/CFS reliably have lower aerobic capacity and a low peak heart rate on exercise testing, and they report delayed, prolonged PEM that took about two weeks to resolve after a 2-day test. Whether performance measurably drops on a second test 24 hours later is now contested: several linked groups and two meta-analyses say yes, mainly at the ventilatory threshold, while a 2026 replication found no change. Invasive testing points to poor cardiac filling and poor oxygen extraction, and newer biopsy work from one Amsterdam group finds fewer oxidative fibres, fewer capillaries and lower mitochondrial capacity in ME/CFS that bed rest does not reproduce, plus post-exertion muscle damage and amyloid-containing deposits in long COVID. Mitochondrial studies in blood cells disagree in direction, and the idea that patient serum alters muscle-cell mitochondria failed a large pre-registered replication (Ryback 2026). Post-exertion omics (metabolomics, proteomics, EVs) show resting differences and recovery-phase changes, but within-person exercise effects are modest and mostly unreplicated.",
   "findings": [
    {
     "subdomain": "PEM definition and timing",
     "finding": "PEM is usually delayed by hours to a day, lasts days to weeks, and after a 2-day CPET took ME/CFS patients a mean of about 13 days to recover versus about 2 days in sedentary controls.",
     "grade": "several",
     "plain": "After overdoing it, people with ME/CFS often feel worse later rather than at once, and the crash can last much longer than ordinary tiredness after exercise.",
     "detail": "Chu 2018 (n=150 survey): 84% reported PEM lasting 24 h or more; onset varied, and only 11% had a consistent delay of at least 24 h. Moore 2023 (80 vs 64, Ithaca 2-day CPET): judged recovery 12.7 ± 1.2 d vs 2.1 ± 0.2 d, range 1-64 d; one patient had not recovered at one year. NIH qualitative work (Stussman 2024, 10 vs 9) found every patient's PEM had a different onset, trajectory and worst symptom, and VAS scales missed changes that interviews caught. Self-report designs and one centre for the recovery data are the main limits.",
     "studies": [
      {
       "cite": "Chu 2018, PLOS ONE",
       "n": "150 Fukuda CFS",
       "design": "patient-centred cross-sectional survey",
       "result": "90% PEM with physical, cognitive and emotional triggers; 84% PEM >=24 h; 11% consistent >=24 h delay",
       "url": "https://doi.org/10.1371/journal.pone.0197811",
       "verified": true
      },
      {
       "cite": "Moore 2023, Medicina",
       "n": "80 ME/CFS vs 64 controls",
       "design": "prospective symptom tracking for 10 days after 2-day CPET",
       "result": "Recovery 12.7 d vs 2.1 d (p<0.0001); <10% of patients took >3 weeks",
       "url": "https://doi.org/10.3390/medicina59030571",
       "verified": true
      },
      {
       "cite": "Stussman 2024, BMJ Neurol Open",
       "n": "10 ME/CFS vs 9 controls",
       "design": "mixed-methods (VAS + qualitative interviews) over 72 h after CPET",
       "result": "Each patient had a unique PEM course; no controls had PEM; interviews outperformed VAS at peak PEM",
       "url": "https://doi.org/10.1136/bmjno-2023-000529",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Stussman 2025, Front Neurol",
       "n": "34 long COVID exercise cohort, 9 ME/CFS, 9 controls; 244 questionnaire cohort",
       "design": "CPET with serial VAS and interviews over 72 h",
       "result": "Self-reported PEM 67% in long COVID questionnaires, but only 2 of 34 long COVID patients showed observable PEM after CPET; ME/CFS PEM was more severe and prolonged",
       "url": "https://doi.org/10.3389/fneur.2025.1534352",
       "verified": true
      }
     ],
     "open_questions": [
      "Is there an objective marker that tracks PEM onset and recovery within a person?",
      "Why does self-reported PEM in long COVID often not reproduce after a laboratory stressor?",
      "Do cognitive and orthostatic triggers produce the same biology as physical exertion?"
     ],
     "hypotheses": [
      "sickness-behaviour",
      "cell-danger-response",
      "mitochondrial-energy"
     ],
     "test_available": "Clinical history and symptom diaries (DSQ-PEM questionnaire); no objective clinical test",
     "id": "muscle-exertion-pem-timing"
    },
    {
     "subdomain": "Exercise capacity (single CPET)",
     "finding": "On a single maximal exercise test, ME/CFS patients have lower peak oxygen uptake (pooled about 5 ml/kg/min below controls) with a low peak heart rate (chronotropic incompetence).",
     "grade": "replicated",
     "plain": "Patients can take in and use less oxygen at maximal effort than healthy people, and their heart rate does not rise as high.",
     "detail": "Large clinical series (VanNess 2003, n=189) and case-control studies (Keller 2024, Walitt 2024 NIH, Charlton 2026) agree. Effort criteria (RER, perceived exertion) are usually met, which argues against low effort. The main confounder is deconditioning; Keller 2024 used VO2-matched pairs and Charlton 2026 compared with 60-day bed rest, and both found patterns that inactivity did not reproduce. A single CPET's peak VO2 overlaps heavily with sedentary controls, so it is not diagnostic on its own.",
     "studies": [
      {
       "cite": "VanNess 2003, Med Sci Sports Exerc",
       "n": "189 CFS",
       "design": "clinical series, single CPET",
       "result": "Peak VO2 below predicted in all impairment groups; peak HR and SBP lower in more impaired groups",
       "url": "https://doi.org/10.1249/01.mss.0000069510.58763.e8",
       "verified": true
      },
      {
       "cite": "Franklin 2018/2021 meta-analysis (Teesside)",
       "n": "pooled studies",
       "design": "systematic review/meta-analysis",
       "result": "Pooled VO2peak 5.2 (95% CI 3.8-6.6) ml/kg/min lower in ME/CFS vs controls",
       "url": "https://research.tees.ac.uk/en/publications/repeated-maximal-exercise-tests-of-peak-oxygen-consumption-in-peo/",
       "verified": false
      },
      {
       "cite": "Walitt 2024, Nat Commun (NIH)",
       "n": "about 17 PI-ME/CFS vs 21 controls",
       "design": "deep phenotyping",
       "result": "Peak VO2 lower (p=0.004), peak HR lower (p=0.07), about 3.3 METs difference; patients performed at a lower % of predicted",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Keller 2024, J Transl Med",
       "n": "84 ME/CFS vs 71 sedentary controls (55 VO2-matched pairs)",
       "design": "2-day CPET, cycle ergometer, 24 h apart",
       "result": "ME/CFS failed to reproduce day-1 values: e.g. ~6% fall in peak VO2/kg and 6.7% fall in VO2 at ventilatory threshold in matched pairs; controls stable; both groups met maximal-effort criteria",
       "url": "https://doi.org/10.1186/s12967-024-05410-5",
       "verified": true
      },
      {
       "cite": "Charlton 2026, Nat Commun",
       "n": "19-23 ME/CFS, 24-25 long COVID, 29-30 matched controls, 24 healthy people after 60-day bed rest",
       "design": "case-control biopsies plus bed-rest comparison",
       "result": "ME/CFS: lower type I fibre proportion and type I-specific atrophy, lower SDH activity, lower OXPHOS capacity, lower capillary-to-fibre ratio; bed rest caused atrophy but no fibre-type shift; OXPHOS tracked VO2max only after bed rest",
       "url": "https://doi.org/10.1038/s41467-026-75725-y",
       "verified": true
      }
     ],
     "open_questions": [
      "How much of low VO2 is preload/venous return versus muscle oxygen use?",
      "Does peak VO2 track severity in severe and housebound patients, who are rarely tested?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "mitochondrial-energy",
      "autonomic-small-fibre",
      "deconditioning"
     ],
     "test_available": "Cardiopulmonary exercise test (CPET) at cardiology/sports-medicine labs; carries PEM risk",
     "id": "muscle-exertion-low-peak-vo2",
     "contradicting": []
    },
    {
     "subdomain": "2-day CPET",
     "finding": "ME/CFS patients perform worse on a second maximal exercise test 24 h after the first, most clearly in workload and VO2 at the ventilatory threshold, but the largest independent replication found no day-2 change.",
     "grade": "contested",
     "plain": "Several labs found that patients cannot repeat their performance a day after a hard test, while healthy people can. A 2026 study using the same design found no drop, so the test's reliability as a PEM marker is in doubt.",
     "detail": "Positive studies: Snell 2013 (51 vs 10 women; 95% classification), van Campen 2020/2021 (82 women by severity; idiopathic chronic fatigue improved on day 2 while ME/CFS declined), Keller 2024 (84 vs 71; declines of ~5-7% in peak VO2 and VO2 at threshold), Hodges 2018 (MS controls improved), Davenport 2026 preprint (84 ME/CFS, 79 long COVID, 71 controls). Meta-analyses: Lim 2020 (workload at VT difference -10.8 day 1 vs -33.0 day 2) and Franklin & Graham 2022 (work at anaerobic threshold -21 W, 78% predictive probability of exceeding a 10 W MCID). Most positive data come from the Workwell/Pacific/Ithaca network and the van Campen clinic, so they are not fully independent. Mancini/Natelson 2026 (58 vs 25) and Lien 2019 (VO2peak day-2 fall not different between groups) did not reproduce the VO2 effect. Effect sizes are small relative to test-retest noise, and threshold detection is operator-dependent.",
     "studies": [
      {
       "cite": "Snell 2013, Phys Ther",
       "n": "51 CFS vs 10 controls, women",
       "design": "2-day CPET",
       "result": "No difference on test 1; on test 2 lower VO2 and workload at peak and threshold; 95.1% classification",
       "url": "https://doi.org/10.2522/ptj.20110368",
       "verified": true
      },
      {
       "cite": "Keller 2024, J Transl Med",
       "n": "84 ME/CFS vs 71 sedentary controls (55 VO2-matched pairs)",
       "design": "2-day CPET, cycle ergometer, 24 h apart",
       "result": "ME/CFS failed to reproduce day-1 values: e.g. ~6% fall in peak VO2/kg and 6.7% fall in VO2 at ventilatory threshold in matched pairs; controls stable; both groups met maximal-effort criteria",
       "url": "https://doi.org/10.1186/s12967-024-05410-5",
       "verified": true
      },
      {
       "cite": "van Campen 2020, Healthcare",
       "n": "82 women (31 mild, 31 moderate, 20 severe)",
       "design": "2-day CPET by severity",
       "result": "All four parameters fell on day 2 (p 0.003 to <0.0001); peak workload fall largest in severe (-19%)",
       "url": "https://doi.org/10.3390/healthcare8030192",
       "verified": true
      },
      {
       "cite": "van Campen 2021, Healthcare",
       "n": "50 ME/CFS vs 51 idiopathic chronic fatigue (women); 26 vs 25 (men)",
       "design": "2-day CPET, disease-control comparison",
       "result": "ME/CFS worsened on day 2; idiopathic chronic fatigue improved",
       "url": "https://doi.org/10.3390/healthcare9060682",
       "verified": true
      },
      {
       "cite": "Hodges 2018, Clin Physiol Funct Imaging",
       "n": "10 CFS, 7 MS, 17 controls",
       "design": "pilot 2-day CPET with disease control",
       "result": "CFS workload fell on day 2; MS and controls improved",
       "url": "https://doi.org/10.1111/cpf.12460",
       "verified": true
      },
      {
       "cite": "Lim 2020, J Clin Med",
       "n": "pooled 2-day CPET studies",
       "design": "meta-analysis",
       "result": "All parameters fell on day 2 in ME/CFS, rose in controls; workload at VT difference grew from -10.8 to -33.0",
       "url": "https://doi.org/10.3390/jcm9124040",
       "verified": true
      },
      {
       "cite": "Franklin & Graham 2022, Fatigue: Biomed Health Behav",
       "n": "4-5 studies per outcome",
       "design": "systematic review and meta-analysis",
       "result": "Day-2 drop in peak work -8.55 W and in work at anaerobic threshold -21 W vs controls",
       "url": "https://research.tees.ac.uk/en/publications/repeated-maximal-exercise-tests-of-peak-oxygen-consumption-in-peo/",
       "verified": false
      },
      {
       "cite": "Davenport 2026, Research Square preprint",
       "n": "84 ME/CFS, 79 long COVID, 71 controls",
       "design": "retrospective 2-day CPET",
       "result": "Both patient groups had reduced VO2 and workload at threshold, larger in women; groups similar",
       "url": "https://doi.org/10.21203/rs.3.rs-8606329/v1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Mancini 2026, Front Physiol",
       "n": "58 ME/CFS vs 25 sedentary controls",
       "design": "2-day CPET replication attempt",
       "result": "No day-2 change in peak VO2 (22.3 to 22.5 ml/kg/min) or VO2 at threshold in either group; patients had lower peak HR and higher perceived exertion; authors say 2-day CPET should not be used to define PEM",
       "url": "https://doi.org/10.3389/fphys.2026.1816082",
       "verified": true
      },
      {
       "cite": "Lien 2019, Physiol Rep",
       "n": "18 vs 15 women",
       "design": "2-day CPET with arterial lactate",
       "result": "VO2peak fell on day 2 in patients, but the change did not differ significantly between groups",
       "url": "https://doi.org/10.14814/phy2.14138",
       "verified": true
      }
     ],
     "open_questions": [
      "Why did Mancini 2026 find no day-2 effect: cohort selection, threshold method, or sample from a different clinic?",
      "What is the test-retest variability of workload at VT in sedentary people, and does the day-2 effect exceed it?",
      "Can a gentler, submaximal provocation give the same information with less harm (Moore 2023: about two weeks to recover)?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "mitochondrial-energy",
      "autonomic-small-fibre"
     ],
     "test_available": "Offered by a few specialist labs (e.g. Workwell-linked) mainly for disability documentation; provokes PEM and is not advised for moderate-severe patients",
     "id": "muscle-exertion-two-day-cpet"
    },
    {
     "subdomain": "Lactate and glycolysis",
     "finding": "Some patients produce more lactate for a given workload, and this worsens on a repeat test, but plasma studies after one maximal test show a smaller total lactate rise that mainly reflects lower achieved workloads.",
     "grade": "several",
     "plain": "Muscles in some patients seem to lean on the quick, lactate-producing fuel route earlier than expected, especially the day after hard effort.",
     "detail": "Lien 2019 (18 vs 15 women): arterial lactate per power output was higher in patients on both tests and rose on day 2 while it fell in controls (interaction p<0.001). Lane 1998: about a third of patients had an abnormal lactate rise on a sub-anaerobic-threshold test, and this subgroup had fewer type 1 fibres (n=105 biopsies). Wong 1992 (31P-MRS) inferred accelerated glycolysis. The Solving ME/CFS re-analysis of Germain 2022 found the post-exercise lactate rise was smaller in patients (log2 +1.02 vs +1.40), which disappears as a signal once effort is considered. Lactate per watt, not peak lactate, is the informative measure.",
     "studies": [
      {
       "cite": "Lien 2019, Physiol Rep",
       "n": "18 vs 15 women",
       "design": "2-day CPET with arterial lactate every 30 s",
       "result": "Higher lactate per power output in patients; lactate increased on day 2 in patients and fell in controls",
       "url": "https://doi.org/10.14814/phy2.14138",
       "verified": true
      },
      {
       "cite": "Lane 1998, J Neurol Neurosurg Psychiatry",
       "n": "105 CFS biopsies",
       "design": "biopsy plus sub-anaerobic threshold exercise test",
       "result": "Abnormal lactate responders had fewer type 1 fibres; no ultrastructural abnormality",
       "url": "https://doi.org/10.1136/jnnp.64.3.362",
       "verified": true
      },
      {
       "cite": "Wong 1992, Chest",
       "n": "22 vs 21",
       "design": "31P-MRS during exercise to exhaustion",
       "result": "Similar pH/PCr patterns but faster changes and lower ATP at exhaustion in CFS; authors infer accelerated glycolysis",
       "url": "https://doi.org/10.1378/chest.102.6.1716",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis of Germain 2022 data",
       "n": "60 vs 45 (404 samples)",
       "design": "within-person change scores, mixed models, permutation",
       "result": "Resting differences real (~150 of 780 metabolites at p<0.05 vs ~35 expected, perm p=0.002); exercise-response differences at chance (perm p=0.12-0.61); survivors: smaller lactate rise (explained by effort), blunted post-exercise glucose rise, rise in oxidised cys-gly",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Is early lactate due to poor oxygen delivery, fibre-type shift, or a mitochondrial/PDH block?",
      "Does lactate per watt separate ME/CFS from deconditioned controls prospectively?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "small-vessel-capillary",
      "low-blood-volume-preload"
     ],
     "test_available": "Research only (lactate measured during CPET in some labs)",
     "id": "muscle-exertion-lactate"
    },
    {
     "subdomain": "Oxygen delivery and extraction",
     "finding": "Invasive exercise testing shows two peripheral limits in ME/CFS: low cardiac filling (preload failure) and, in a high-flow subgroup, poor systemic oxygen extraction by tissue.",
     "grade": "several",
     "plain": "During exercise, either not enough blood returns to the heart, or the blood that reaches muscle passes through without giving up enough oxygen.",
     "detail": "Joseph 2021 (Systrom lab, 160 ME/CFS vs 36 controls from 1,516 iCPETs): right atrial pressure 1.9 vs 8.3 mmHg, peak VO2 80% vs 101% predicted; the high-flow tertile had impaired extraction (0.74 vs 0.86-0.88), consistent with peripheral shunting; 31% had small-fibre neuropathy on skin biopsy. Melamed 2019: impaired extraction was the sole limit in 12.5% of unexplained exertional intolerance. Vermeulen 2014 (non-invasive, 178 CFS women) estimated lower peak extraction and a steep cardiac output/VO2 slope. McCully 1999 (NIRS) found slower muscle oxygen delivery. Invasive data come largely from one centre; capillary rarefaction in Charlton 2026 offers a structural explanation for low extraction.",
     "studies": [
      {
       "cite": "Joseph 2021, Chest",
       "n": "160 ME/CFS vs 36 controls",
       "design": "invasive CPET plus skin biopsy, retrospective",
       "result": "Low RAP (preload failure); impaired O2 extraction in high-flow tertile; 31% SFN",
       "url": "https://doi.org/10.1016/j.chest.2021.01.082",
       "verified": true
      },
      {
       "cite": "Melamed 2019, Eur J Appl Physiol",
       "n": "313 iCPETs for unexplained exertional intolerance",
       "design": "retrospective iCPET",
       "result": "Impaired systemic O2 extraction sole limit in 12.5%; higher mixed-venous saturation at peak",
       "url": "https://doi.org/10.1007/s00421-019-04222-6",
       "verified": true
      },
      {
       "cite": "Vermeulen 2014, J Transl Med",
       "n": "178 CFS women, 172 idiopathic fatigue, 11 healthy women (plus men)",
       "design": "CPET with non-invasive cardiac output (Nexfin)",
       "result": "Peak extraction 10.8 vs 13.5 ml/100 ml in women; dQ/dVO2 >6 L/L in 70% of patients",
       "url": "https://doi.org/10.1186/1479-5876-12-20",
       "verified": true
      },
      {
       "cite": "McCully 1999, Clin Sci",
       "n": "20 vs 12",
       "design": "NIRS plus 31P-MRS",
       "result": "Slower post-exercise and post-ischaemia muscle oxygen resaturation in CFS",
       "url": "https://doi.org/10.1042/cs0970603",
       "verified": true
      },
      {
       "cite": "Appelman 2024, Nat Commun",
       "n": "25 vs 21",
       "design": "NIRS during CPET",
       "result": "Muscle deoxygenation responses lower in long COVID",
       "url": "https://doi.org/10.1038/s41467-023-44432-3",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is poor extraction due to microvascular shunting, capillary loss, or mitochondria that cannot use oxygen?",
      "Independent invasive replication outside the Systrom lab is lacking.",
      "Do extraction subgroups predict response to pyridostigmine or volume expansion?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "small-vessel-capillary",
      "autonomic-small-fibre",
      "vascular-endothelial",
      "mitochondrial-energy"
     ],
     "test_available": "Invasive CPET (a few centres, e.g. Brigham/Harvard); NIRS research only",
     "id": "muscle-exertion-oxygen-extraction"
    },
    {
     "subdomain": "31P-MRS muscle energetics",
     "finding": "31P-MRS studies from Newcastle found exaggerated muscle acidosis and up to ~4-fold slower pH recovery after repeated exercise in a subset of patients, but other MRS studies found no consistent group abnormality.",
     "grade": "contested",
     "plain": "Magnetic scans of calf muscle during exercise suggest that some patients' muscles become more acidic and clear acid slowly, but not all studies see this.",
     "detail": "Jones 2010 (16 vs 8): proton efflux immediately post-exercise 1.1 vs 3.6 mmol/L/min, time to maximal efflux 25.6 vs 3.8 s, and the normal link between efflux and pH nadir was lost, possibly via autonomic dysfunction. Jones 2012 (18 vs 12): 10 of 18 had low PCr depletion; the other 8 showed more acidosis over three bouts, no adaptation and about 4-fold longer pH recovery. Lane 1998 found lower end-exercise pH and slower ATP synthesis only in the lactate-responder subgroup. Barnes 1993 (46 patients) found no consistent abnormality overall, with 12 outliers; Wong 1992 found similar end-points; a 2025 7 T study found no resting muscle differences. Small samples, subgroup analyses and heterogeneous criteria dominate.",
     "studies": [
      {
       "cite": "Jones 2010, J Intern Med",
       "n": "16 vs 8",
       "design": "31P-MRS plantar flexion at 35% MVC plus HRV",
       "result": "Suppressed proton efflux and delayed peak efflux; correlation with pH nadir lost in CFS",
       "url": "https://doi.org/10.1111/j.1365-2796.2009.02160.x",
       "verified": true
      },
      {
       "cite": "Jones 2012, Eur J Clin Invest",
       "n": "18 vs 12",
       "design": "31P-MRS over three exercise bouts",
       "result": "Subgroup with normal PCr depletion had more acidosis, no adaptation, ~4-fold slower pH recovery",
       "url": "https://doi.org/10.1111/j.1365-2362.2011.02567.x",
       "verified": true
      },
      {
       "cite": "Lane 1998, Neuromuscul Disord",
       "n": "10 SATET+ vs 9 SATET- vs 13 controls",
       "design": "forearm 31P-MRS",
       "result": "Lower end-exercise pH and slower ATP resynthesis in lactate-responder subgroup only",
       "url": "https://doi.org/10.1016/s0960-8966(98)00021-2",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Barnes 1993, J Neurol Neurosurg Psychiatry",
       "n": "46 CFS vs controls and mitochondrial myopathy",
       "design": "31P-MRS",
       "result": "No consistent abnormality of glycolysis, mitochondrial metabolism or pH regulation; 12 of 46 outside normal range",
       "url": "https://doi.org/10.1136/jnnp.56.6.679",
       "verified": true
      },
      {
       "cite": "Wong 1992, Chest",
       "n": "22 vs 21",
       "design": "31P-MRS gastrocnemius",
       "result": "No group differences in pH, PCr or Pi at rest, exhaustion or recovery; lower ATP at exhaustion",
       "url": "https://doi.org/10.1378/chest.102.6.1716",
       "verified": true
      },
      {
       "cite": "Godlewska 2025, Mol Psychiatry",
       "n": "24 ME/CFS, 25 long COVID, 24 controls",
       "design": "7 T MRS brain and calf muscle at rest",
       "result": "Resting muscle metabolites did not differ (brain lactate raised in ME/CFS)",
       "url": "https://doi.org/10.1038/s41380-025-03108-8",
       "verified": true
      }
     ],
     "open_questions": [
      "Would a pre-registered, larger repeat-bout 31P-MRS study with matched activity levels reproduce the Newcastle subgroup?",
      "Is slow pH recovery a perfusion problem or a muscle-intrinsic one?",
      "Does the MRS phenotype change during PEM?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "autonomic-small-fibre",
      "sodium-calcium-overload",
      "small-vessel-capillary"
     ],
     "test_available": "Research MRI only",
     "id": "muscle-exertion-31p-mrs-ph"
    },
    {
     "subdomain": "Muscle sodium MRI",
     "finding": "23Na-MRI in 6 women with ME/CFS and 6 controls found higher resting muscle sodium in all lower-leg compartments and a larger sodium rise after exercise, inversely related to grip strength.",
     "grade": "early",
     "plain": "A special MRI scan found more salt inside leg muscles of patients, which in theory could disturb muscle energy and calcium handling. The study was very small.",
     "detail": "Petter 2022: anterior compartment 12.20 ± 1.66 vs 9.38 ± 0.71 mM (p=0.0034); medial gastrocnemius rose 30% vs 17% at 12 min after plantar flexion. Sodium correlated with lower handgrip strength (R2=0.38). This fits the Wirth/Scheibenbogen model in which hypoperfusion overloads the Na+/H+ exchanger, then Na+/Ca2+ exchange, loading muscle with calcium and damaging mitochondria. No independent replication; n=12 total.",
     "studies": [
      {
       "cite": "Petter 2022, J Transl Med",
       "n": "6 vs 6 women",
       "design": "23Na-MRI lower leg before and after plantar flexion",
       "result": "Higher baseline sodium in all compartments; larger exercise rise; correlates with weaker grip",
       "url": "https://doi.org/10.1186/s12967-022-03616-z",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication in a larger, sex-balanced cohort with deconditioned and disease controls.",
      "Does muscle sodium change during PEM or with treatments that improve perfusion?"
     ],
     "hypotheses": [
      "sodium-calcium-overload",
      "small-vessel-capillary",
      "vascular-endothelial"
     ],
     "test_available": "Research MRI only",
     "id": "muscle-exertion-muscle-sodium"
    },
    {
     "subdomain": "Muscle biopsies (pre-2020)",
     "finding": "Older ME/CFS muscle biopsy studies mostly found normal or nonspecific histology, without the fibre atrophy and type 2 shift expected from inactivity, with abnormalities confined to subgroups.",
     "grade": "contested",
     "plain": "Muscle samples taken in the 1990s mostly looked normal or only mildly abnormal under the microscope.",
     "detail": "Lane 1998 (105 quadriceps biopsies): type 1 predominance (23%) was more common than type 2 predominance (3%); atrophy in 10%; no ultrastructural abnormality in 41 EM samples; the abnormal-lactate subgroup had fewer type 1 fibres. Grau 1992 (n=20): only minor nonspecific changes in 9. Wassif 1994 (n=10): normal ischaemic lactate responses. Behan 1991 reported mitochondrial structural changes and Bowles 1993 enteroviral RNA in muscle (41/158 postviral fatigue vs 2/152 controls), neither reproduced with modern methods. Criteria, techniques and selection varied widely.",
     "studies": [
      {
       "cite": "Lane 1998, J Neurol Neurosurg Psychiatry",
       "n": "105",
       "design": "quadriceps needle biopsy, histometry, EM",
       "result": "Changes not those of inactivity; no ultrastructural abnormality; lactate responders fewer type 1 fibres",
       "url": "https://doi.org/10.1136/jnnp.64.3.362",
       "verified": true
      },
      {
       "cite": "Grau 1992, Clin Neuropathol",
       "n": "20",
       "design": "histopathology",
       "result": "Minor nonspecific changes in 9/20",
       "url": "https://pubmed.ncbi.nlm.nih.gov/1473316/",
       "verified": true
      },
      {
       "cite": "Bowles 1993, J Med",
       "n": "158 postviral fatigue vs 152 control muscles",
       "design": "molecular hybridisation",
       "result": "Enterovirus RNA in 41/158 vs 2/152; histology otherwise normal",
       "url": "https://pubmed.ncbi.nlm.nih.gov/8409778/",
       "verified": true
      },
      {
       "cite": "Behan 1991, Acta Neuropathol",
       "n": "about 50",
       "design": "electron microscopy",
       "result": "Mitochondrial structural abnormalities reported",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Behan+1991+mitochondrial+abnormalities+postviral+fatigue+syndrome",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Wassif 1994, Ann Clin Biochem",
       "n": "10 CFS vs 10 alcohol misuse",
       "design": "ischaemic lactate/ammonia test and biopsy histomorphometry",
       "result": "No distinctive CFS abnormality",
       "url": "https://doi.org/10.1177/000456329403100507",
       "verified": true
      }
     ],
     "open_questions": [
      "Re-examine archived biopsies with modern immunostaining (capillaries, immune cells, amyloid)."
     ],
     "hypotheses": [
      "viral-persistence",
      "mitochondrial-energy",
      "deconditioning"
     ],
     "test_available": "Diagnostic muscle biopsy (neuromuscular clinics) mainly to exclude myopathy",
     "id": "muscle-exertion-old-biopsies"
    },
    {
     "subdomain": "Muscle biopsies after exertion",
     "finding": "In long COVID with PEM, muscle biopsies taken one day after a maximal exercise test showed lower mitochondrial enzyme activity, more fibre necrosis, more T-cell infiltration and more amyloid-containing deposits than before exercise and than in controls.",
     "grade": "early",
     "plain": "When patients with long COVID were biopsied before and after a hard exercise test, their muscle looked more damaged and inflamed afterwards, and had more abnormal protein clumps.",
     "detail": "Appelman 2024 (25 vs 21, Amsterdam): lower VO2max and gas exchange threshold; more glycolytic fibres; SDH activity fell after exercise in patients; large necrotic areas in 36% of patients after exercise (group p=0.09, so not firmly significant); CD3+ T cells present in patients but absent in controls; CD68 macrophages higher. Biopsies were scored as present/absent because sizes varied, and a first biopsy can itself cause regeneration at the second site. It is long COVID, not ME/CFS, and the amyloid-deposit finding has not been reproduced by another lab. The study prompted published criticism and replies in Nat Commun 2025.",
     "studies": [
      {
       "cite": "Appelman 2024, Nat Commun",
       "n": "25 long COVID vs 21 controls",
       "design": "longitudinal case-control; vastus lateralis biopsies before and 1 day after a maximal CPET",
       "result": "Lower exercise capacity; after exercise, lower SDH activity, more atrophic fibres and focal necrosis, more T-cell infiltration, more amyloid-containing deposits in patients",
       "url": "https://doi.org/10.1038/s41467-023-44432-3",
       "verified": true
      },
      {
       "cite": "Charlton 2025, Trends Endocrinol Metab",
       "n": "review",
       "design": "opinion article from the same group",
       "result": "Argue for intrinsic mitochondrial dysfunction, endothelial abnormalities and glycolytic fibre shift as drivers; PEM trigger unknown",
       "url": "https://doi.org/10.1016/j.tem.2024.11.008",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does the same post-exertion pathology occur in ME/CFS (registered study NCT05225688 includes ME/CFS)?",
      "Are the amyloid-containing deposits fibrin(ogen)-derived and do they occlude capillaries?",
      "Is necrosis specific to PEM or a nonspecific response to unaccustomed maximal effort?"
     ],
     "hypotheses": [
      "microclots",
      "mitochondrial-energy",
      "innate-hyperactivation",
      "small-vessel-capillary",
      "viral-persistence"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-appelman-pem-biopsy"
    },
    {
     "subdomain": "Fibre type and capillaries",
     "finding": "ME/CFS muscle has a lower proportion of type I (oxidative) fibres, type I-specific atrophy, lower mitochondrial capacity and fewer capillaries per fibre, a pattern that 60 days of strict bed rest in healthy people did not reproduce.",
     "grade": "early",
     "plain": "Patients' muscles have fewer endurance-type fibres and fewer tiny blood vessels. Healthy volunteers kept in bed for two months did not develop the same pattern, so lack of activity alone does not seem to explain it.",
     "detail": "Charlton 2026 (Amsterdam group; 19-23 ME/CFS, 24-25 long COVID, 29-30 controls, 24 bed rest): type I proportion lower (p<0.001), type IIa/IIx higher; ME/CFS-only lower SDH; both patient groups lower OXPHOS; capillary-to-fibre ratio lower in ME/CFS than in long COVID and controls (p<0.001), whereas bed rest caused atrophy with unchanged capillary-to-fibre ratio and fibre type. OXPHOS correlated with VO2max in controls and after bed rest but not in patients. Same lab as Appelman; bed-rest cohort was a different (younger, male) population studied separately. Lane 1998 reported type 1 predominance more often than type 2 in older ME/CFS biopsies, which does not fit; differences in criteria and methods may explain this.",
     "studies": [
      {
       "cite": "Charlton 2026, Nat Commun",
       "n": "19-23 ME/CFS, 24-25 long COVID, 29-30 matched controls, 24 healthy people after 60-day bed rest",
       "design": "case-control biopsies plus bed-rest comparison",
       "result": "ME/CFS: lower type I fibre proportion and type I-specific atrophy, lower SDH activity, lower OXPHOS capacity, lower capillary-to-fibre ratio; bed rest caused atrophy but no fibre-type shift; OXPHOS tracked VO2max only after bed rest",
       "url": "https://doi.org/10.1038/s41467-026-75725-y",
       "verified": true
      },
      {
       "cite": "Appelman 2024, Nat Commun",
       "n": "25 long COVID vs 21 controls",
       "design": "longitudinal case-control; vastus lateralis biopsies before and 1 day after a maximal CPET",
       "result": "Lower exercise capacity; after exercise, lower SDH activity, more atrophic fibres and focal necrosis, more T-cell infiltration, more amyloid-containing deposits in patients",
       "url": "https://doi.org/10.1038/s41467-023-44432-3",
       "verified": true
      },
      {
       "cite": "Bizjak 2024, Int J Mol Sci",
       "n": "small CFS, post-COVID and control groups",
       "design": "high-resolution respirometry and EM of muscle",
       "result": "Complex I OXPHOS lower in post-COVID vs controls; CFS showed more advanced mitochondrial structural changes than post-COVID",
       "url": "https://doi.org/10.3390/ijms25031675",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Lane 1998, J Neurol Neurosurg Psychiatry",
       "n": "105",
       "design": "biopsy histometry",
       "result": "Type 1 predominance (23%) more common than type 2 predominance (3%)",
       "url": "https://doi.org/10.1136/jnnp.64.3.362",
       "verified": true
      }
     ],
     "open_questions": [
      "Independent replication of capillary rarefaction in ME/CFS muscle.",
      "Is the glycolytic shift a cause of exercise intolerance or an adaptation to poor oxygen delivery?",
      "Do severe, bedbound patients show the same profile?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "mitochondrial-energy",
      "vascular-endothelial",
      "deconditioning"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-fibre-capillary-bedrest"
    },
    {
     "subdomain": "Muscle mitochondria",
     "finding": "WASF3 protein is raised in ME/CFS muscle, and in cells and mice it disrupts respiratory supercomplex assembly and lowers exercise capacity via ER stress.",
     "grade": "early",
     "plain": "One protein, WASF3, was found at high levels in patients' muscle; in experiments it jammed the parts of mitochondria that make energy.",
     "detail": "Wang/Hwang 2023 (NIH): started from one patient with high WASF3; transgenic mice overexpressing WASF3 ran less, with lower complex IV; endotoxin-induced ER stress raised WASF3; the ER-stress inhibitor 4-PBA lowered WASF3 and improved the patient's cell respiration. A cohort of ME/CFS muscle biopsies showed raised WASF3 and ER stress markers (small cohort; group n not given in abstract). Glass 2025 also found ER-stress proteins in post-exercise EVs tracking PEM severity. No independent replication of the WASF3 biopsy finding.",
     "studies": [
      {
       "cite": "Wang 2023, PNAS",
       "n": "index patient; transgenic mice; small ME/CFS biopsy cohort",
       "design": "mechanistic case study plus cohort biopsies",
       "result": "WASF3 disrupts supercomplexes; raised WASF3 and ER stress in ME/CFS muscle",
       "url": "https://doi.org/10.1073/pnas.2302738120",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication of raised muscle WASF3 in a larger, activity-matched cohort.",
      "Does patient serum induce WASF3 in healthy muscle cells?",
      "Would ER-stress modulators help in a trial?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "er-stress",
      "innate-hyperactivation"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-muscle-mito-wasf3"
    },
    {
     "subdomain": "Cultured patient muscle cells",
     "finding": "Muscle cells grown from ME/CFS patients failed to activate AMPK and raise glucose uptake after electrical pulse stimulation, and secreted less IL-6, suggesting a defect that persists outside the body.",
     "grade": "early",
     "plain": "Patients' muscle cells grown in a dish did not respond to simulated exercise the normal way, which hints at a built-in problem rather than one caused only by something in the blood.",
     "detail": "Brown 2015 (Newcastle): control cultures increased AMPK phosphorylation and glucose uptake after 16 h EPS; CFS cultures did not, though insulin-stimulated uptake was preserved; IL-6 secretion lower. Sample sizes small. Tomas 2019 (same group) found no differences in individual complex activity in patient myotubes (n=6), so the defect, if real, is in signalling rather than respiratory chain capacity.",
     "studies": [
      {
       "cite": "Brown 2015, PLOS ONE",
       "n": "small (CFS vs control myoblast donors)",
       "design": "in vitro EPS of cultured myotubes",
       "result": "No AMPK activation or glucose uptake rise after EPS; lower IL-6",
       "url": "https://doi.org/10.1371/journal.pone.0122982",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Tomas 2019, PeerJ",
       "n": "myotubes n=6; PBMC 13 CFS vs 6 controls",
       "design": "permeabilised-cell extracellular flux",
       "result": "No differences in complex I, II, IV activity or FAO/glutaminolysis-supported respiration",
       "url": "https://doi.org/10.7717/peerj.6500",
       "verified": true
      }
     ],
     "open_questions": [
      "Replication with more donors and blinded processing.",
      "Is the defect epigenetic or genetic?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "genetic-neuronal"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-cultured-myotubes"
    },
    {
     "subdomain": "Mitochondria in blood cells",
     "finding": "Mitochondrial function in patients' blood cells has been reported as lower (PBMC maximal respiration, T-cell glycolysis), higher (lymphoblast maximal respiration with Complex V inefficiency) or normal, depending on lab and cell type.",
     "grade": "contested",
     "plain": "Studies of energy production in white blood cells disagree with each other, so it is unclear whether blood cells show an energy defect.",
     "detail": "Tomas 2017: lower PBMC maximal respiration (p<=0.003), but control groups were small (fresh n=3, frozen n=12, vs 25 and 38 patients). Mandarano 2020 (53 vs 45, Hanson lab): reduced glycolysis in CD4 and CD8 T cells at rest and reduced CD8 membrane potential, with normal mitochondrial respiration in most measures. Missailidis 2020/2021 (51 CCC patients; immortalised lymphoblasts): higher maximal respiration and spare capacity but lower ATP synthesis fraction (Complex V inefficiency), with TCA, PPP and beta-oxidation enzymes up; claimed high discrimination, unreplicated. Vermeulen 2010 and Tomas 2019 found normal PBMC OXPHOS or complex activity. Directions conflict between studies, freezing affects results, and blood cells may not reflect muscle.",
     "studies": [
      {
       "cite": "Tomas 2017, PLOS ONE",
       "n": "CFS fresh 25/frozen 38 vs control fresh 3/frozen 12",
       "design": "Seahorse PBMC",
       "result": "Lower maximal respiration and other OXPHOS parameters",
       "url": "https://doi.org/10.1371/journal.pone.0186802",
       "verified": true
      },
      {
       "cite": "Mandarano 2020, J Clin Invest",
       "n": "53 vs 45",
       "design": "Seahorse on isolated CD4/CD8 T cells",
       "result": "Reduced T-cell glycolysis; reduced CD8 membrane potential",
       "url": "https://doi.org/10.1172/jci132185",
       "verified": true
      },
      {
       "cite": "Missailidis 2020, Int J Mol Sci",
       "n": "51 ME/CFS lymphoblast lines vs matched controls",
       "design": "Seahorse, proteomics",
       "result": "Complex V inefficiency; elevated maximal OCR and spare capacity; mTORC1 hyperactivation",
       "url": "https://doi.org/10.3390/ijms21031074",
       "verified": true
      },
      {
       "cite": "Missailidis 2021, Int J Mol Sci",
       "n": "up to 34 vs 31 lines",
       "design": "transcriptomics/proteomics",
       "result": "Elevated TCA, PPP, FAO enzymes: dysregulated substrate supply",
       "url": "https://doi.org/10.3390/ijms22042046",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Vermeulen 2010, J Transl Med",
       "n": "15 vs 15 women",
       "design": "PBMC OXPHOS plus 2-day CPET",
       "result": "PBMC oxidative phosphorylation normal despite worse day-2 performance",
       "url": "https://doi.org/10.1186/1479-5876-8-93",
       "verified": true
      },
      {
       "cite": "Tomas 2019, PeerJ",
       "n": "13 CFS vs 6 controls (PBMC)",
       "design": "permeabilised cells",
       "result": "No complex activity differences",
       "url": "https://doi.org/10.7717/peerj.6500",
       "verified": true
      },
      {
       "cite": "Fernandez-Guerra 2021, Biomolecules",
       "n": "6 vs 6",
       "design": "PBMC bioenergetics and proteomics",
       "result": "Exploratory; small",
       "url": "https://doi.org/10.3390/biom11070961",
       "verified": true
      }
     ],
     "open_questions": [
      "A multi-lab, blinded, pre-registered PBMC Seahorse study with fresh cells and adequate controls.",
      "Do blood-cell findings correlate with muscle mitochondrial measures in the same people?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "immune-exhaustion",
      "genetic-neuronal"
     ],
     "test_available": "Research only; commercial 'mitochondrial function' blood tests are not validated",
     "id": "muscle-exertion-immune-cell-mito"
    },
    {
     "subdomain": "Pyruvate dehydrogenase",
     "finding": "Fluge 2016 proposed impaired pyruvate dehydrogenase (PDH) function from low TCA-fuelling amino acids and raised PDK mRNA in blood cells; this has not been independently confirmed.",
     "grade": "early",
     "plain": "One group suggested that the step that lets sugar enter mitochondria to be burned cleanly is partly blocked, pushing cells toward lactate. Others have not yet confirmed it.",
     "detail": "Fluge 2016 (200 vs 102): lower serum amino acids feeding the TCA cycle (mainly women), raised 3-methylhistidine in men, PDK1/2/4, SIRT4 and PPARdelta mRNA up in PBMC. Mughal 2025 found PDK3/PDK4 up in 3D muscle exposed to patient serum (n=4 ME/CFS). The serum-transfer part of Fluge 2016 failed replication (Ryback 2026). Solving ME/CFS re-analysis of resting plasma metabolomics found low acylcarnitines and fatty acids (lower fat-fuel supply) rather than a pile-up pattern, and a 5-cohort aromatic-lactate deficit, which point to related but different steps. No study has measured PDH activity directly in ME/CFS muscle.",
     "studies": [
      {
       "cite": "Fluge 2016, JCI Insight",
       "n": "200 ME/CFS vs 102 controls (serum amino acids); 12 vs 12 (myoblast serum assay)",
       "design": "case-control metabolomics, PBMC qPCR, serum-transfer cell assay",
       "result": "Lower TCA-fuelling amino acids (mainly women); PDK1/2/4, SIRT4, PPARdelta mRNA up in PBMC; severe-patient serum raised myoblast respiration and lactate secretion",
       "url": "https://doi.org/10.1172/jci.insight.89376",
       "verified": true
      },
      {
       "cite": "Mughal 2025, Biofabrication",
       "n": "4 ME/CFS, 5 long COVID, 4 controls",
       "design": "3D engineered muscle exposed to serum",
       "result": "PDK3/PDK4 up by qPCR; glycolysis/TCA genes up",
       "url": "https://doi.org/10.1088/1758-5090/adf66c",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (resting metabolomics, several cohorts)",
       "n": "Germain 2022 60 vs 45 plus 5 cohorts for aromatic lactates",
       "design": "re-analysis of public metabolomics",
       "result": "Resting pattern fits low fat-derived fuel supply, not a PDH/fat-oxidation block with acylcarnitine build-up; aromatic lactates -0.53 SD",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Direct PDH activity and phosphorylation in muscle biopsies.",
      "Replication of the amino-acid pattern in an independent cohort with diet control."
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "metabolic-trap-ido",
      "serum-factor"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-pdh-block"
    },
    {
     "subdomain": "Serum factors and muscle cells",
     "finding": "The claim that ME/CFS serum changes healthy myoblast mitochondrial respiration failed a pre-registered replication of 67 patients vs 53 controls, though a small 3D engineered-muscle study reported weaker contraction with patient serum.",
     "grade": "refuted",
     "plain": "An influential idea was that something in patients' blood harms muscle cells' energy production. A large, careful repeat of the original experiment found no difference.",
     "detail": "Ryback 2026: blinded, randomised, 1,926 wells, primary maximal OCR +2.50 pmol/min (95% CI -1.62 to 6.62); recomputed participant-level d=0.14 (CI -0.22 to 0.50), excluding the original d=1.32. Limits: mostly mild-moderate (only 6 severe), resting samples, batch partly collinear with group, one myoblast donor, single readout. Mughal 2025 (4/5/4 donors, IBEC) found reduced contractile force at 48 h and mitochondrial fragmentation later, with pseudoreplication risk. Prusty 2026 found patient IgG fragments endothelial (not muscle) mitochondria, with MS controls. The refutation applies to the 2D myoblast respiration readout, not to all serum-factor ideas.",
     "studies": [
      {
       "cite": "Fluge 2016, JCI Insight",
       "n": "12 vs 12 (9 severe)",
       "design": "serum on healthy myoblasts",
       "result": "Higher respiration and lactate secretion with patient serum",
       "url": "https://doi.org/10.1172/jci.insight.89376",
       "verified": true
      },
      {
       "cite": "Mughal 2025, Biofabrication",
       "n": "4 ME/CFS, 5 long COVID, 4 controls",
       "design": "3D muscle tissue force under electrical stimulation",
       "result": "Lower force after 48 h patient serum; fragmented mitochondria by 96-144 h",
       "url": "https://doi.org/10.1088/1758-5090/adf66c",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Ryback 2026, PLOS ONE",
       "n": "67 ME vs 53 controls, all female",
       "design": "pre-registered, blinded replication of Fluge 2016; healthy myoblasts in 20% serum 6 days, Seahorse",
       "result": "No difference in maximal respiration (+2.50 pmol/min, 95% CI -1.62 to 6.62, p=0.23) or any secondary measure; powered for d=0.52 vs original d=1.32",
       "url": "https://doi.org/10.1371/journal.pone.0341334",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis of Ryback 2026 supplementary data",
       "n": "67 vs 53",
       "design": "participant-level recomputation",
       "result": "d=0.14 overall; severe subgroup (n=6) d=0.002",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Do sera drawn during PEM or from severe patients behave differently?",
      "Would differentiated myotubes, force readouts or IgG fractions show an effect that whole serum in 2D did not?",
      "Can a blinded two-lab sample exchange reproduce the 3D force result?"
     ],
     "hypotheses": [
      "serum-factor",
      "autoimmunity",
      "mitochondrial-energy"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-serum-myoblast-refuted"
    },
    {
     "subdomain": "Exercise-induced gene expression",
     "finding": "After moderate exercise, leukocyte mRNA for metabolite-detecting (ASIC3, P2X4/5, TRPV1), adrenergic and immune genes rose for up to 48 h in most CFS patients but not in controls or MS patients.",
     "grade": "early",
     "plain": "In one lab's studies, a 25-minute bout of moderate exercise switched on 'muscle-fatigue sensing' genes in patients' white blood cells for two days, and this tracked how tired and sore they felt.",
     "detail": "Light 2012 (48 CFS, 18 FM, 49 controls; 25 min at 70% age-predicted HR, blood at 0.5, 8, 24, 48 h, blinded qPCR of 13 genes): 71% of CFS had increases correlating with fatigue and pain; the other 29% showed decreased alpha-2A receptor mRNA and more orthostatic intolerance. White 2012 (22 CFS, 20 MS, 23 controls) added a disease control. All from the Utah Light lab with a small targeted gene panel; no independent replication. Vu 2024 single-cell RNA-seq after exercise found minimal immune changes other than platelets.",
     "studies": [
      {
       "cite": "Light 2012, J Intern Med",
       "n": "48 CFS, 18 FM, 49 controls",
       "design": "pre/post moderate exercise qPCR",
       "result": "Post-exercise rise in sensory/adrenergic/cytokine gene mRNA for 48 h in 71% of CFS; none in controls",
       "url": "https://doi.org/10.1111/j.1365-2796.2011.02405.x",
       "verified": true
      },
      {
       "cite": "White 2012, Psychosom Med",
       "n": "22 CFS, 20 MS, 23 controls",
       "design": "pre/post moderate exercise qPCR",
       "result": "Larger rises in P2X4, TRPV1, CD14 and adrenergic receptors in CFS than controls; MS differed",
       "url": "https://doi.org/10.1097/psy.0b013e31824152ed",
       "verified": true
      },
      {
       "cite": "Light 2009, J Pain",
       "n": "small pilot (n from memory)",
       "design": "pilot",
       "result": "First report of post-exercise gene rises",
       "url": "https://doi.org/10.1016/j.jpain.2009.06.003",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Vu 2024, Cell Rep Med",
       "n": "ME/CFS vs controls, before and after exercise",
       "design": "single-cell RNA-seq of PBMC",
       "result": "Minimal post-exercise changes outside platelets (improper platelet activation)",
       "url": "https://doi.org/10.1016/j.xcrm.2023.101373",
       "verified": true
      }
     ],
     "open_questions": [
      "Independent replication with a pre-registered gene panel.",
      "Do leukocyte receptor transcripts reflect muscle afferent signalling at all?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-exercise-gene-expression"
    },
    {
     "subdomain": "Post-exertion metabolomics",
     "finding": "Plasma metabolomics across a 2-day CPET shows clear resting differences in ME/CFS, but once each person is compared with themselves the exercise response differs little from controls.",
     "grade": "contested",
     "plain": "Blood chemistry differs between patients and healthy people even at rest. Whether hard exercise changes it in a disease-specific way is less clear.",
     "detail": "Germain 2022 reported growing patient-control divergence (women: 6 to 54 metabolites at q<0.05 across four timepoints) and glutamate/TCA-linked recovery pathways. The Solving ME/CFS re-analysis reproduced those counts but found day-1 and day-2 differences correlate at 0.85 and the within-person response is at chance; alpha-ketoglutarate did not reproduce. Surviving modest signals: blunted post-exercise glucose rise (p=0.018, survives lactate adjustment), oxidised cys-gly rise in patients (q=0.033), lower day-2 N-acetylarginine and C6-DC acylcarnitine. Che 2025 (56 vs 52, Lipkin) reported TCA, beta-oxidation and urea-cycle abnormalities worse 24 h after exercise, but with only pre/24 h timepoints. Five of the most impaired male patients lacked post-exercise samples in Germain 2022.",
     "studies": [
      {
       "cite": "Germain 2022, JCI Insight",
       "n": "60 ME/CFS vs 45 controls, 4 timepoints",
       "design": "2-day CPET plasma metabolomics (1,157 metabolites)",
       "result": "Reported growing patient-control disparity over the two days, with lipid, energy and glutamate-linked pathways in recovery",
       "url": "https://doi.org/10.1172/jci.insight.157621",
       "verified": true
      },
      {
       "cite": "Che 2025, npj Metab Health Dis",
       "n": "56 ME/CFS vs 52 controls (47 matched pairs)",
       "design": "plasma metabolomics/proteomics and stimulated immune responses before and 24 h after exercise",
       "result": "Exaggerated innate responses; impaired energy pathways; many abnormalities worse after exercise and correlated with symptoms",
       "url": "https://doi.org/10.1038/s44324-025-00079-w",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis of Germain 2022 data",
       "n": "60 vs 45 (404 samples)",
       "design": "within-person change scores, mixed models, permutation",
       "result": "Resting differences real (~150 of 780 metabolites at p<0.05 vs ~35 expected, perm p=0.002); exercise-response differences at chance (perm p=0.12-0.61); survivors: smaller lactate rise (explained by effort), blunted post-exercise glucose rise, rise in oxidised cys-gly",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Does a within-person analysis of Che 2025 hold up?",
      "Plasma may be the wrong compartment; paired muscle microdialysis or biopsy metabolomics are lacking."
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "innate-hyperactivation",
      "cell-danger-response"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-post-exertion-metabolomics"
    },
    {
     "subdomain": "Post-exertion proteomics",
     "finding": "Plasma proteomics of 79 ME/CFS and 53 sedentary controls across a 2-day CPET found the largest patient-specific changes in the 24 h recovery phase, with suppressed T/B-cell and IL-17 signalling and raised glycolysis proteins.",
     "grade": "contested",
     "plain": "The biggest differences in blood proteins appeared during the recovery day, when PEM begins, rather than straight after exercise.",
     "detail": "Germain 2025 (SomaScan 7K, 6,361 proteins; 96 F, 36 M): persistent dysregulation of immune, metabolic and neuromuscular pathways; protein-VO2max/AT relationships present in controls were disrupted in patients; sex-specific responses. Che 2025 proteomics added complement activation and extracellular-matrix ligands after exercise. The Solving ME/CFS proteome re-analysis found resting muscle-derived proteins lower in 3 SomaScan cohorts (including Germain) and UK Biobank (d -0.50). Pathway enrichment across thousands of aptamers is vulnerable to multiplicity; no independent post-exertion proteomics cohort yet.",
     "studies": [
      {
       "cite": "Germain 2025, Mol Cell Proteomics",
       "n": "79 ME/CFS vs 53 controls",
       "design": "longitudinal SomaScan 7K across 2-day CPET",
       "result": "Recovery-phase maladaptation; suppressed lymphocyte and IL-17 signalling; glycolysis up; links to muscle pain and sore throat",
       "url": "https://doi.org/10.1016/j.mcpro.2025.101467",
       "verified": true
      },
      {
       "cite": "Che 2025, npj Metab Health Dis",
       "n": "56 vs 52",
       "design": "multi-omics pre/24 h post exercise",
       "result": "Complement activation, ECM disruption, redox imbalance worse post-exercise",
       "url": "https://doi.org/10.1038/s44324-025-00079-w",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (proteome)",
       "n": "Germain 79/51 per-person",
       "design": "within-person change",
       "result": "0 proteins with a patient-specific exercise response at FDR; pathway claims not reproduced per person",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Replication of recovery-phase protein changes in an independent cohort.",
      "Which changes are effort-dependent (adjust for achieved workload)?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "innate-hyperactivation",
      "complement-thromboinflammation",
      "mitochondrial-energy"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-post-exertion-proteomics"
    },
    {
     "subdomain": "Extracellular vesicles after exercise",
     "finding": "Extracellular-vesicle protein cargo responds to maximal exercise differently in ME/CFS (blunted in women; TCA proteins down and complement up in men), and recovery-phase changes correlate with PEM severity.",
     "grade": "early",
     "plain": "Tiny packages that cells release into the blood carry different proteins after exercise in patients than in healthy people.",
     "detail": "Giloteaux 2024 (18 vs 17 women; pre, 15 min, 24 h; TMT proteomics): patients' EV changes differed and correlated with symptom severity, involving coagulation, muscle contraction, cytoskeleton, immune and brain proteins. Glass 2025 (10 vs 12 men): TCA-cycle proteins down and complement up at 15 min; ER-stress and protein-folding changes in recovery correlated with PEM; EV-VO2 correlations seen in controls were absent in patients. Both from the Hanson lab, small, many comparisons; no transfer experiment shows patient EVs act on cells.",
     "studies": [
      {
       "cite": "Giloteaux 2024, J Extracell Vesicles",
       "n": "18 vs 17 women",
       "design": "EV proteomics pre/15 min/24 h post CPET",
       "result": "Different EV response in patients; correlates with severity",
       "url": "https://doi.org/10.1002/jev2.12403",
       "verified": true
      },
      {
       "cite": "Glass 2025, Clin Transl Med",
       "n": "10 vs 12 men",
       "design": "EV proteomics pre/15 min/24 h post CPET",
       "result": "TCA down, complement up post-exercise; ER-stress changes track PEM",
       "url": "https://doi.org/10.1002/ctm2.70346",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent replication, and functional tests of patient EVs on muscle or endothelial cells.",
      "Tissue of origin of the EVs (muscle, platelet, endothelium)."
     ],
     "hypotheses": [
      "complement-thromboinflammation",
      "er-stress",
      "mitochondrial-energy",
      "cell-danger-response"
     ],
     "test_available": "Research only",
     "id": "muscle-exertion-ev-cargo"
    },
    {
     "subdomain": "Muscle strength and fatigability",
     "finding": "Repeated maximal handgrip testing shows lower strength, faster decline across repetitions and poorer recovery after 60 minutes in ME/CFS, correlating with severity and PEM.",
     "grade": "several",
     "plain": "Squeezing a grip meter ten times in a row, twice an hour apart, shows patients' strength fading faster and recovering less than healthy people's. It is cheap and quick.",
     "detail": "Jäkel 2021 (105 ME/CFS, 18 cancer-related fatigue, 66 controls; Charité): lower Fmax and Fmean (p<0.0001), higher fatigue ratio, lower recovery ratio (Fmean2/Fmean1); lower grip linked to severity, PEM, muscle pain and post-exertion CK/LDH. Paffrath 2024 (144 women with post-COVID, 78 meeting CCC): grip impaired across post-COVID, but linked to key symptoms only in ME/CFS. Walitt 2024 (NIH) found normal MVC but shorter time to failure at 50% MVC (p=0.0002) and faster decline on repetitive grip, which the authors attributed to 'effort preference', an interpretation that has been disputed. Paul 1999 (10 vs 10) showed slower quadriceps strength recovery up to 24 h. Most data come from one centre; effort and motivation cannot be fully excluded from voluntary tests.",
     "studies": [
      {
       "cite": "Jäkel 2021, J Transl Med",
       "n": "105 ME/CFS, 18 CRF, 66 controls",
       "design": "repeat maximal HGS (10 pulls x 2, 60 min apart)",
       "result": "Lower strength, higher fatigue ratio, lower recovery ratio; correlates with severity and PEM",
       "url": "https://doi.org/10.1186/s12967-021-02774-w",
       "verified": true
      },
      {
       "cite": "Paffrath 2024, J Clin Med",
       "n": "144 post-COVID women (78 ME/CFS)",
       "design": "repeat HGS",
       "result": "Impaired HGS in all; associations with PEM, fatigue, cognition only in ME/CFS",
       "url": "https://doi.org/10.3390/jcm13072153",
       "verified": true
      },
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "16-20 per group",
       "design": "grip MVC, time to failure, repetitive grip",
       "result": "Normal MVC; shorter time to failure; faster decline on repetitive grip",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Paul 1999, Eur J Neurol",
       "n": "10 vs 10",
       "design": "quadriceps fatiguing protocol with recovery to 24 h",
       "result": "Similar decline during exercise; recovery prolonged in CFS at 200 min and 24 h",
       "url": "https://doi.org/10.1046/j.1468-1331.1999.610063.x",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent validation outside Charité with deconditioned and depressed controls.",
      "Can repeat HGS track PEM day by day or serve as a trial endpoint?",
      "How much reflects central drive versus peripheral muscle fatigue (Walitt found no central or peripheral fatigue markers)?"
     ],
     "hypotheses": [
      "sodium-calcium-overload",
      "mitochondrial-energy",
      "sickness-behaviour"
     ],
     "test_available": "Yes: handgrip dynamometer (cheap; offered by some ME/CFS clinics, e.g. Charité protocol)",
     "id": "muscle-exertion-grip-fatigability"
    },
    {
     "subdomain": "Muscle proteins in blood",
     "finding": "Muscle-derived proteins (myoglobin, troponins, CA3 and similar) are lower in ME/CFS blood at rest across three SomaScan cohorts and UK Biobank, not explained by step counts or walking pace.",
     "grade": "several",
     "plain": "Proteins that leak from muscle into blood are slightly lower in patients, which suggests less muscle mass or slower muscle turnover rather than ongoing damage.",
     "detail": "Solving ME/CFS re-analysis: muscle-damage set d -0.50 (95% CI -0.76 to -0.24, q=0.002, I2 0.07) across Hoel (-0.85), Germain (-0.43) and Walitt (-0.34); UK Biobank competitive shift negative; not mediated by walking pace; unrelated to steps, Bell score or illness duration within patients. Lean mass was not available, so reduced muscle mass from inactivity cannot be excluded. Resting CK and myoglobin are expected to be low-normal.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (proteome)",
       "n": "Hoel 50/24, Germain 78/51, Walitt 21/21, plus UK Biobank",
       "design": "meta-analysis of public proteomics",
       "result": "Muscle protein set d -0.50, consistent across cohorts",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does the signal survive adjustment for DXA lean mass?",
      "Does it relate to type I fibre atrophy or capillary loss in biopsies from the same people?"
     ],
     "hypotheses": [
      "deconditioning",
      "mitochondrial-energy",
      "small-vessel-capillary"
     ],
     "test_available": "Not clinically meaningful; DXA lean mass is available",
     "id": "muscle-exertion-muscle-proteins-blood"
    },
    {
     "subdomain": "Deconditioning versus disease",
     "finding": "Several designs (aerobic-capacity-matched controls, idiopathic-fatigue and MS comparison groups, and a 60-day bed-rest model) indicate that deconditioning alone does not explain the exercise and muscle abnormalities in ME/CFS.",
     "grade": "several",
     "plain": "It is often said patients are just unfit from resting. Studies that compared them with equally unfit people or with healthy volunteers kept in bed found differences that inactivity did not produce.",
     "detail": "Keller 2024: day-2 declines persisted in 55 pairs matched for peak VO2. van Campen 2021 and Hodges 2018: idiopathic chronic fatigue and MS patients improved on day 2. Lane 1998: biopsy changes were not those of inactivity. Charlton 2026: bed rest caused atrophy with preserved fibre type and capillary-to-fibre ratio; patients showed the opposite pattern; OXPHOS-VO2max coupling present after bed rest but absent in patients. Vermeulen 2014: a high cardiac output/VO2 slope argues against central deconditioning. These are mostly cross-sectional and from enthusiast centres; Mancini 2026 found no day-2 decline at all.",
     "studies": [
      {
       "cite": "Keller 2024, J Transl Med",
       "n": "84 ME/CFS vs 71 sedentary controls (55 VO2-matched pairs)",
       "design": "2-day CPET, cycle ergometer, 24 h apart",
       "result": "ME/CFS failed to reproduce day-1 values: e.g. ~6% fall in peak VO2/kg and 6.7% fall in VO2 at ventilatory threshold in matched pairs; controls stable; both groups met maximal-effort criteria",
       "url": "https://doi.org/10.1186/s12967-024-05410-5",
       "verified": true
      },
      {
       "cite": "Charlton 2026, Nat Commun",
       "n": "19-23 ME/CFS, 24-25 long COVID, 29-30 matched controls, 24 healthy people after 60-day bed rest",
       "design": "case-control biopsies plus bed-rest comparison",
       "result": "ME/CFS: lower type I fibre proportion and type I-specific atrophy, lower SDH activity, lower OXPHOS capacity, lower capillary-to-fibre ratio; bed rest caused atrophy but no fibre-type shift; OXPHOS tracked VO2max only after bed rest",
       "url": "https://doi.org/10.1038/s41467-026-75725-y",
       "verified": true
      },
      {
       "cite": "van Campen 2021, Healthcare",
       "n": "50 vs 51 women",
       "design": "2-day CPET, idiopathic chronic fatigue control",
       "result": "ICF improved on day 2; ME/CFS declined",
       "url": "https://doi.org/10.3390/healthcare9060682",
       "verified": true
      },
      {
       "cite": "Charlton 2026, Br J Sports Med",
       "n": "narrative",
       "design": "review/editorial",
       "result": "Argues cardiac deconditioning is a myth in long COVID PEM",
       "url": "https://doi.org/10.1136/bjsports-2025-111387",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Mancini 2026, Front Physiol",
       "n": "58 ME/CFS vs 25 sedentary controls",
       "design": "2-day CPET replication attempt",
       "result": "No day-2 change in peak VO2 (22.3 to 22.5 ml/kg/min) or VO2 at threshold in either group; patients had lower peak HR and higher perceived exertion; authors say 2-day CPET should not be used to define PEM",
       "url": "https://doi.org/10.3389/fphys.2026.1816082",
       "verified": true
      }
     ],
     "open_questions": [
      "A prospective study following new post-infectious patients from onset, measuring muscle before inactivity accumulates."
     ],
     "hypotheses": [
      "deconditioning",
      "small-vessel-capillary",
      "mitochondrial-energy"
     ],
     "test_available": "n/a",
     "id": "muscle-exertion-deconditioning"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "mitochondrial-energy",
     "for": "Lower SDH/OXPHOS and type I atrophy in ME/CFS muscle (Charlton 2026); post-exertion SDH drop in long COVID (Appelman 2024); WASF3 (Wang 2023); 31P-MRS subgroups (Jones 2010/2012); early lactate per watt (Lien 2019); AMPK defect in cultured myotubes (Brown 2015).",
     "against": "Ryback 2026 refutes the serum-myoblast respiration effect; blood-cell mitochondrial findings conflict in direction (Tomas vs Missailidis) or are null (Vermeulen 2010, Tomas 2019); OXPHOS does not correlate with VO2max in patients (Charlton 2026), so mitochondria may not be the limiting step; several MRS studies null (Barnes 1993, Godlewska 2025)."
    },
    {
     "id": "small-vessel-capillary",
     "for": "Lower capillary-to-fibre ratio in ME/CFS muscle not reproduced by bed rest (Charlton 2026); impaired O2 extraction in high-flow iCPET subgroup (Joseph 2021); slower NIRS reoxygenation (McCully 1999).",
     "against": "Capillary finding from one lab; long COVID patients in the same study did not show it as strongly; extraction data largely single-centre."
    },
    {
     "id": "low-blood-volume-preload",
     "for": "Low right atrial pressure during invasive CPET in 160 patients (Joseph 2021); low peak HR across CPET studies.",
     "against": "Preload failure explains low peak output but not why muscle pathology worsens a day later or why PEM is delayed."
    },
    {
     "id": "sodium-calcium-overload",
     "for": "Higher muscle sodium on 23Na-MRI, inversely correlated with grip (Petter 2022); grip fatigability (Jäkel 2021); review by Scheibenbogen & Wirth 2025.",
     "against": "Sodium MRI n=6 vs 6, unreplicated; no direct muscle calcium measurement in patients."
    },
    {
     "id": "serum-factor",
     "for": "Fluge 2016 myoblast effect; Mughal 2025 3D muscle force loss (n=4); Prusty 2026 IgG fragments endothelial mitochondria (non-muscle).",
     "against": "Ryback 2026 pre-registered replication (67 vs 53) null, excluding the original effect size."
    },
    {
     "id": "microclots",
     "for": "Amyloid-containing deposits in long COVID muscle increased after PEM induction (Appelman 2024).",
     "against": "Not yet shown in ME/CFS muscle; single lab; the composition of the deposits is not established."
    },
    {
     "id": "innate-hyperactivation",
     "for": "T-cell and macrophage infiltration in long COVID muscle after exertion (Appelman 2024); exaggerated innate responses and complement after exercise (Che 2025); complement up in male EVs (Glass 2025).",
     "against": "Germain 2025 found suppressed rather than activated lymphocyte signalling in recovery; single-cell RNA after exercise showed minimal immune changes (Vu 2024)."
    },
    {
     "id": "cell-danger-response",
     "for": "Delayed, prolonged PEM (Moore 2023) and recovery-phase molecular changes (Germain 2025) fit a stress response that persists after the stimulus.",
     "against": "No study has measured purinergic/cell-danger signalling directly in muscle during PEM."
    },
    {
     "id": "autonomic-small-fibre",
     "for": "31% SFN and shunting physiology in iCPET (Joseph 2021); loss of normal pH-efflux coupling linked to autonomic function (Jones 2010); adrenergic receptor mRNA changes after exercise (Light 2012).",
     "against": "Denervation severity did not correlate with exertional measures (Joseph 2021)."
    },
    {
     "id": "sickness-behaviour",
     "for": "Walitt 2024 framed grip and effort findings as altered effort preference.",
     "against": "Effort criteria are met in most CPETs; objective muscle, capillary and post-exertion biopsy changes (Appelman, Charlton) are not explained by behaviour; the effort-preference interpretation has been formally criticised."
    },
    {
     "id": "deconditioning",
     "for": "(New id: exercise and muscle abnormalities are secondary to inactivity.) Low peak VO2 overlaps with sedentary controls; lower muscle proteins in blood could reflect reduced mass; Mancini 2026 found no day-2 decline.",
     "against": "VO2-matched pairs still declined on day 2 (Keller 2024); bed rest does not reproduce fibre-type shift, capillary loss or OXPHOS uncoupling (Charlton 2026); disease controls improve on day 2 (van Campen 2021, Hodges 2018)."
    },
    {
     "id": "er-stress",
     "for": "(New id: endoplasmic reticulum stress impairs mitochondrial supercomplexes and energy production.) WASF3 and ER-stress markers raised in ME/CFS muscle (Wang 2023); ER-stress EV proteins track PEM severity (Glass 2025).",
     "against": "Both small and unreplicated; no trial of ER-stress modulators."
    },
    {
     "id": "metabolic-trap-ido",
     "for": "Fluge 2016 amino-acid pattern implies altered fuel handling.",
     "against": "Kynurenine pathway metabolites show no patient-specific exercise response in the Solving ME/CFS re-analysis of Germain 2022."
    },
    {
     "id": "viral-persistence",
     "for": "Enterovirus RNA in muscle in older studies (Bowles 1993).",
     "against": "Not reproduced with modern methods; Appelman 2024 did not report persistent virus as the driver of post-exertion pathology."
    }
   ],
   "glossary": [
    {
     "term": "PEM (post-exertional malaise)",
     "plain": "A worsening of symptoms after physical, mental or emotional effort, often delayed by hours to a day and lasting days or longer."
    },
    {
     "term": "CPET",
     "plain": "Cardiopulmonary exercise test: cycling or treadmill to exhaustion while breathing gases and heart rate are measured."
    },
    {
     "term": "2-day CPET",
     "plain": "Two CPETs 24 hours apart, used to see whether performance drops on the second day."
    },
    {
     "term": "Peak VO2",
     "plain": "The most oxygen the body can take up and use per minute at maximal effort; the standard measure of aerobic capacity."
    },
    {
     "term": "Ventilatory (anaerobic) threshold",
     "plain": "The point during exercise when breathing rises faster than oxygen use because muscles lean more on lactate-producing fuel use."
    },
    {
     "term": "Chronotropic incompetence",
     "plain": "The heart rate fails to rise as much as expected during exercise."
    },
    {
     "term": "Invasive CPET (iCPET)",
     "plain": "CPET with catheters in the heart and an artery, measuring filling pressures, cardiac output and oxygen extraction directly."
    },
    {
     "term": "Oxygen extraction",
     "plain": "How much oxygen tissues remove from the blood that flows through them."
    },
    {
     "term": "31P-MRS",
     "plain": "A magnetic resonance scan that measures phosphorus-containing energy molecules (PCr, ATP) and acidity inside working muscle."
    },
    {
     "term": "23Na-MRI",
     "plain": "An MRI scan tuned to sodium, used to measure salt content in tissue."
    },
    {
     "term": "Type I / type II fibres",
     "plain": "Type I fibres are slow, endurance, mitochondria-rich; type II are faster and more reliant on glycolysis."
    },
    {
     "term": "SDH (succinate dehydrogenase)",
     "plain": "A mitochondrial enzyme whose staining is used as a marker of mitochondrial content in muscle sections."
    },
    {
     "term": "OXPHOS",
     "plain": "Oxidative phosphorylation: the mitochondrial process that uses oxygen to make most of the cell's ATP."
    },
    {
     "term": "Seahorse assay",
     "plain": "A plate-based instrument measuring how fast cells use oxygen and make acid, used to estimate mitochondrial function."
    },
    {
     "term": "PDH (pyruvate dehydrogenase)",
     "plain": "The enzyme that lets the product of sugar breakdown enter mitochondria; if blocked, cells make more lactate."
    },
    {
     "term": "PBMC",
     "plain": "Peripheral blood mononuclear cells: lymphocytes and monocytes isolated from a blood sample."
    },
    {
     "term": "Extracellular vesicles (EVs)",
     "plain": "Small membrane packages released by cells into blood, carrying proteins and RNA."
    },
    {
     "term": "Handgrip fatigability",
     "plain": "The fall in grip strength across repeated maximal squeezes, and how well it recovers after rest."
    },
    {
     "term": "WASF3",
     "plain": "A protein that, when raised, disrupts the assembly of mitochondrial energy-producing complexes."
    }
   ],
   "key_reviews": [
    {
     "cite": "Scheibenbogen & Wirth 2025, J Cachexia Sarcopenia Muscle",
     "url": "https://doi.org/10.1002/jcsm.13669",
     "why": "Collects the evidence for skeletal muscle disturbance and the sodium/calcium overload model (advocacy-leaning, from the model's authors)."
    },
    {
     "cite": "Charlton 2025, Trends Endocrinol Metab",
     "url": "https://doi.org/10.1016/j.tem.2024.11.008",
     "why": "Opinion piece from the Amsterdam biopsy group on muscle adaptations and PEM in long COVID."
    },
    {
     "cite": "Lim 2020, J Clin Med",
     "url": "https://doi.org/10.3390/jcm9124040",
     "why": "Meta-analysis of 2-day CPET; workload at ventilatory threshold is the most discriminating measure."
    },
    {
     "cite": "Franklin & Graham 2022, Fatigue: Biomed Health Behav",
     "url": "https://research.tees.ac.uk/en/publications/repeated-maximal-exercise-tests-of-peak-oxygen-consumption-in-peo/",
     "why": "Meta-analysis estimating clinically meaningful day-2 drop in work at anaerobic threshold (-21 W)."
    },
    {
     "cite": "Stevens 2018, Front Pediatr",
     "url": "https://doi.org/10.3389/fped.2018.00242",
     "why": "Methods paper for administering 2-day CPET in ME/CFS."
    },
    {
     "cite": "Fanò-Illic 2026, Diagnostics",
     "url": "https://doi.org/10.3390/diagnostics16071019",
     "why": "Recent review of skeletal muscle involvement in ME/CFS (title/abstract seen only)."
    },
    {
     "cite": "Solving ME/CFS re-analyses (PEM, serum-check, proteome)",
     "url": "/#data",
     "why": "Within-person re-analysis of Germain 2022 metabolomics; participant-level check of Ryback 2026; muscle-protein meta-analysis."
    }
   ],
   "gaps": [
    "No ME/CFS (as opposed to long COVID) study has biopsied muscle before and after induced PEM; the registered Amsterdam study NCT05225688 and Systrom's OMF-funded biopsy study have not yet reported.",
    "No independent replication of 2-day CPET from a group outside the Workwell/Ithaca/van Campen network that also reports test-retest variability in sedentary controls; Mancini 2026 null needs explaining.",
    "Severe and housebound patients are almost absent from exercise, biopsy and MRS studies; a provocation-free design (e.g. orthostatic or small-muscle stressor) is needed.",
    "No study pairs muscle biopsy, 31P-MRS, sodium MRI and invasive CPET in the same people to locate the limiting step in the oxygen cascade.",
    "Serum-transfer studies have not used sera drawn during PEM, fractionation (IgG, complement, EVs), or blinded multi-lab sample exchange.",
    "No pre-registered multi-site PBMC or muscle respirometry study with adequate controls exists.",
    "Lean mass has not been measured alongside the low muscle-protein blood signal.",
    "Handgrip fatigability needs external validation against deconditioned, depressed and other-fatigue controls before use as a trial endpoint.",
    "Whether the amyloid-containing deposits in long COVID muscle also occur in ME/CFS, and what they are made of, is untested."
   ]
  },
  {
   "domain": "neuro",
   "title": "Nervous system and brain",
   "overview": "The most consistent nervous-system findings in ME/CFS are physiological rather than anatomical. Brain blood flow drops more than normal when patients are upright, often with over-breathing (low CO2), and resting heart-rate variability is lower; both are seen across several independent labs. Small-fibre nerve loss on skin biopsy is common in referral clinics (roughly 30-55%), but the main controlled NIH study found no difference, and nobody has yet shown that nerve loss explains symptoms. Brain imaging gives many positive but small and mostly unreplicated results: raised ventricular lactate (mostly one group), raised brain temperature (one study), TSPO-PET neuroinflammation (one positive and one negative ME/CFS study), and newer diffusion MRI signals. Structural MRI, routine sleep studies and markers of nerve injury are mostly normal or inconsistent. The NIH 'effort preference' interpretation has been seriously challenged, because patients in that study often could not complete the hard task. Claims about craniocervical instability rest on uncontrolled clinic series and case reports.",
   "findings": [
    {
     "id": "neuro-cbf-tilt",
     "subdomain": "Cerebral perfusion / orthostatic",
     "finding": "Blood flow to the brain falls much more than normal during head-up tilt in ME/CFS (about 26% vs 7% in controls), including in patients whose heart rate and blood pressure stay normal.",
     "grade": "replicated",
     "plain": "When patients are tilted upright, less blood reaches the brain than in healthy people, even if their pulse and blood pressure look normal. That can explain light-headedness and brain fog when standing.",
     "detail": "van Campen 2020 measured extracranial carotid plus vertebral flow by Doppler during a 30-min tilt in 429 patients vs 44 controls. End-tilt drop was 7% in controls and 26% in patients overall: 24% with a normal HR/BP response, 28% with delayed orthostatic hypotension and 29% with POTS. About 90% exceeded the control range. Transcranial Doppler studies (Medow 2014; Novak 2026, 88% of 170 ME/CFS) agree. A 2025 systematic review (118 studies) found reduced CBF in 12 of 26 ME/CFS-only studies and a larger fall when ME/CFS and orthostatic intolerance coexist. Caveats: most data come from referral clinics; controls are few; and CBF velocity (transcranial Doppler) is not the same as volume flow.",
     "studies": [
      {
       "cite": "van Campen 2020, Clin Neurophysiol Pract",
       "n": "429 vs 44",
       "design": "case-control, tilt with extracranial Doppler",
       "result": "End-tilt CBF -26% vs -7%; ~90% of patients abnormal, incl. normal HR/BP responders",
       "url": "https://doi.org/10.1016/j.cnp.2020.01.003",
       "verified": true
      },
      {
       "cite": "Novak 2026, PLoS One",
       "n": "170 ME/CFS, 143 long COVID, 73 HC, 290 hEDS",
       "design": "retrospective autonomic-lab cohort, TCD + capnography",
       "result": "Reduced orthostatic CBF velocity in 88% ME/CFS and 92% long COVID",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "Medow 2014, J Appl Physiol",
       "n": "15 vs 11",
       "design": "case-control tilt with TCD",
       "result": "CBFv fell 22.5% vs 8.7% in controls",
       "url": "https://doi.org/10.1152/japplphysiol.00527.2014",
       "verified": true
      },
      {
       "cite": "Christopoulos 2025, J Transl Med",
       "n": "118 studies, 9185 participants",
       "design": "systematic review",
       "result": "CBF reduced in ME/CFS and in OI; larger reduction when both present (4/11 studies)",
       "url": "https://doi.org/10.1186/s12967-025-06954-w",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "deep phenotyping incl. 40-min tilt",
       "result": "No group differences in orthostatic hypotension, tachycardia or tilt-related stopping (CBF not measured)",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the CBF fall cause the cognitive symptoms, or only accompany them?",
      "How much is driven by hypocapnia versus low stroke volume?",
      "Is it specific to ME/CFS versus deconditioning, hEDS or other dysautonomias?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre",
      "vascular-endothelial"
     ],
     "test_available": "Tilt with extracranial Doppler (few centres, e.g. Netherlands) or transcranial Doppler in some autonomic labs; mostly research or specialist only"
    },
    {
     "id": "neuro-hypocapnia",
     "subdomain": "Cerebral perfusion / orthostatic",
     "finding": "Many patients over-breathe when upright and lower their end-tidal CO2. This drives part of the fall in brain blood flow, and correcting it with phenylephrine restored blood flow and working memory in one small trial.",
     "grade": "several",
     "plain": "Standing makes some patients breathe off too much carbon dioxide. Low CO2 narrows brain blood vessels, so less blood reaches the brain. A drug that tightens blood vessels prevented this and improved a memory test in a small study.",
     "detail": "Medow 2014: on tilt, ETCO2 was 33.9 vs 42.8 Torr in 15 CFS vs 11 controls. Phenylephrine prevented the hypocapnia, cut the CBFv fall from 22.5% to 8.1%, and returned n-back reaction time to supine levels. Yamazaki 2026: 40.8% of 49 adults met the criteria for postural orthostatic syndrome of hypocapnia (POSH) on a lean test with capnography, more often than POTS. Novak 2026 defines a hypocapnic cerebral hypoperfusion phenotype. In long COVID (O'Brien 2026, n=50), 64% had supine or orthostatic hypocapnia. Solving ME/CFS re-analysis: hypocapnia on tilt is reported by 5 groups and the CBF fall by 4. Treatment evidence is still single-group and acute.",
     "studies": [
      {
       "cite": "Medow 2014, J Appl Physiol",
       "n": "15 vs 11",
       "design": "case-control + acute phenylephrine crossover",
       "result": "ETCO2 33.9 vs 42.8 Torr on tilt; phenylephrine normalised CBFv and n-back",
       "url": "https://doi.org/10.1152/japplphysiol.00527.2014",
       "verified": true
      },
      {
       "cite": "Yamazaki 2026, Physiol Rep",
       "n": "49",
       "design": "cross-sectional, lean test with capnography + Daxor blood volume",
       "result": "POSH in 40.8%; lean-test abnormalities not related to blood volume",
       "url": "https://doi.org/10.14814/phy2.71087",
       "verified": true
      },
      {
       "cite": "O'Brien 2026, J Clin Med",
       "n": "50 long COVID",
       "design": "clinic cohort, NASA lean test + ETCO2",
       "result": "Hypocapnia 64%; POTS 32%; breathing retraining improved symptoms, not ETCO2",
       "url": "https://doi.org/10.3390/jcm15186970",
       "verified": true
      },
      {
       "cite": "Medow & Stewart 2024, Am J Physiol Regul Integr Comp Physiol",
       "n": "small",
       "design": "physiology",
       "result": "Phenylephrine alters CBFv-BP phase synchronisation in ME/CFS with OI",
       "url": "https://doi.org/10.1152/ajpregu.00071.2024",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is hypocapnia primary (respiratory/chemoreflex) or compensation for low venous return?",
      "Would capnography-guided treatment (compression, midodrine, breathing retraining) improve function over weeks?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Capnography during a 10-min stand or lean test: cheap; available in some respiratory/autonomic clinics"
    },
    {
     "id": "neuro-pots-prevalence",
     "subdomain": "Autonomic",
     "finding": "POTS occurs in a minority of ME/CFS patients, and published rates vary widely (about 6-43%) with the setting, the age group and the test protocol.",
     "grade": "contested",
     "plain": "Some, but not most, people with ME/CFS meet the heart-rate criteria for POTS. How many depends a lot on who was tested and how.",
     "detail": "van Campen 2020: 120 of 429 tilt-tested patients (28%) had POTS. Novak 2026: POTS in 19% of ME/CFS and 22% of long COVID, but widespread mild autonomic failure in 89-95%. Adolescents (Leone 2026): PoTS in 7/18 ME/CFS vs 0/18 controls on a 10-min stand, although 83% reported OI symptoms. In the NIH cohort (Walitt 2024, n=17), tilt showed no excess tachycardia. Solving ME/CFS re-analysis of the literature: POTS prevalence is inconsistent (6-43%) and lists it among the contradictory findings. Orthostatic intolerance symptoms are far more common than POTS defined by heart rate.",
     "studies": [
      {
       "cite": "van Campen 2020, Clin Neurophysiol Pract",
       "n": "429",
       "design": "tilt cohort",
       "result": "POTS 28%, delayed OH 14%, normal HR/BP 58%",
       "url": "https://doi.org/10.1016/j.cnp.2020.01.003",
       "verified": true
      },
      {
       "cite": "Novak 2026, PLoS One",
       "n": "170 ME/CFS",
       "design": "retrospective autonomic-lab cohort",
       "result": "POTS 19%, neurogenic OH 15%, mild-moderate autonomic failure 89%",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "Leone 2026, Front Pediatr",
       "n": "18 vs 18 adolescents",
       "design": "case-control, 10-min passive stand",
       "result": "PoTS 39% vs 0%; OI history 83% vs 17%",
       "url": "https://doi.org/10.3389/fped.2026.1836407",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "case-control tilt",
       "result": "No excess orthostatic tachycardia vs controls",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Do POTS and non-POTS ME/CFS differ in mechanism (e.g. hyperadrenergic vs low-flow)?",
      "How much do medication, test length and time since onset change the rate?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "low-blood-volume-preload",
      "autoimmunity"
     ],
     "test_available": "10-min active stand or tilt table test: widely available clinically"
    },
    {
     "id": "neuro-hrv",
     "subdomain": "Autonomic",
     "finding": "Resting heart rate is higher and high-frequency heart-rate variability lower in ME/CFS. This fits reduced vagal (parasympathetic) tone.",
     "grade": "replicated",
     "plain": "The 'rest and digest' nerve signal to the heart looks weaker. The heart beats a little faster at rest, and its beat-to-beat variation is reduced.",
     "detail": "Nelson 2019 meta-analysis (64 case-control studies): resting HR +4.1 bpm, HR on tilt SMD 0.92, resting LF/HF SMD 0.39 higher, HF-HRV SMD -0.34, and peak HR 13.8 bpm lower. Walitt 2024: 24-h Holter SDNNi, rMSSD and pNN50 were lower, HF and LF power reduced, and Valsalva baroreflex-cardiovagal function was lower. Effects are moderate. They are not specific to ME/CFS, and deconditioning, medications and sleep loss confound them. HRV is not a diagnostic test.",
     "studies": [
      {
       "cite": "Nelson 2019, Medicine (Baltimore)",
       "n": "64 studies",
       "design": "systematic review and meta-analysis",
       "result": "RHR +4.1 bpm; HF-HRV SMD -0.34; HRtilt SMD 0.92; HRmax -13.8 bpm",
       "url": "https://doi.org/10.1097/MD.0000000000017600",
       "verified": true
      },
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "14 vs 19 (Holter)",
       "design": "case-control",
       "result": "Lower 24-h HRV time and frequency domains; reduced baroreflex-cardiovagal function",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is low HRV cause, consequence or a marker of deconditioning/sleep disruption?",
      "Can wearable HRV usefully track PEM risk in individuals?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "neuroinflammation",
      "sickness-behaviour"
     ],
     "test_available": "24-h Holter HRV (clinical); consumer wearables give rough trends"
    },
    {
     "id": "neuro-nasa-lean",
     "subdomain": "Autonomic",
     "finding": "On the 10-minute NASA lean test, a subgroup of recent-onset patients shows a rising heart rate with an abnormally narrow pulse pressure. Autonomic specialists say the test has not been validated against tilt or active stand.",
     "grade": "several",
     "plain": "The lean test (standing against a wall for 10 minutes while pulse and blood pressure are recorded) is a cheap way to see orthostatic problems. It is useful for spotting a pattern, but it is not an approved test for diagnosing POTS.",
     "detail": "Lee 2020 (Bateman Horne Center): 150 ME/CFS (75 ill <4 y, 75 ill >10 y) vs 75 controls. At least 5 min was needed to see changes. The <4-y group had higher HR, a narrower pulse pressure and more OI symptoms, interpreted as inadequate ventricular filling. Yamazaki 2026 found lean-test abnormalities (most often hypocapnia) in 55% of 49 patients, unrelated to measured blood volume. Teuschl 2025 (European autonomic experts) argue that NLT criteria were borrowed from 1970s NASA physiology, that the leaning posture mixes active stand and passive tilt, and that the NLT 'may currently not be used as a diagnostic tool'.",
     "studies": [
      {
       "cite": "Lee 2020, J Transl Med",
       "n": "150 vs 75",
       "design": "case-control",
       "result": "<4-y patients: higher HR and narrowed pulse pressure vs >10-y and HC",
       "url": "https://doi.org/10.1186/s12967-020-02481-y",
       "verified": true
      },
      {
       "cite": "Yamazaki 2026, Physiol Rep",
       "n": "49",
       "design": "cross-sectional",
       "result": "55% had >=1 lean-test abnormality (POSH 41%); not related to blood volume",
       "url": "https://doi.org/10.14814/phy2.71087",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Teuschl 2025, Clin Auton Res",
       "n": "n/a",
       "design": "expert commentary",
       "result": "No validation vs tilt/active stand; should not be used for autonomic diagnosis",
       "url": "https://doi.org/10.1007/s10286-024-01097-2",
       "verified": true
      }
     ],
     "open_questions": [
      "Head-to-head diagnostic accuracy of NLT vs tilt in ME/CFS",
      "Does narrow pulse pressure on NLT predict response to volume or compression?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "NASA lean test: any clinic or at home with a BP cuff (for monitoring, not formal diagnosis)"
    },
    {
     "id": "neuro-sfn-biopsy",
     "subdomain": "Small-fibre neuropathy",
     "finding": "Skin biopsies show reduced small-nerve-fibre density in about 31-53% of ME/CFS patients from specialist referral cohorts, but the controlled NIH study found no difference from healthy volunteers.",
     "grade": "contested",
     "plain": "In some clinics, a third to a half of patients have lost some of the thinnest nerves in the skin. Those nerves control sweating and blood vessels. But a carefully matched NIH study did not find this, so how common and how important it is remains uncertain.",
     "detail": "Joseph 2021 (Systrom/Oaklander, Harvard): 31% of 160 ME/CFS biopsies were consistent with SFN (epidermal innervation <=5th percentile of predicted). Denervation severity did not correlate with exertional measures. Novak 2026 (same network): SFN in 53% of ME/CFS and 67% of long COVID; hEDS had more severe neurodegeneration. Azcue 2025 (30 ME/CFS, 30 long COVID, 30 HC) found corneal fibre tortuosity and heat-detection changes rather than a clear length-dependent pattern. Walitt 2024: no difference in small-fibre density. Major caveats: the positive cohorts were referred for exertional intolerance or dysautonomia; biopsy normative cut-offs and patchy, non-length-dependent SFN make rates sensitive to method; and ME/CFS-specific matched-control biopsy studies are few. Solving ME/CFS re-analysis of Finnish registry data: an SFN diagnosis is about 8-10 times more common than expected in post-viral fatigue (crude, shared with fibromyalgia).",
     "studies": [
      {
       "cite": "Joseph 2021, Chest",
       "n": "160 ME/CFS (36 iCPET controls)",
       "design": "retrospective iCPET + skin biopsy",
       "result": "31% SFN; severity not correlated with exertional limits",
       "url": "https://doi.org/10.1016/j.chest.2021.01.082",
       "verified": true
      },
      {
       "cite": "Novak 2026, PLoS One",
       "n": "170 ME/CFS, 143 LC",
       "design": "retrospective autonomic-lab cohort",
       "result": "SFN 53% ME/CFS, 67% LC",
       "url": "https://doi.org/10.1371/journal.pone.0341278",
       "verified": true
      },
      {
       "cite": "Azcue 2025, Eur J Neurol",
       "n": "30 ME/CFS, 30 LC, 30 HC",
       "design": "case-control, QST, Sudoscan, corneal confocal microscopy",
       "result": "Corneal fibre tortuosity higher (AUC 0.72); heat detection impaired",
       "url": "https://doi.org/10.1111/ene.70016",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "case-control, thigh + leg biopsies",
       "result": "Small nerve fibre density not different",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "What is the SFN rate in unselected, community ME/CFS with blinded, matched controls?",
      "Does fibre loss explain any symptom (OI, pain, exertional intolerance)?",
      "Is it autoimmune, and does IVIG change it? (no ME/CFS RCT)"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "autoimmunity",
      "connective-tissue"
     ],
     "test_available": "3 mm skin punch biopsy for IENFD via neurology (clinically available); corneal confocal microscopy in some centres"
    },
    {
     "id": "neuro-sfn-longcovid",
     "subdomain": "Small-fibre neuropathy",
     "finding": "Long COVID patients referred for neuropathy evaluation often have small-fibre neuropathy on skin biopsy, including loss of autonomic fibres to sweat glands and hair muscles.",
     "grade": "several",
     "plain": "Similar nerve damage turns up in long COVID, especially in patients with pain or nerve-type symptoms.",
     "detail": "Oaklander 2022 (NIH/MGH): 10 of 16 skin biopsies (63%) in referred long COVID were abnormal; 52% average improvement over 1.4 years, often after immunotherapy (uncontrolled). Falco 2026: piloerector and sweat-gland nerve density reduced in 31 painful long COVID vs 42 controls, even with normal IENFD, but not correlated with COMPASS-31 symptom scores. These are selected samples with neuropathic complaints, so the results do not give a general prevalence.",
     "studies": [
      {
       "cite": "Oaklander 2022, Neurol Neuroimmunol Neuroinflamm",
       "n": "17",
       "design": "case series",
       "result": "63% of biopsies confirm SFN",
       "url": "https://doi.org/10.1212/NXI.0000000000001146",
       "verified": true
      },
      {
       "cite": "Falco 2026, Front Hum Neurosci",
       "n": "31 vs 42",
       "design": "retrospective case-control histology",
       "result": "Autonomic fibre density reduced; no correlation with symptoms",
       "url": "https://doi.org/10.3389/fnhum.2025.1719705",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Prevalence in unselected long COVID",
      "Does SFN predict who develops ME/CFS after COVID?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "autoimmunity",
      "viral-persistence"
     ],
     "test_available": "Skin biopsy (IENFD, sweat-gland nerve density in specialist labs)"
    },
    {
     "id": "neuro-mri-structure",
     "subdomain": "Brain structure",
     "finding": "Conventional structural MRI (grey matter volume, routine clinical reads) shows no consistent abnormality in ME/CFS. Reported regional volume differences rarely replicate across groups.",
     "grade": "contested",
     "plain": "Standard brain scans usually look normal. Studies that report bigger or smaller brain regions mostly disagree with each other.",
     "detail": "Shan 2020 systematic review (63 imaging studies to 2018): the only findings reported by multiple teams were extra brain-area recruitment during cognitive tasks (11 studies), brainstem abnormalities (9), sluggish BOLD responses, reduced serotonin transporter and regional hypometabolism. Volumetric findings were inconsistent. Lee 2023 meta-analysis (65 studies, 1529 patients) reported insular and thalamic hypoactivity but noted poor inter-rater reliability. Walitt 2024: brain MRI unremarkable. Kimura 2026 (38 ME/CFS, 27 LC, 47 HC): amygdala subfield differences did not survive correction. For contrast, the UK Biobank pre/post scans (Douaud 2022) show that SARS-CoV-2 infection itself produces small grey-matter changes, although that sample was not long COVID or ME/CFS.",
     "studies": [
      {
       "cite": "Shan 2020, J Transl Med",
       "n": "63 studies",
       "design": "systematic review",
       "result": "Few consistent findings; brainstem and task recruitment most repeated",
       "url": "https://doi.org/10.1186/s12967-020-02506-6",
       "verified": true
      },
      {
       "cite": "Lee 2023, Autoimmun Rev",
       "n": "65 studies; 1529 vs 1715",
       "design": "systematic review and meta-analysis",
       "result": "Insula and thalamus hypoactivity; frontal cortex most often reported",
       "url": "https://doi.org/10.1016/j.autrev.2023.103484",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "case-control 3T MRI incl. DTI",
       "result": "Brain imaging unremarkable",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Kimura 2026, Front Neurol",
       "n": "38 ME/CFS, 27 LC, 47 HC",
       "design": "case-control volumetry",
       "result": "No amygdala subfield difference after correction",
       "url": "https://doi.org/10.3389/fneur.2026.1913553",
       "verified": true
      }
     ],
     "open_questions": [
      "Large, multi-site, pre-registered MRI with harmonised protocols has not been done",
      "Do subtle changes track severity or duration longitudinally?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "genetic-neuronal"
     ],
     "test_available": "Clinical MRI (useful only to exclude other diagnoses)"
    },
    {
     "id": "neuro-white-matter-diffusion",
     "subdomain": "Brain structure",
     "finding": "Advanced diffusion MRI models report widespread white-matter differences in ME/CFS that conventional DTI misses, and the pattern may differ between post-infectious and gradual-onset cases.",
     "grade": "early",
     "plain": "Newer scan analyses suggest subtle changes in the brain's wiring, possibly swelling or inflammation. Standard analyses of the same scans find little.",
     "detail": "Yu 2026 (Shan group, 67 vs 67 matched): a neuroinflammation-imaging (NII) model gave lower hindered and restricted water fractions and higher fibre fraction across association, commissural and projection tracts. The authors read this as oedema, cellular infiltration and axonal reorganisation, while conventional DTI showed minimal differences. Yu 2025 (same group): post-infectious cases had higher axial diffusivity, while gradual-onset cases had lower AD in the corpus callosum. Singh 2026 (Griffith, 37 ME/CFS, 19 LC, 27 HC) found DTI/DKI alterations in the cingulum, SMA and corpus callosum. The model-based metrics are not validated against histology in ME/CFS, the analyses carry many degrees of freedom, and independent replication is missing.",
     "studies": [
      {
       "cite": "Yu 2026, Hum Brain Mapp",
       "n": "67 vs 67",
       "design": "case-control diffusion MRI (NII model)",
       "result": "Widespread NII differences; conventional DTI minimal",
       "url": "https://doi.org/10.1002/hbm.70505",
       "verified": true
      },
      {
       "cite": "Yu 2025, Sci Rep",
       "n": "PI and GO subgroups vs sedentary HC",
       "design": "case-control diffusion MRI",
       "result": "Opposite axial diffusivity patterns by onset type",
       "url": "https://doi.org/10.1038/s41598-025-09379-z",
       "verified": true
      },
      {
       "cite": "Singh 2026, Front Med",
       "n": "37 ME/CFS, 19 LC, 27 HC",
       "design": "case-control DTI/DKI",
       "result": "Microstructural changes in cingulum, SMA, corpus callosum",
       "url": "https://doi.org/10.3389/fmed.2026.1824498",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "15-direction DTI",
       "result": "No reported imaging abnormality",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Independent replication with shared analysis code",
      "Correlation with TSPO-PET or MRS markers in the same people"
     ],
     "hypotheses": [
      "neuroinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-brainstem",
     "subdomain": "Brain structure",
     "finding": "Brainstem differences (smaller volume, altered signal or connectivity) are among the more often repeated imaging findings in ME/CFS, though the methods differ between studies.",
     "grade": "several",
     "plain": "The brainstem controls breathing, heart rate and arousal. Several groups using different scans have reported differences there.",
     "detail": "Shan 2020 counted brainstem abnormalities in 9 studies from different teams and modalities. Bader 2026 (preprint, 26 vs 27): brainstem volume 1.46 vs 1.55% of eTIV (FDR p=0.039), with deep grey matter and whole-brain volume unchanged. Inderyas 2026 (7T fMRI, 32 ME/CFS): altered cuneiform-nucleus to medulla connectivity during Stroop. The Griffith (Barnden/Thapaliya) group produced several of the brainstem reports, so the evidence is not fully independent. This imaging work does not support the mechanical 'craniocervical compression' model, which is a separate claim (see neuro-cci).",
     "studies": [
      {
       "cite": "Bader 2026, medRxiv (preprint)",
       "n": "26 vs 27",
       "design": "case-control volumetry + ASL + MRS under hypoxia",
       "result": "Brainstem volume reduced",
       "url": "https://doi.org/10.64898/2026.08.10.26359935",
       "verified": true
      },
      {
       "cite": "Inderyas 2026, J Transl Med",
       "n": "32 ME/CFS, 19 LC, 27 HC",
       "design": "7T task fMRI",
       "result": "Altered brainstem functional connectivity during cognitive fatigue",
       "url": "https://doi.org/10.1186/s12967-026-07708-y",
       "verified": true
      },
      {
       "cite": "Shan 2020, J Transl Med",
       "n": "9 studies",
       "design": "systematic review",
       "result": "Brainstem abnormalities repeatedly reported",
       "url": "https://doi.org/10.1186/s12967-020-02506-6",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Which brainstem nuclei, and do they map onto autonomic or respiratory findings (hypocapnia, low HRV)?"
     ],
     "hypotheses": [
      "brainstem-craniocervical",
      "neuroinflammation",
      "autonomic-small-fibre"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-fmri-post-exertion",
     "subdomain": "Functional MRI",
     "finding": "After a single 30-minute exercise bout, women with ME/CFS performed worse on a demanding cognitive task and recruited more parietal and cingulate cortex. Controls improved.",
     "grade": "early",
     "plain": "Exercise made patients' thinking worse the next day, and their brains had to work harder at the same task. This is one of the few direct brain measurements of post-exertional malaise.",
     "detail": "Cook 2017: 15 vs 15 women, scanned 1 week before and 24 h after 30 min of cycling at 70% peak HR. Group x time interactions showed worse PASAT performance in patients and increased activation in bilateral inferior/superior parietal and cingulate cortex, correlated with symptom change. Finger tapping did not differ. Patients cycled at lower watts, so absolute workload differed. Related work: Schönberg 2024 (34 vs 34) found that BOLD signals in ME/CFS did not adapt across repeated task blocks as they did in controls, and Shan 2020 counted extra task recruitment as a finding repeated by several teams. A direct replication of the exercise-provocation design has not been published.",
     "studies": [
      {
       "cite": "Cook 2017, Brain Behav Immun",
       "n": "15 vs 15",
       "design": "pre/post exercise fMRI case-control",
       "result": "Post-exercise cognitive decline and increased parietal/cingulate activation in patients",
       "url": "https://doi.org/10.1016/j.bbi.2017.02.009",
       "verified": true
      },
      {
       "cite": "Schönberg 2024, J Cereb Blood Flow Metab",
       "n": "34 vs 34",
       "design": "task fMRI case-control",
       "result": "Absent BOLD adaptation across repeated SDMT blocks",
       "url": "https://doi.org/10.1177/0271678X241270528",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication with workload matched relative to individual capacity",
      "Time course over 24-72 h of PEM"
     ],
     "hypotheses": [
      "neuroinflammation",
      "mitochondrial-energy",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-motor-fatigability",
     "subdomain": "Functional MRI",
     "finding": "During a sustained grip task, ME/CFS patients fatigue earlier while their brain motor-network activity fails to ramp up as it does in controls (NIH; reduced temporoparietal-junction activation).",
     "grade": "early",
     "plain": "When squeezing repeatedly, patients tire sooner. Their brains do not step up the drive to the muscle the way healthy brains do.",
     "detail": "Walitt 2024 reported decreased right temporoparietal junction activation during repeated 50% MVC grip in fMRI and a faster decline in force. The authors linked it to 'effort preference', which is disputed. Bedard 2026 (NIH, 15 vs 19, EEG+EMG+fMRI) found that maximum force was equal, but patients reached fatigue much earlier and showed minimal change in brain and muscle activity across blocks. Healthy controls increased activity until fatigue. Interpretation: a central contribution to fatigability. Both studies come from the same NIH programme and have small samples.",
     "studies": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "case-control task fMRI",
       "result": "Reduced TPJ activation; earlier force decline",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Bedard 2026, Neuroimage Clin",
       "n": "15 vs 19",
       "design": "case-control multimodal EEG/EMG/fMRI",
       "result": "Earlier fatigue; brain output did not increase to drive muscle",
       "url": "https://doi.org/10.1016/j.nicl.2026.104041",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is the lack of central ramp-up primary or protective (driven by peripheral afferent feedback)?",
      "Independent replication outside NIH"
     ],
     "hypotheses": [
      "sickness-behaviour",
      "neuroinflammation",
      "mitochondrial-energy"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-mrs-lactate",
     "subdomain": "MR spectroscopy",
     "finding": "Brain lactate measured by MR spectroscopy is raised in ME/CFS: ventricular CSF lactate in three cohorts from one group, and thalamic or regional lactate in two other groups. It is also raised in depression, so it is not specific.",
     "grade": "several",
     "plain": "Brain scans that measure chemicals show more lactate, a by-product of energy production without enough oxygen, in patients' brains. The same change appears in depression.",
     "detail": "Mathew 2009: ventricular lactate +348% vs healthy and +297% vs anxiety disorder (16 vs 15 vs 14). Shungu 2012 (third cohort, 15 vs 13): raised lactate and lower cortical glutathione, not different from major depression. Natelson 2017 replicated higher lactate, lower GSH and lower CBF in pooled CFS. Mueller 2020 (Younger group, whole-brain EPSI, 15 vs 15): raised LAC/CR in some regions, overlapping with raised temperature. Bader 2026 (Vienna, 26 vs 27, preprint): thalamic Lac/tCr 0.171 vs 0.135 at normoxia, with no further rise under hypoxia, unlike controls. Bradley 2016 found raised ventricular lactate in adolescent depression. Small samples, MRS quantification at 3T near detection limits, and the lack of specificity limit interpretation.",
     "studies": [
      {
       "cite": "Mathew 2009, NMR Biomed",
       "n": "16 CFS, 14 GAD, 15 HC",
       "design": "case-control 1H MRSI",
       "result": "Ventricular lactate +348% vs HC",
       "url": "https://doi.org/10.1002/nbm.1315",
       "verified": true
      },
      {
       "cite": "Shungu 2012, NMR Biomed",
       "n": "15 CFS, 15 MDD, 13 HC",
       "design": "case-control MRSI + ASL + 31P",
       "result": "Raised lactate, lower GSH, lower rCBF; CFS ~ MDD",
       "url": "https://doi.org/10.1002/nbm.2772",
       "verified": true
      },
      {
       "cite": "Natelson 2017, J Neurol Sci",
       "n": "CFS +/- psychiatric comorbidity vs HC",
       "design": "case-control multimodal",
       "result": "Replicated raised lactate, lower GSH and CBF; 13/26 had >=2 abnormal brain measures",
       "url": "https://doi.org/10.1016/j.jns.2017.02.046",
       "verified": true
      },
      {
       "cite": "Bader 2026, medRxiv (preprint)",
       "n": "26 vs 27",
       "design": "case-control MRS with hypoxic challenge",
       "result": "Thalamic lactate raised at rest, no rise with hypoxia",
       "url": "https://doi.org/10.64898/2026.08.10.26359935",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Bradley 2016, Eur Psychiatry",
       "n": "23 MDD vs 29 HC",
       "design": "case-control MRSI",
       "result": "Raised ventricular lactate in adolescent depression (shows non-specificity)",
       "url": "https://doi.org/10.1016/j.eurpsy.2015.08.009",
       "verified": true
      }
     ],
     "open_questions": [
      "Does brain lactate rise further after exertion (PEM)?",
      "Is it from hypoperfusion, mitochondrial limitation or glial activation?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "neuroinflammation",
      "small-vessel-capillary"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-brain-temperature",
     "subdomain": "MR spectroscopy",
     "finding": "MRS thermometry found raised temperature in several brain regions (insula, putamen, frontal cortex, thalamus, cerebellum) in women with ME/CFS, not explained by body temperature.",
     "grade": "early",
     "plain": "One study found parts of the brain running slightly warmer in patients, which could signal low-grade inflammation. It has not been repeated.",
     "detail": "Mueller 2020: 15 vs 15 women, whole-brain echo-planar spectroscopic imaging. Temperature was raised in the right insula, putamen, frontal cortex, thalamus and cerebellum (p<0.05, many regions tested), along with raised choline/creatine in the left anterior cingulate. The method responded to an experimental inflammatory challenge (typhoid vaccine) in 44 of 47 regions, but whole-brain temperature did not change (Plank 2022), so its sensitivity is modest. No independent ME/CFS replication was found.",
     "studies": [
      {
       "cite": "Mueller 2020, Brain Imaging Behav",
       "n": "15 vs 15",
       "design": "case-control whole-brain MRSI",
       "result": "Raised regional brain temperature; raised CHO/CR",
       "url": "https://doi.org/10.1007/s11682-018-0029-4",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Plank 2022, Neuroimage Clin",
       "n": "20 healthy",
       "design": "RCT crossover, typhoid vaccine",
       "result": "Regional but not whole-brain temperature rose (method validity context)",
       "url": "https://doi.org/10.1016/j.nicl.2022.103053",
       "verified": true
      }
     ],
     "open_questions": [
      "Independent replication",
      "Correlation with TSPO-PET in the same people"
     ],
     "hypotheses": [
      "neuroinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-pet-tspo-mecfs",
     "subdomain": "PET neuroinflammation",
     "finding": "TSPO-PET evidence of microglial activation in ME/CFS is contested: one Japanese study found 45-199% higher binding in limbic and brainstem regions, and a Dutch study using the same tracer found none.",
     "grade": "contested",
     "plain": "A special scan for brain immune cells showed inflammation in one small study and none in another of the same size. The question is open.",
     "detail": "Nakatomi 2014: [11C]-(R)-PK11195, 9 vs 10. BPND was higher in the cingulate, hippocampus, amygdala, thalamus, midbrain and pons, and correlated with cognitive impairment and pain. Raijmakers 2021: same tracer, 9 CFS, 10 Q-fever fatigue and 9 matched women, stricter exclusion. No difference, and a negative correlation with severity in CFS. Both used the cerebellum as reference region, which can bias results if the reference is also affected. PK11195 has a low signal-to-noise ratio, and second-generation tracers need TSPO genotyping. Stanford DPA-713 data exist only as a conference abstract (not verified). A Belgian [18F]DPA714 study is in protocol (Dooms 2026).",
     "studies": [
      {
       "cite": "Nakatomi 2014, J Nucl Med",
       "n": "9 vs 10",
       "design": "case-control PET",
       "result": "BPND +45-199% in limbic/brainstem regions",
       "url": "https://doi.org/10.2967/jnumed.113.131045",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Raijmakers 2021, Neurol Neuroimmunol Neuroinflamm",
       "n": "9 CFS, 10 QFS, 9 HC",
       "design": "case-control PET",
       "result": "No difference in BPND",
       "url": "https://doi.org/10.1212/NXI.0000000000001113",
       "verified": true
      }
     ],
     "open_questions": [
      "Adequately powered study with a second- or third-generation tracer, arterial input and genotyping",
      "Does TSPO signal change during PEM?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-pet-tspo-longcovid",
     "subdomain": "PET neuroinflammation",
     "finding": "In long COVID, one [11C]PBR28 PET study found widespread raised TSPO signal that correlated with vascular markers, and a [11C]PK11195 study found no group difference.",
     "grade": "contested",
     "plain": "Long COVID brain-inflammation scans also disagree. One found widespread signal and another found none.",
     "detail": "VanElzakker 2024: 12 PASC vs 43 normative controls. Raised PBR28 in the mid and anterior cingulate, corpus callosum, thalamus, basal ganglia and periventricular regions, correlated with plasma vascular-dysfunction analytes. The comparison against a pooled normative control set and the small n are limits. Tuomaala 2026: 14 LC vs 11 HC vs 13 MS. TSPO DVR was not raised in LC, though higher in people imaged within 16 months, and limbic DVR correlated with depression and anxiety. An earlier Braga 2023 report in long COVID with depression/cognitive symptoms was not verified this session.",
     "studies": [
      {
       "cite": "VanElzakker 2024, Brain Behav Immun",
       "n": "12 vs 43",
       "design": "case-control PET",
       "result": "Widespread raised PBR28; correlates with vascular markers",
       "url": "https://doi.org/10.1016/j.bbi.2024.04.015",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Tuomaala 2026, J Neurol",
       "n": "14 LC, 11 HC, 13 MS",
       "design": "case-control PET",
       "result": "No TSPO elevation vs HC; time-since-infection effect",
       "url": "https://doi.org/10.1007/s00415-026-13842-w",
       "verified": true
      }
     ],
     "open_questions": [
      "Does neuroinflammation fade with time since infection?",
      "Is it seen in long COVID that meets ME/CFS criteria specifically?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "viral-persistence",
      "vascular-endothelial"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-cbf-rest",
     "subdomain": "Cerebral perfusion",
     "finding": "Resting (supine) cerebral blood flow by ASL or SPECT is reported as lower in some ME/CFS studies, but the results are inconsistent and often confounded by unmeasured orthostatic intolerance.",
     "grade": "contested",
     "plain": "Lying-down brain blood flow is sometimes lower, but not consistently. The clearest effect appears when patients are upright.",
     "detail": "Shungu 2012 (ASL) found lower rCBF in the left anterior cingulate and right lingual gyrus vs controls, but not vs depression. Natelson 2017 pooled CFS had lower CBF. Christopoulos 2025 review: only 12 of 26 ME/CFS-only studies found reduced CBF, and most did not record OI status. Bader 2026: whole-brain CBF responses to hypoxia were similar in mean but far more variable in patients (variance ratio up to 6.9).",
     "studies": [
      {
       "cite": "Shungu 2012, NMR Biomed",
       "n": "15 vs 13",
       "design": "ASL case-control",
       "result": "Lower regional CBF in 2 regions",
       "url": "https://doi.org/10.1002/nbm.2772",
       "verified": true
      },
      {
       "cite": "Christopoulos 2025, J Transl Med",
       "n": "26 ME/CFS-only studies",
       "design": "systematic review",
       "result": "12/26 reduced",
       "url": "https://doi.org/10.1186/s12967-025-06954-w",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Bader 2026, medRxiv (preprint)",
       "n": "26 vs 27",
       "design": "ASL under normoxia/hypoxia",
       "result": "No mean group difference; higher variability in patients",
       "url": "https://doi.org/10.64898/2026.08.10.26359935",
       "verified": true
      }
     ],
     "open_questions": [
      "Resting CBF stratified by OI and hypocapnia status"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial",
      "low-blood-volume-preload"
     ],
     "test_available": "research only (ASL MRI)"
    },
    {
     "id": "neuro-csf-catechol",
     "subdomain": "Cerebrospinal fluid",
     "finding": "In the NIH cohort, CSF levels of the catechol metabolites DOPA, DOPAC and DHPG were lower in post-infectious ME/CFS. Noradrenaline and dopamine did not differ.",
     "grade": "early",
     "plain": "Spinal fluid showed lower breakdown products of dopamine and noradrenaline, which hints at reduced activity of these brain chemical systems. It comes from one small study.",
     "detail": "Walitt 2024: CSF DOPA, DOPAC and DHPG were reduced; noradrenaline, cys-DOPA and dopamine were unchanged. Excluding participants on central-acting drugs did not change the result. Catechols correlated with perceived symptoms and, in patients, with only one cognitive test (WCST). CSF metabolomics also showed lower glutamate, tryptophan, spermidine and citrate. n was about 17 vs 21, with many outcomes and no correction across domains. Solving ME/CFS re-analysis: plasma VMA showed nothing, which fits a central rather than peripheral signal but is weak support.",
     "studies": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "~17 vs 21",
       "design": "case-control CSF",
       "result": "Lower DOPA, DOPAC, DHPG",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication in an independent CSF cohort",
      "Relation to orthostatic phenotype and HRV"
     ],
     "hypotheses": [
      "neuroinflammation",
      "sickness-behaviour",
      "autonomic-small-fibre"
     ],
     "test_available": "research only (lumbar puncture with catechol panel)"
    },
    {
     "id": "neuro-csf-immune-proteome",
     "subdomain": "Cerebrospinal fluid",
     "finding": "CSF cytokine and proteome studies report differences (e.g. raised eotaxin/CCL11, distinct immunotypes), but no CSF protein signature has replicated, and ME/CFS and fibromyalgia CSF proteomes were indistinguishable.",
     "grade": "contested",
     "plain": "Spinal-fluid tests have hinted at immune changes, but different studies point in different directions, and none gives a reliable marker.",
     "detail": "Baraniuk 2005: pooled and individual CSF proteomics (about 10-20 per group) gave a 5-protein set with 80% concordance; pilot only. Hornig 2016 (online 2015): 32 cases vs 40 MS vs 19 controls, 51-plex. CCL11 was raised and the IL-1 network disturbed, with interpretation hindered by the small control group. Schutzer 2023: 2083 proteins quantified; no protein differed between ME/CFS with and without fibromyalgia. Bastos 2025 (40 cases, CSF in cases only) found two MMP-defined clusters. Bragée 2026 (31 cases, no CSF controls) found complement/coagulation pathways associated with severity and POTS. Solving ME/CFS re-analysis: the Walitt CSF SomaScan set gives 0 FDR hits, and CSF neuronal markers do not replicate.",
     "studies": [
      {
       "cite": "Hornig 2016, Mol Psychiatry",
       "n": "32 vs 19 HC (40 MS)",
       "design": "case-control 51-plex",
       "result": "CCL11 up; IL-1 network disturbance",
       "url": "https://doi.org/10.1038/mp.2015.29",
       "verified": true
      },
      {
       "cite": "Baraniuk 2005, BMC Neurol",
       "n": "10 CFS/10 GWI/10 HC pooled; 9 vs 12",
       "design": "pilot proteomics",
       "result": "5-protein set, 80% concordance",
       "url": "https://doi.org/10.1186/1471-2377-5-22",
       "verified": true
      },
      {
       "cite": "Bastos 2025, J Immunol",
       "n": "40 cases (CSF), 41 HC (plasma)",
       "design": "cases-only CSF clustering",
       "result": "Two MMP-based CSF immunotypes",
       "url": "https://doi.org/10.1093/jimmun/vkaf087",
       "verified": true
      },
      {
       "cite": "Bragée 2026, Sci Rep",
       "n": "31 cases",
       "design": "cases-only CSF proteomics",
       "result": "Complement/coagulation pathways linked to severity; neutrophil degranulation with POTS",
       "url": "https://doi.org/10.1038/s41598-026-46965-1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Schutzer 2023, Ann Med",
       "n": "15 vs 15 (ME/CFS +/- FM)",
       "design": "CSF proteomics, 2083 proteins",
       "result": "No proteins differed: indistinguishable",
       "url": "https://doi.org/10.1080/07853890.2023.2208372",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (Walitt 2024 CSF SomaScan, GEO GSE251790)",
       "n": "~17 vs 21",
       "design": "re-analysis",
       "result": "0 FDR hits; no case-control leak-score difference",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "A CSF study with adequate healthy controls (n>50) and pre-registered analysis",
      "Paired pre/post-exertion CSF"
     ],
     "hypotheses": [
      "neuroinflammation",
      "innate-hyperactivation",
      "complement-thromboinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-csf-metabolomics",
     "subdomain": "Cerebrospinal fluid",
     "finding": "Targeted CSF metabolomics found raised serine and phospholipids and reduced 5-methyltetrahydrofolate in ME/CFS, with different metabolite responses to exercise than in controls.",
     "grade": "early",
     "plain": "Spinal fluid chemistry suggests changes in a vitamin/amino-acid pathway (folate and serine), possibly affecting brain fats. These are single-group results.",
     "detail": "Baraniuk 2025 (Sci Rep; separate non-exercise and post-exercise LP cohorts): ME/CFS showed raised serine, sarcosine, creatine, purines and sphingomyelins and lower 5MTHF, dimethylglycine and choline. After exercise, metabolites were consumed in ME/CFS but generated in controls. The design is cross-sectional with separate cohorts per condition, not paired. Earlier Baraniuk 2021 work found little non-exercise ME/CFS vs GWI difference. NIH found lower CSF glutamate, tryptophan and spermidine (Walitt 2024), so the two sets point in different directions.",
     "studies": [
      {
       "cite": "Baraniuk 2025, Sci Rep",
       "n": "ME/CFS vs sedentary controls, rest and post-exercise cohorts",
       "design": "case-control targeted metabolomics",
       "result": "Serine up, 5MTHF down; divergent exercise response",
       "url": "https://doi.org/10.1038/s41598-025-91324-1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "~17 vs 21",
       "design": "CSF metabolomics",
       "result": "Different pattern: lower glutamate, tryptophan, spermidine, citrate",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Replication; paired pre/post-exercise sampling in the same people"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "metabolic-trap-ido"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-injury-markers",
     "subdomain": "Cerebrospinal fluid",
     "finding": "Markers of neuronal or glial injury (NfL, GFAP, tau, UCH-L1) are not raised in ME/CFS blood or CSF.",
     "grade": "several",
     "plain": "Blood and spinal-fluid tests that pick up nerve-cell damage in diseases like MS or dementia are normal in ME/CFS. That argues against ongoing destruction of brain tissue.",
     "detail": "Walitt 2024 measured Simoa Neurology 4-Plex (NfL, tau, GFAP, UCH-L1) in plasma and CSF: no group difference. Solving ME/CFS re-analysis of SomaScan/Olink proteomics across about 150 patients: NEFL and GFAP flat or inconsistent (neuronal_glial set -0.25, p=0.07). Long COVID TSPO-PET (Tuomaala 2026) also measured serum NfL/GFAP, without a clear LC effect. A normal result does not exclude functional or neuroinflammatory changes without cell death.",
     "studies": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "~17 vs 21",
       "design": "case-control Simoa",
       "result": "No difference in plasma or CSF NfL/GFAP/tau/UCH-L1",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (proteomics)",
       "n": "~150 patients across cohorts",
       "design": "re-analysis",
       "result": "NEFL/GFAP not different",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do levels rise transiently during PEM or in severe/bedbound patients?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "genetic-neuronal"
     ],
     "test_available": "Serum NfL/GFAP increasingly available clinically (mainly to exclude other disease)"
    },
    {
     "id": "neuro-cci",
     "subdomain": "Craniocervical / intracranial pressure",
     "finding": "Uncontrolled clinic series report high rates of craniocervical 'obstruction', low-lying tonsils and signs of raised intracranial pressure in severe ME/CFS, and surgical case reports describe improvement. There are no controlled imaging studies or trials.",
     "grade": "early",
     "plain": "Some clinics report that many severe patients have crowding where the skull meets the neck, or high spinal-fluid pressure, and a few patients improved after neck surgery. Without comparison groups or controlled trials, it is unknown whether these findings are more common than in healthy people or whether surgery helps.",
     "detail": "Bragée 2020 (Swedish referral clinic, 229 CCC patients, retrospective): hypermobility 50%, enlarged optic-nerve-sheath ratio 55%, possible IH signs 83%, tonsils below McRae line 56%, and cervical 'craniocervical obstructions' 80% of 125 scanned. No controls; MRI reads were not blinded and the radiological thresholds are not validated. Jolley 2026 (34 cases, no controls): LP opening pressure >=20 cmH2O in 38%; that group reported more relief after LP (80% vs 19%, unblinded). Rowe/Edwards case reports (3+1 patients) describe improvement after cervical decompression. Solving ME/CFS re-analysis of Finnish registry data: CSF-leak codes are not specific (36% of LPs in ME/CFS vs 32% in fibromyalgia, 45% in hypermobility). Neurosurgical series (Ruhoy 2025, n=717) are in connective-tissue-disorder cohorts, not ME/CFS. The evidence quality is low, and surgery carries serious risk.",
     "studies": [
      {
       "cite": "Bragée 2020, Front Neurol",
       "n": "229 (205 brain MRI, 125 c-spine MRI)",
       "design": "retrospective uncontrolled series",
       "result": "80% craniocervical obstruction; 83% possible IH signs; 56% low tonsils",
       "url": "https://doi.org/10.3389/fneur.2020.00828",
       "verified": true
      },
      {
       "cite": "Jolley 2026, Front Med",
       "n": "34",
       "design": "uncontrolled case series",
       "result": "38% opening pressure >=20 cmH2O; more post-LP relief in high-OP group",
       "url": "https://doi.org/10.3389/fmed.2026.1869714",
       "verified": true
      },
      {
       "cite": "Edwards 2023, Front Neurol",
       "n": "1 (follow-up of 3-case series)",
       "design": "case report",
       "result": "Symptoms returned with recurrent stenosis and improved after re-operation",
       "url": "https://doi.org/10.3389/fneur.2023.1284062",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (FinnGen/FinRegistry)",
       "n": "~1,600 G93.3 cases",
       "design": "registry co-occurrence",
       "result": "CSF-leak codes equally common in fibromyalgia/hypermobility; not ME/CFS-specific",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Blinded, controlled upright/dynamic MRI comparing ME/CFS, hypermobile and healthy controls",
      "Any randomised or sham-controlled evaluation of surgery or CSF drainage"
     ],
     "hypotheses": [
      "brainstem-craniocervical",
      "connective-tissue"
     ],
     "test_available": "Clinical cervical MRI is available; upright/flexion MRI and CCI surgery are offered privately with no controlled evidence"
    },
    {
     "id": "neuro-effort-preference",
     "subdomain": "Effort and motivation",
     "finding": "The NIH claim that altered 'effort preference' defines post-infectious ME/CFS rests on the EEfRT (OR 1.65 for choosing hard tasks). Patients completed only 65% of the hard tasks they chose vs 96% for controls, which confounds preference with ability.",
     "grade": "contested",
     "plain": "The NIH team said patients chose the harder button-pressing task less often and called this 'effort preference'. Critics showed that many patients could not physically finish the hard task, so the choice more likely reflects ability than wanting to avoid effort.",
     "detail": "Walitt 2024: 15 PI-ME/CFS vs 16 HV on EEfRT. Controls were 1.65x more likely to choose hard (95% CI 1.03-2.65, p=0.04), and no group difference in task-related fatigue response was reported. Kirvin-Quamme 2025 re-analysed mapMECFS data. Hard-task completion was 65% (SD 37) in patients vs 96% (SD 8) in controls; 7/15 patients completed fewer hard tasks than any control. Choice correlated with completion (rs 0.38). This fails the EEfRT's own ability check (Treadway 2009). Davenport 2025 (Nat Commun Matters Arising) also criticised the single-day CPET, matching for deconditioning and generalisation from n=17. The NIH reply defended single CPET on safety grounds. The effort result also sits on p=0.04 with no multiplicity correction.",
     "studies": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "15 vs 16 (EEfRT)",
       "design": "case-control",
       "result": "OR 1.65 for hard-task choice in controls",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Walitt 2025 reply, Nat Commun",
       "n": "n/a",
       "design": "reply",
       "result": "Defends single CPET and interpretation",
       "url": "https://doi.org/10.1038/s41467-025-64539-z",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Kirvin-Quamme 2025, Front Psychol",
       "n": "15 vs 16 (re-analysis)",
       "design": "secondary analysis of public data",
       "result": "Hard-task completion 65% vs 96%; choice confounded with ability",
       "url": "https://doi.org/10.3389/fpsyg.2025.1593269",
       "verified": true
      },
      {
       "cite": "Davenport 2025, Nat Commun (Matters Arising)",
       "n": "n/a",
       "design": "critique",
       "result": "Single CPET cannot assess PEM; deconditioning/effort conclusions not supported",
       "url": "https://doi.org/10.1038/s41467-025-64538-0",
       "verified": true
      }
     ],
     "open_questions": [
      "An effort-based decision task calibrated to each participant's ability",
      "Relation of motor fatigability to peripheral muscle findings"
     ],
     "hypotheses": [
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "neuro-cognition",
     "subdomain": "Cognition",
     "finding": "Meta-analysis shows moderate-to-large deficits in information-processing speed, attention, working memory and reaction time in ME/CFS, with intact vocabulary and reasoning. Performance worsens with upright posture and after exertion.",
     "grade": "replicated",
     "plain": "'Brain fog' shows up on tests as slower processing and difficulty holding information, especially over sustained effort, rather than loss of intelligence or knowledge.",
     "detail": "Cockshell & Mathias 2010: 50 studies, deficits mainly in attention, memory and reaction time, with fine motor speed, vocabulary and reasoning spared. On tilt, n-back reaction time rose from 96 to 148 ms in CFS but not controls (Medow 2014). Post-exercise, PASAT performance fell (Cook 2017). The NIH study found no differences on any of 15 neuropsychological tests despite large subjective complaints (n=17), which shows that small samples and unprovoked testing can miss deficits. Reaction-time variability and sustained-attention tasks are more sensitive than standard batteries.",
     "studies": [
      {
       "cite": "Cockshell & Mathias 2010, Psychol Med",
       "n": "50 studies",
       "design": "meta-analysis",
       "result": "Moderate-large deficits in processing speed, attention, memory, RT",
       "url": "https://doi.org/10.1017/S0033291709992054",
       "verified": true
      },
      {
       "cite": "Medow 2014, J Appl Physiol",
       "n": "15 vs 11",
       "design": "case-control tilt",
       "result": "Orthostatic worsening of n-back; reversed by phenylephrine",
       "url": "https://doi.org/10.1152/japplphysiol.00527.2014",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "case-control, 15 tests",
       "result": "No group differences in objective performance",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "A standard provocation protocol (upright, post-exertion) for clinical cognitive testing",
      "Contribution of sleep, medication and orthostatic hypoperfusion"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "neuroinflammation",
      "sickness-behaviour"
     ],
     "test_available": "Clinical neuropsychology; computerised reaction-time tests; tests done upright or after exertion are research only"
    },
    {
     "id": "neuro-sleep",
     "subdomain": "Sleep",
     "finding": "Objective sleep in adult ME/CFS shows modestly longer sleep latency, more wake after sleep onset and lower sleep efficiency. Standard polysomnography usually finds no clinical disorder that explains non-restorative sleep.",
     "grade": "several",
     "plain": "Sleep studies show patients take longer to fall asleep and wake more, but the differences are small. They do not explain why sleep feels so unrefreshing.",
     "detail": "Mohamed 2023 meta-analysis: 20 adult studies (426 vs 375) showed more time in bed, longer SOL and WASO, lower efficiency, less N2, more N3 and longer REM latency. In 4 adolescent studies (242 vs 235), total sleep was longer. Heterogeneity was high, and the stage-level findings are inconsistent with older reviews (Jackson & Bruck 2012: few PSG differences; microstructure, such as instability and alpha intrusion, more promising). Walitt 2024: PSG without clinically relevant findings. Solving ME/CFS re-analysis of Finnish registries: hypersomnia codes are enriched about 20-50x and sleep apnoea precedes diagnosis (HR 15), probably reflecting diagnostic work-up as well as biology. Primary sleep disorders still need excluding clinically.",
     "studies": [
      {
       "cite": "Mohamed 2023, Sleep Med Rev",
       "n": "24 studies; 668 vs 610",
       "design": "systematic review and meta-analysis",
       "result": "Lower efficiency, longer SOL/WASO in adults",
       "url": "https://doi.org/10.1016/j.smrv.2023.101771",
       "verified": true
      },
      {
       "cite": "Jackson & Bruck 2012, J Clin Sleep Med",
       "n": "review",
       "design": "narrative review",
       "result": "Few PSG differences; microstructure/HRV in sleep promising",
       "url": "https://doi.org/10.5664/jcsm.2276",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17",
       "design": "clinical PSG",
       "result": "No clinically relevant findings",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "open_questions": [
      "What makes sleep non-restorative: autonomic arousal, microstructure, or glymphatic function?",
      "No ME/CFS trials of orexin antagonists or low-dose sedating agents"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "neuroinflammation",
      "hpa-endocrine"
     ],
     "test_available": "Clinical polysomnography (to exclude apnoea, PLMs, narcolepsy)"
    },
    {
     "id": "neuro-sensory",
     "subdomain": "Sensory sensitivity",
     "finding": "Self-reported light and sound sensitivity is common in ME/CFS. One ERP study found normal early sensory gating (P50) but a reduced P300, pointing to higher-order processing rather than early filtering.",
     "grade": "early",
     "plain": "Many patients find light and noise hard to tolerate. The single brain-wave study suggests that the basic filtering of sounds is normal and that later attention steps are affected.",
     "detail": "Kumar 2026: 31 vs 30, auditory paired-click and oddball paradigms. Patients reported more sensory problems; the P50 suppression index did not differ, while P300 amplitude was reduced. Severe and very severe patients report extreme sensitivity (Vink 2026 review), but they are largely absent from laboratory studies. There are no controlled studies of photophobia thresholds, hyperacusis audiometry or migraine-type central sensitisation specific to ME/CFS. The genetic overlap with chronic pain and migraine (Solving ME/CFS genetics re-analysis) is consistent with shared central sensitisation.",
     "studies": [
      {
       "cite": "Kumar 2026, Front Med",
       "n": "31 vs 30",
       "design": "case-control ERP",
       "result": "Normal P50 gating; reduced P300",
       "url": "https://doi.org/10.3389/fmed.2026.1842841",
       "verified": true
      },
      {
       "cite": "Vink & Vink-Niese 2026, J Clin Med",
       "n": "review",
       "design": "narrative review of severe ME",
       "result": "Extreme light/sound sensitivity described in severe ME",
       "url": "https://doi.org/10.3390/jcm15020805",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Quantitative sensory thresholds in severe patients (home-based measurement)",
      "Overlap with migraine and fibromyalgia central sensitisation"
     ],
     "hypotheses": [
      "neuroinflammation",
      "genetic-neuronal",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "autonomic-small-fibre",
     "for": "Reduced vagal HRV (meta-analysis), orthostatic CBF fall and hypocapnia across labs, SFN in 31-53% of referral cohorts, autonomic-fibre loss in long COVID, POTS in a minority.",
     "against": "SFN not found in the controlled NIH cohort; SFN severity does not correlate with exertional limits or symptom scores; HRV changes are non-specific and confounded by deconditioning."
    },
    {
     "id": "low-blood-volume-preload",
     "for": "Orthostatic CBF fall even without tachycardia; narrow pulse pressure on lean test in recent-onset patients; preload failure in 92% of iCPET-tested ME/CFS (Novak 2026).",
     "against": "Yamazaki 2026: measured total and red-cell volume did not predict lean-test abnormalities; hypocapnia, not volume, was the most common abnormality."
    },
    {
     "id": "neuroinflammation",
     "for": "Nakatomi TSPO-PET; MRS raised lactate, choline and brain temperature; diffusion NII signals; VanElzakker PBR28 in long COVID; CSF cytokine changes (Hornig).",
     "against": "Raijmakers TSPO-PET null; Tuomaala long COVID TSPO null; NfL/GFAP normal; CSF proteomes indistinguishable from fibromyalgia and no replicated CSF signature."
    },
    {
     "id": "brainstem-craniocervical",
     "for": "Brainstem imaging differences repeatedly reported (volume, connectivity); uncontrolled series with high rates of craniocervical findings and raised opening pressure in a subset.",
     "against": "No controlled or blinded imaging of craniocervical anatomy; no trials of surgery; CSF-leak codes not specific to ME/CFS in registry data; imaging brainstem changes need not be mechanical."
    },
    {
     "id": "sickness-behaviour",
     "for": "Central fatigability (brain fails to ramp up motor drive), lower CSF catechol metabolites, altered task activation after exertion: consistent with a brain-level 'sickness' state.",
     "against": "The 'effort preference' reading is confounded by inability to complete hard tasks; central changes could equally be downstream of peripheral pathology."
    },
    {
     "id": "mitochondrial-energy",
     "for": "Raised brain lactate at rest and failure to raise it further under hypoxia (Bader 2026); lower cortical glutathione; post-exertion cognitive decline.",
     "against": "Brain lactate also raised in depression; no 31P-MRS energetics replicated; samples small."
    },
    {
     "id": "connective-tissue",
     "for": "Hypermobility in 50% of a severe-clinic series; hEDS cohorts show more severe neurodegeneration and share the autonomic phenotype.",
     "against": "Clinic series are uncontrolled; overlap may reflect referral patterns."
    },
    {
     "id": "genetic-neuronal",
     "for": "Solving ME/CFS re-analysis: DecodeME heritability enriched in brain tissues and neurons, with shared architecture with chronic pain and migraine.",
     "against": "Structural MRI largely normal; no neuronal injury markers; no locus is an autonomic gene."
    }
   ],
   "glossary": [
    {
     "term": "Orthostatic intolerance (OI)",
     "plain": "Symptoms (dizziness, fog, fatigue, nausea) that come on when upright and ease when lying down."
    },
    {
     "term": "POTS",
     "plain": "Postural orthostatic tachycardia syndrome: heart rate rises by 30+ bpm (40+ in teenagers) within 10 minutes of standing, without a big blood-pressure drop, plus symptoms."
    },
    {
     "term": "Hypocapnia / POSH",
     "plain": "Low carbon dioxide from over-breathing. POSH means CO2 drops when upright; low CO2 narrows brain blood vessels."
    },
    {
     "term": "Heart-rate variability (HRV)",
     "plain": "Beat-to-beat variation in heart rhythm; higher high-frequency HRV reflects stronger vagal (calming) nerve activity."
    },
    {
     "term": "NASA lean test",
     "plain": "10 minutes leaning with shoulders on a wall while pulse and blood pressure are taken each minute. Cheap, but not validated for formal diagnosis."
    },
    {
     "term": "Small-fibre neuropathy (SFN)",
     "plain": "Loss of the thinnest nerves in skin and organs, which carry pain/temperature and control sweating and blood vessels; diagnosed by counting fibres in a 3 mm skin biopsy (IENFD)."
    },
    {
     "term": "MR spectroscopy (MRS)",
     "plain": "An MRI method that measures chemicals such as lactate, choline and glutathione in brain tissue, and can estimate tissue temperature."
    },
    {
     "term": "TSPO-PET",
     "plain": "A PET scan using a tracer that binds a protein raised in activated microglia and astrocytes, used as a marker of brain inflammation."
    },
    {
     "term": "Diffusion MRI / DTI",
     "plain": "MRI that tracks water movement along nerve fibres to infer white-matter structure."
    },
    {
     "term": "CSF",
     "plain": "Cerebrospinal fluid, which bathes the brain and spinal cord; sampled by lumbar puncture."
    },
    {
     "term": "NfL / GFAP",
     "plain": "Proteins released when neurons (NfL) or astrocytes (GFAP) are damaged; raised in MS, stroke and dementia."
    },
    {
     "term": "EEfRT",
     "plain": "Effort-Expenditure for Rewards Task: repeated choices between an easy and a hard button-pressing task for money; used by NIH to define 'effort preference'."
    },
    {
     "term": "Craniocervical instability (CCI)",
     "plain": "Proposed abnormal movement at the skull-neck junction that compresses the brainstem; mainly discussed in hypermobile patients."
    }
   ],
   "key_reviews": [
    {
     "cite": "Shan 2020, J Transl Med: Neuroimaging characteristics of ME/CFS, systematic review",
     "url": "https://doi.org/10.1186/s12967-020-02506-6",
     "why": "Sorts which brain-imaging findings are repeated across teams and which are single reports."
    },
    {
     "cite": "Christopoulos 2025, J Transl Med: Mapping cerebral blood flow in ME/CFS and OI",
     "url": "https://doi.org/10.1186/s12967-025-06954-w",
     "why": "Up-to-date systematic review of cerebral blood flow, including the OI confound."
    },
    {
     "cite": "Nelson 2019, Medicine: altered cardiac autonomic regulation in ME/CFS, meta-analysis",
     "url": "https://doi.org/10.1097/MD.0000000000017600",
     "why": "Quantifies resting HR and HRV differences across 64 studies."
    },
    {
     "cite": "Mohamed 2023, Sleep Med Rev: Objective sleep measures in CFS, meta-analysis",
     "url": "https://doi.org/10.1016/j.smrv.2023.101771",
     "why": "Best summary of PSG/actigraphy differences, adults vs adolescents."
    },
    {
     "cite": "Cockshell & Mathias 2010, Psychol Med: Cognitive functioning in CFS, meta-analysis",
     "url": "https://doi.org/10.1017/S0033291709992054",
     "why": "Defines the cognitive deficit profile."
    },
    {
     "cite": "VanElzakker 2019, Front Neurol: Neuroinflammation and cytokines in ME/CFS, critical review of methods",
     "url": "https://doi.org/10.3389/fneur.2018.01033",
     "why": "Explains the pitfalls of TSPO-PET, MRS and cytokine studies of 'neuroinflammation'."
    },
    {
     "cite": "Kirvin-Quamme 2025, Front Psychol: Unwilling or unable?",
     "url": "https://doi.org/10.3389/fpsyg.2025.1593269",
     "why": "Clearest data-based critique of the NIH effort-preference claim."
    }
   ],
   "gaps": [
    "No adequately powered (n>50 per group), multi-site TSPO-PET study in ME/CFS using a modern tracer and arterial input.",
    "No blinded, controlled imaging study of craniocervical anatomy in ME/CFS vs hypermobile and healthy controls, and no controlled trial of CCI surgery or CSF drainage.",
    "No skin-biopsy SFN prevalence study in unselected community ME/CFS with matched controls and blinded reading.",
    "Brain measures before and after exertion (MRS lactate, fMRI, CSF) in the same people across the 24-72 h PEM window are almost absent; Cook 2017 has not been directly replicated.",
    "No CSF study with a large healthy-control group and a pre-registered analysis; most recent CSF cohorts lack controls.",
    "No trial of treatments that target orthostatic hypocapnia or cerebral hypoperfusion (capnography-guided breathing, midodrine, compression) measuring function over weeks.",
    "Severe and very severe patients are largely excluded from imaging, CSF and sensory studies.",
    "Few studies stratify by orthostatic phenotype (POTS vs hypocapnia vs normal HR/BP), which may explain much of the inconsistency in perfusion and imaging results.",
    "No effort-based decision study in ME/CFS that calibrates task difficulty to each person's ability."
   ]
  },
  {
   "domain": "immune",
   "title": "Immune system",
   "overview": "Immunology is the oldest and most heavily worked area of ME/CFS research, and the one with the widest gap between positive reports and independent replication. Reduced natural-killer-cell killing is the most repeated finding (meta-analytic Hedges g about 0.96 across 28 papers), yet the largest standardised multi-site attempt (MCAM, 174 vs 86) and two careful phenotyping studies found nothing, so the honest grade is 'contested and not usable as a test'. Several newer cell-level findings are more consistent in direction than in detail: CD8 T-cell exhaustion programmes, shifts in B-cell maturation (more naive, fewer switched-memory), monocyte dysregulation, and an exaggerated innate response to microbial stimulation that worsens after exercise. Cytokines look disturbed in aggregate (TNF, IL-2, IL-4, TGF-beta, CRP in meta-analysis) but no single cytokine survives as a marker, TGF-beta turned out to be largely a blood-handling artefact, and the clearest pattern is that abnormalities depend on illness duration (Hornig 2015) and severity (Montoya 2017) rather than on diagnosis alone. The autoimmunity strand has had the clearest negative resolution: GPCR autoantibodies against beta2/M3/M4 receptors are not reproduced by unbiased antigen profiling, removing them by immunoadsorption fails against sham, rituximab failed in a 151-patient RCT, and BC007's larger trial failed; what survives is narrower and interesting - patient IgG does things to endothelial mitochondria and, in long COVID, transfers pain and fatigue-like phenotypes to mice, and plasma-cell depletion with daratumumab produced striking but uncontrolled responses in 6 of 10 patients. Common-variant genetics (DecodeME) shows no enrichment in any blood immune cell type, which pushes most of these immune findings toward being consequences or state markers rather than inherited causes.",
   "findings": [
    {
     "id": "immune-nk-cytotoxicity",
     "subdomain": "NK cells",
     "finding": "Natural-killer-cell cytotoxicity is reduced in ME/CFS in meta-analysis of the older literature (Hedges g ~0.96), but the largest standardised multi-site study and two deep-phenotyping studies found no difference.",
     "grade": "contested",
     "plain": "The most-repeated immune finding in ME/CFS is that one type of white blood cell kills target cells less well. When labs tried to turn it into a standardised test, the difference disappeared.",
     "detail": "Baraniuk, Eaton-Fitch and Marshall-Gradisnik 2024 pooled 28 papers and 55 effector:target data points: cytotoxicity about half of control levels, overall Hedges g 0.96 (0.75-1.18), heterogeneity high and explained by E:T ratio and method. Against this: the CDC Multi-Site Clinical Assessment (MCAM) sub-study used an assay validated for overnight-shipped blood in 174 ME/CFS vs 86 healthy controls and found 34.1% vs 33.6% cytotoxicity (p=0.79), with no association with symptom domains; Theorell 2017 found no reproducible difference in two cohorts (48 patients) across granule content, exocytosis, killing or adrenaline sensitivity; Cliff 2019 found no difference in NK numbers, subsets or in vitro responsiveness in 251 patients (54 severe) vs 107 controls. The assay requires fresh blood and is highly method-sensitive, so publication-era and logistics effects are plausible explanations for the discrepancy. Treat reduced NK function as a real signal in some cohorts with some assays, not as a disease marker.",
     "studies": [
      {
       "cite": "Baraniuk, Eaton-Fitch & Marshall-Gradisnik 2024, Frontiers in Immunology",
       "n": "28 papers, 55 E:T data points",
       "design": "meta-analysis",
       "result": "Hedges g 0.96 (0.75-1.18); cytotoxicity ~50% of controls",
       "url": "https://doi.org/10.3389/fimmu.2024.1440643",
       "verified": true
      },
      {
       "cite": "Klimas 1990, J Clin Microbiol",
       "n": "30 patients vs controls",
       "design": "case-control",
       "result": "Low NK cytotoxicity per CD56 cell despite raised CD56 numbers; reduced mitogen responses and IFN-gamma",
       "url": "https://doi.org/10.1128/jcm.28.6.1403-1410.1990",
       "verified": true
      },
      {
       "cite": "Brenu 2011, J Transl Med",
       "n": "95 vs 50",
       "design": "case-control",
       "result": "NK and CD8 cytotoxicity reduced, CD56bright reduced, granzyme A/K down, perforin up",
       "url": "https://doi.org/10.1186/1479-5876-9-81",
       "verified": true
      },
      {
       "cite": "Eaton-Fitch 2019, Systematic Reviews",
       "n": "17 studies",
       "design": "systematic review",
       "result": "Cytotoxicity consistently reduced; other NK outcomes equivocal",
       "url": "https://doi.org/10.1186/s13643-019-1202-6",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Querec 2023 (MCAM), J Transl Med",
       "n": "174 ME/CFS vs 86 HC vs 10 ill controls",
       "design": "multi-site case-control, shipped-sample validated assay",
       "result": "34.1% vs 33.6% cytotoxicity, p=0.79; no symptom association; 'not ready for clinical implementation'",
       "url": "https://doi.org/10.1186/s12967-023-03958-2",
       "verified": true
      },
      {
       "cite": "Theorell 2017, Frontiers in Immunology",
       "n": "48 patients, two cohorts, matched HC",
       "design": "case-control, deep cytotoxic-lymphocyte phenotyping",
       "result": "No reproducible difference in number, granules, exocytosis, killing, cytokines or adaptive NK expansion",
       "url": "https://doi.org/10.3389/fimmu.2017.00723",
       "verified": true
      },
      {
       "cite": "Cliff 2019, Frontiers in Immunology",
       "n": "251 ME/CFS (54 severe) vs 107 HC vs 46 MS",
       "design": "case-control, UK ME/CFS Biobank",
       "result": "No differences in NK numbers, subsets or in vitro responsiveness",
       "url": "https://doi.org/10.3389/fimmu.2019.00796",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the deficit appear only in fresh same-day assays, i.e. is it partly a pre-analytic artefact?",
      "Is reduced cytotoxicity confined to a severity or herpesvirus-exposure subgroup?",
      "Does NK function change during PEM (no paired rest/post-exertion study exists)?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "herpes-reactivation",
      "viral-persistence"
     ],
     "test_available": "Private NK-function assays exist but need a same-day fresh sample; not validated for diagnosis (MCAM explicitly says so)"
    },
    {
     "id": "immune-trpm3",
     "subdomain": "NK cells",
     "finding": "Reduced TRPM3 ion-channel current and calcium influx in patients' NK cells has been reported repeatedly, including a two-site study (36 vs 42), but only ever by one research group.",
     "grade": "early",
     "plain": "One Australian group finds that a calcium channel on immune cells works poorly in patients, which they argue explains weak NK killing. No other lab has tested it.",
     "detail": "Sasso 2026 (Front Med) ran whole-cell patch-clamp on isolated NK cells at two laboratory sites with independent investigators: 36 CCC-defined ME/CFS vs 42 healthy controls, significant reduction in all TRPM3 parameters with no site effect. Earlier work in the same programme: Eaton-Fitch 2022 (impaired TRPM3-dependent calcium influx, restored in vitro by naltrexone), Eaton-Fitch 2021 (TRPM3/PIP2 co-localisation), Magawa 2026 (deficient TRPM3-linked mitochondrial calcium influx), plus a post-COVID extension (Magawa 2026, Eur J Immunol) and LDN restoration in long COVID NK cells (Sasso 2025). The 'two sites' are both within the same group's network, so this is internal consistency rather than independent replication; patch-clamp of isolated NK cells is a demanding, low-throughput method available in very few labs. Against it indirectly: DecodeME common-variant genetics shows no enrichment in any blood immune cell type, and the low-dose naltrexone RCT in post-COVID ME/CFS (n=137) was negative, which weakens the therapeutic corollary.",
     "studies": [
      {
       "cite": "Sasso 2026, Frontiers in Medicine",
       "n": "36 vs 42",
       "design": "two-site case-control patch-clamp",
       "result": "Significant reduction in all TRPM3 current parameters; no effect of laboratory site",
       "url": "https://doi.org/10.3389/fmed.2025.1703924",
       "verified": true
      },
      {
       "cite": "Eaton-Fitch 2022, J Transl Med",
       "n": "small case-control",
       "design": "calcium imaging",
       "result": "Impaired TRPM3-dependent Ca2+ influx; restored in vitro by naltrexone",
       "url": "https://doi.org/10.1186/s12967-022-03297-8",
       "verified": true
      },
      {
       "cite": "Magawa 2026, BMC Immunology",
       "n": "case-control",
       "design": "mitochondrial Ca2+ imaging in NK cells",
       "result": "Deficient TRPM3-linked mitochondrial Ca2+ influx",
       "url": "https://doi.org/10.1186/s12865-026-00849-1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl)",
       "n": "503,739 PBMCs, 58 people; 5 cohorts",
       "design": "re-analysis of public scRNA, methylation, repertoires",
       "result": "No immune-cell subtype, gene or methylation site separates cases from controls reproducibly; nothing passed FDR in more than one dataset",
       "url": "",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (downloads, DecodeME MAGMA)",
       "n": "15,579 cases vs 259,909 controls",
       "design": "GWAS enrichment",
       "result": "No enrichment in any of 29 immune cell types; signal is brain/neuronal",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Can a lab with no stake in the hypothesis reproduce the patch-clamp result blind?",
      "Is reduced TRPM3 current a cause of poor cytotoxicity or a downstream consequence of cell state/handling?",
      "Does TRPM3 function track symptoms within a person over time?"
     ],
     "hypotheses": [
      "sodium-calcium-overload",
      "immune-exhaustion",
      "autonomic-small-fibre"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-cd8-exhaustion",
     "subdomain": "T cells",
     "finding": "CD8+ effector-memory T cells in ME/CFS show transcriptional and chromatin features of exhaustion, with supporting flow cytometry and an independent cell-free RNA signature.",
     "grade": "several",
     "plain": "Killer T cells in patients look 'worn out', like cells that have been fighting an infection for a long time.",
     "detail": "Iu 2024 (PNAS) integrated scRNA-seq, bulk RNA-seq and ATAC-seq across sorted T-cell subsets (336,269 T-lineage cells from 28 patients and 30 sedentary controls at baseline and after symptom provocation), finding upregulated exhaustion transcription factors, altered chromatin accessibility and metabolic reprogramming most marked in CD8+ effector-memory and innate-like T cells, validated by flow cytometry in an extended cohort (15 patients, 11 controls, female, age/BMI matched). Gardella 2025 independently found T-cell-exhaustion and cytokine-signalling signatures in plasma cell-free RNA (93 vs 75). Supporting but not identical: Mandarano 2020 found reduced CD8+ mitochondrial membrane potential and reduced glycolysis at rest in 53 vs 45. The main caveat is that the Cornell scRNA cohorts are not independent of each other and a re-analysis of the same public scRNA data found no exhausted-CD8 composition difference once cell typing was redone, so the effect may live in gene programmes rather than cell counts.",
     "studies": [
      {
       "cite": "Iu 2024, PNAS",
       "n": "28 vs 30 (scRNA/ATAC); 15 vs 11 flow validation",
       "design": "multiomic case-control with exercise provocation",
       "result": "Exhaustion TFs up in CD8+ Tem, altered chromatin and metabolism; higher frequency of exhaustion markers by flow",
       "url": "https://doi.org/10.1073/pnas.2415119121",
       "verified": true
      },
      {
       "cite": "Gardella 2025, PNAS",
       "n": "93 vs 75",
       "design": "plasma cfRNA sequencing + ML",
       "result": "Cytokine-signalling and T-cell-exhaustion network signatures; classifier AUC 0.81",
       "url": "https://doi.org/10.1073/pnas.2507345122",
       "verified": true
      },
      {
       "cite": "Mandarano 2020, J Clin Invest",
       "n": "53 vs 45",
       "design": "case-control immunometabolism",
       "result": "Reduced CD8+ mitochondrial membrane potential; reduced resting glycolysis in CD4+ and CD8+",
       "url": "https://doi.org/10.1172/JCI132185",
       "verified": true
      },
      {
       "cite": "Yin 2024, Nature Immunology (long COVID)",
       "n": "deep immune profiling, 8 months post-infection",
       "design": "case-control omics + serology",
       "result": "Exhausted SARS-CoV-2-specific CD8+ T cells, systemic inflammation, uncoordinated T/B responses",
       "url": "https://doi.org/10.1038/s41590-023-01724-6",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl, GSE214283)",
       "n": "30 vs 28, 503,739 PBMCs",
       "design": "re-analysis with CellTypist + marker gating",
       "result": "Exhausted-CD8 gating null; 0 genes at q<0.05 at baseline in all 15 cell types",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Is the exhaustion programme driven by a persisting antigen, and if so which?",
      "Does checkpoint blockade or metabolic rescue change anything in vivo (never tried)?",
      "Why do composition-level and programme-level analyses of the same data disagree?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "viral-persistence",
      "herpes-reactivation"
     ],
     "test_available": "research only (specialist flow cytometry)"
    },
    {
     "id": "immune-tcell-severity",
     "subdomain": "T cells",
     "finding": "T-cell abnormalities differ by severity: mild/moderate patients show cytotoxic/immunosenescence features, severe patients show activated lymphocytes and higher stimulated pro-inflammatory cytokines.",
     "grade": "early",
     "plain": "Mildly and severely affected patients show different immune pictures, which may mean different disease mechanisms rather than one disease of different intensity.",
     "detail": "Lee, Cliff and colleagues (UK ME/CFS Biobank; medRxiv 2025, J Transl Med Dec 2025) compared 43 mild/moderate with 53 severe patients by flow cytometry. Mild/moderate: increased cytotoxic effector molecules and more CD28-CD57- early-immunosenescent cells, a pattern associated with persistent viral infection. Severe: more CD69+ and CD38+ activated circulating lymphocytes and more IFN-gamma, TNF and IL-17 after in vitro stimulation, consistent with non-specific ongoing inflammation. Changes spanned CD8+ T cells, MAIT cells and NK cells. This is a within-patient-group comparison without a healthy-control contrast for the severity axis, and severe patients differ in sampling conditions (home visits, deconditioning, medication), so confounding by illness state is not excluded.",
     "studies": [
      {
       "cite": "Lee JS 2025, J Transl Med (also medRxiv 2025.01.02.24319359)",
       "n": "43 mild/moderate vs 53 severe",
       "design": "case-case flow cytometry, UK ME/CFS Biobank",
       "result": "Immunosenescence/cytotoxic markers in mild-moderate; activation (CD69+, CD38+) and higher stimulated IFN-gamma/TNF/IL-17 in severe",
       "url": "https://doi.org/10.1186/s12967-025-07507-x",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do these two patterns define stable subgroups or stages of one illness?",
      "Does the severe-patient activation pattern reflect being bedbound rather than disease mechanism?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-mait",
     "subdomain": "T cells",
     "finding": "Increased mucosal-associated invariant T (MAIT) cell proportions and a shift in CD8+ memory subsets were found in the largest single ME/CFS flow-cytometry cohort, but the finding has not been reproduced in independent data.",
     "grade": "contested",
     "plain": "One large study found more of a gut-associated T-cell type in patients, especially severe ones. Re-analyses of other datasets do not show it.",
     "detail": "Cliff 2019 (251 patients incl. 54 severe, 107 controls, 46 MS) reported increased effector-memory CD8+ and decreased terminally differentiated effector CD8+ T cells, plus a significantly increased proportion of MAIT cells, most marked in severe patients, with no differences in herpesvirus seroprevalence. Iu 2024 also flagged 'certain innate T cells' as among the most dysregulated. Against: a re-analysis across five cohorts and three data types (scRNA of 503,739 PBMCs, four methylation cohorts, B- and T-cell repertoires) found MAIT, gamma-delta T, Th17 and exhausted-CD8 gating all null, and blood RNA cohorts do not show reduced Th17 (meta p=0.39).",
     "studies": [
      {
       "cite": "Cliff 2019, Frontiers in Immunology",
       "n": "251 vs 107 vs 46 MS",
       "design": "case-control flow cytometry",
       "result": "MAIT proportion increased (most in severe); CD8+ Tem up, TEMRA down",
       "url": "https://doi.org/10.3389/fimmu.2019.00796",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl; downloads)",
       "n": "5 cohorts; 58-people scRNA; 5 blood RNA cohorts",
       "design": "re-analysis",
       "result": "MAIT, gamma-delta, Th17, ABC B cells and pDC interferon all null; Th17 meta p=0.39",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Is the MAIT signal a gut-mucosa readout, and does it track gut findings in the same people?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "immune-exhaustion"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-tcr-repertoire",
     "subdomain": "T cells",
     "finding": "T-cell receptor repertoires do not distinguish ME/CFS from controls, arguing against a dominant shared antigen-driven clonal expansion.",
     "grade": "refuted",
     "plain": "If one infection or self-target were driving the disease, you would expect to see armies of identical T cells. Sequencing says they are not there.",
     "detail": "Dibble 2024 (BMC Research Notes) sequenced alpha/delta, beta and gamma TCR loci in 40 samples each from severe ME, mild/moderate ME, MS disease controls and healthy controls, and applied P-SVM classification: the method worked on simulated data but could not partition the real samples significantly. A re-analysis of UK ME/CFS Biobank T-cell repertoires (304 samples) likewise found nothing passing FDR in more than one dataset, with only exploratory herpesvirus-related trends (higher share of EBV-specific TCR-beta in severe patients' CD8, 0.88% vs 0.45%, p=0.06). A near-genome-wide candidate, the TRA locus (Ueland 2022), has not replicated either.",
     "studies": [
      {
       "cite": "Dibble 2024, BMC Research Notes",
       "n": "4 groups x 40",
       "design": "bulk TCR repertoire sequencing + machine learning",
       "result": "No statistically significant partitioning; 'findings do not support altered T-cell receptor diversity'",
       "url": "https://doi.org/10.1186/s13104-023-06616-4",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl)",
       "n": "304 T-cell repertoire samples",
       "design": "re-analysis, UK ME/CFS Biobank",
       "result": "Nothing reproducible; EBV-specific TCR-beta share higher in severe CD8 at p=0.06 only",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would antigen-specific sorting (rather than bulk repertoire) reveal expansions invisible in bulk?",
      "Could tissue (muscle, gut, brainstem) repertoires differ while blood looks normal?"
     ],
     "hypotheses": [
      "autoimmunity",
      "viral-persistence"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-bcell-maturation",
     "subdomain": "B cells",
     "finding": "The NIH deep-phenotyping study found more naive and fewer switched-memory B cells in post-infectious ME/CFS, consistent with chronic antigenic stimulation; supporting signals exist but are weak.",
     "grade": "early",
     "plain": "Patients seem to have more 'untrained' B cells and fewer that have already matured, as if the immune system keeps being prodded by something.",
     "detail": "Walitt 2024 (Nat Commun) did intensive inpatient phenotyping of a small, rigorously selected post-infectious ME/CFS cohort (17 PI-ME/CFS vs 21 healthy volunteers for immune measures) and reported increased naive and decreased switched-memory B cells, interpreted as chronic antigenic stimulation and linked to STAT4-TLR9 B-cell pathway signals; PBMC gene-expression and metabolic differences were sex-dependent. A Solving ME/CFS re-analysis found plasma-cell/immunoglobulin transcripts lower in 2 of 4 blood RNA cohorts (meta d -0.42, p=0.06), which points the same way without reaching significance. Earlier B-cell phenotype work (Mensah 2016, 2018) found no reproducible subset differences but raised CD24 expression on naive and memory B cells. The cohort is tiny and much of Walitt 2024's framing (effort preference) is contested, so this should be read as a lead, not an established feature.",
     "studies": [
      {
       "cite": "Walitt 2024, Nature Communications",
       "n": "17 PI-ME/CFS vs 21 HV (immune measures; 16 vs 20 for switched memory)",
       "design": "deep phenotyping case-control",
       "result": "Naive B cells increased, switched-memory B cells decreased; interpreted as chronic antigenic stimulation",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome)",
       "n": "4 blood RNA cohorts",
       "design": "meta-analysis of public data",
       "result": "Plasma-cell/immunoglobulin transcripts lower in 2 of 4 cohorts, meta d -0.42, p=0.06",
       "url": "",
       "verified": true
      },
      {
       "cite": "Mensah 2016, Clin Exp Immunol",
       "n": "cross-sectional case-control",
       "design": "extended B-cell immunophenotyping",
       "result": "No reproducible subset differences; higher CD24 expression within naive and memory B cells",
       "url": "https://doi.org/10.1111/cei.12749",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl)",
       "n": "58-person scRNA; 123-person BCR repertoire",
       "design": "re-analysis",
       "result": "No B-cell composition difference survived; ABC B-cell call lower only at p=0.03, not replicated",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the naive/switched-memory shift replicate in a larger, less selected cohort?",
      "Is it explained by medication, inactivity or infection history?"
     ],
     "hypotheses": [
      "autoimmunity",
      "viral-persistence",
      "immune-exhaustion"
     ],
     "test_available": "Lymphocyte subset flow cytometry is widely available, but the naive/memory B-cell split is not a validated marker"
    },
    {
     "id": "immune-bcr-repertoire",
     "subdomain": "B cells",
     "finding": "Reported skewing of the B-cell receptor repertoire (IGHV3-30/3-33 overuse) partially replicates in mild/moderate patients but shows no clonality, somatic-hypermutation or autoimmunity signature.",
     "grade": "contested",
     "plain": "An early report said patients over-use certain antibody genes. A careful follow-up found a trace of that but none of the signs you would expect if antibodies were driving the disease.",
     "detail": "Sato 2021 (Brain Behav Immun) reported biased IGHV/IGHD/IGHJ usage in ME/CFS peripheral B cells with ROC separation from controls. Ryback and Cowan 2025 (Front Immunol) sequenced BCR heavy-chain repertoires in 25 mild/moderate ME/CFS, 36 severe, 21 healthy controls and 28 MS: no increased clonality and no differential somatic hypermutation versus healthy controls or MS; IGHV3-30 usage was replicated in mild/moderate patients only, but with no evidence of ongoing antigen-driven responses within IGHV3-30 repertoires and no infection or autoimmunity signature; a skewed IgM:IgG ratio in mild/moderate patients was the one new preliminary observation. A Solving ME/CFS re-analysis of UK ME/CFS Biobank repertoires (123 people) found IgM IGHV3-30/3-33 usage essentially identical (9.34% vs 9.37%, p=0.96).",
     "studies": [
      {
       "cite": "Ryback & Cowan 2025, Frontiers in Immunology",
       "n": "25 mild/mod + 36 severe vs 21 HC vs 28 MS",
       "design": "AIRR-seq case-control with disease control",
       "result": "No increased clonality or SHM; IGHV3-30 replicated only in mild/moderate; IgM:IgG ratio skewed",
       "url": "https://doi.org/10.3389/fimmu.2025.1489312",
       "verified": true
      },
      {
       "cite": "Sato 2021, Brain Behav Immun",
       "n": "case-control",
       "design": "BCR repertoire sequencing",
       "result": "Biased IGHV gene usage; ROC separation suggested",
       "url": "https://doi.org/10.1016/j.bbi.2021.03.023",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl)",
       "n": "123 people, UK ME/CFS Biobank",
       "design": "re-analysis of B-cell repertoires",
       "result": "IGHV3-30/3-33 IgM usage 9.34% vs 9.37%, p=0.96 - claim fails",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Is the IgM:IgG skew in mild/moderate disease real and does it predict anything?"
     ],
     "hypotheses": [
      "autoimmunity",
      "viral-persistence"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-rituximab",
     "subdomain": "B cells",
     "finding": "B-cell depletion with rituximab failed in a 151-patient placebo-controlled trial, after positive small phase II results.",
     "grade": "refuted",
     "plain": "A drug that wipes out antibody-making B cells looked promising in small studies, then failed completely in a proper large trial.",
     "detail": "Fluge 2011 (PLOS ONE, n=30 randomised) reported major or moderate response in 10/15 on rituximab vs 2/15 on saline (p=0.003), with delayed onset 2-7 months. RituxME (Fluge 2019, Ann Intern Med) randomised 151 CCC patients to induction plus 4 maintenance infusions over 12 months or placebo: response rates 26.0% rituximab vs 35.1% placebo (difference -9.2 points, 95% CI -5.5 to 23.3 for placebo advantage, p=0.22), no difference in fatigue score over 24 months or any secondary endpoint, with more serious adverse events on drug (26.0% vs 18.9%). The high placebo response rate (35%) is the key number for reading every uncontrolled immunotherapy report in this field. A 6-year follow-up showed SF-36 physical function 45.5 vs 43.1, i.e. both groups improved similarly.",
     "studies": [
      {
       "cite": "Fluge 2019 (RituxME), Annals of Internal Medicine",
       "n": "151 randomised (77 vs 74)",
       "design": "multicentre double-blind placebo-controlled RCT",
       "result": "Response 26.0% vs 35.1% placebo, p=0.22; all secondary endpoints null",
       "url": "https://doi.org/10.7326/M18-1451",
       "verified": true
      },
      {
       "cite": "Fluge 2011, PLOS ONE",
       "n": "30 randomised",
       "design": "double-blind placebo-controlled phase II",
       "result": "10/15 vs 2/15 responders (p=0.003); primary 3-month endpoint negative",
       "url": "https://doi.org/10.1371/journal.pone.0026358",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does rituximab's failure exclude autoantibodies, or only short-lived B-cell-dependent ones (long-lived plasma cells are CD20-negative)?"
     ],
     "hypotheses": [
      "autoimmunity"
     ],
     "test_available": "Not indicated"
    },
    {
     "id": "immune-daratumumab",
     "subdomain": "Plasma cells",
     "finding": "Plasma-cell depletion with daratumumab produced marked, in some cases sustained, improvement in 6 of 10 patients in an open-label pilot, with low baseline NK count predicting non-response.",
     "grade": "early",
     "plain": "A myeloma drug that kills the cells making long-lived antibodies helped six of ten patients, some of them a lot and for years. It was a small study with no placebo group.",
     "detail": "Fluge 2025 (Front Med, corrected 2025; EudraCT 2022-000281-18): 10 female moderate-to-severe patients, 12-week run-in, then subcutaneous daratumumab 1800 mg at weeks 0/2/4/6 (last four patients received three additional injections from week 14). No serious adverse events. All 10: mean SF-36 physical function 25.9 to 55.0 at 8-9 months (p=0.002), DSQ-SF 72.3 to 43.1 (p=0.002). Six responders: SF-36 PF 32.2 to 78.3, DSQ-SF 71.1 to 24.3, and five of the six sustained improvement with mean SF-36 PF 88 toward end of follow-up and step counts above 10,000/day for periods. Serum IgG fell 54% in improvers vs 40% in non-improvers. Low baseline blood NK-cell count was significantly associated with lack of response (responders 238 vs 97 cells/microlitre, rho 0.77). The design cannot separate drug effect from the 35%+ open-label/placebo response documented in RituxME, and n=10 with a post-hoc predictor is hypothesis-generating; an RCT is planned.",
     "studies": [
      {
       "cite": "Fluge 2025, Frontiers in Medicine",
       "n": "10 (open-label)",
       "design": "prospective open-label pilot, anti-CD38",
       "result": "6/10 marked improvement; SF-36 PF 25.9 to 55.0 (p=0.002); 5 sustained 12-24 months; NK count predicted response",
       "url": "https://doi.org/10.3389/fmed.2025.1607353",
       "verified": true
      },
      {
       "cite": "Rekeland 2020 (CycloME), Frontiers in Medicine",
       "n": "40 (open-label)",
       "design": "open-label phase II cyclophosphamide",
       "result": "55% response; SF-36 PF 33.0 to 51.5 at 18 months; 68% of responders still in remission at 4 years; HLA-DQB1*03:03/C*07:04 83% vs 43% response; 11 SAEs in 8 patients",
       "url": "https://doi.org/10.3389/fmed.2020.00162",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Fluge 2019 (RituxME), Annals of Internal Medicine",
       "n": "151",
       "design": "RCT",
       "result": "35% response on placebo - the benchmark any open-label 'response rate' must beat",
       "url": "https://doi.org/10.7326/M18-1451",
       "verified": true
      }
     ],
     "open_questions": [
      "Will the planned randomised trial reproduce it?",
      "If long-lived plasma cells matter, why did immunoadsorption (which removes the antibodies themselves) fail against sham?",
      "Is the NK-count predictor real or a chance correlation in n=10?"
     ],
     "hypotheses": [
      "autoimmunity",
      "serum-factor"
     ],
     "test_available": "Lymphocyte subsets (NK count) and serum IgG are routine; the treatment is research/trial only"
    },
    {
     "id": "immune-cytokine-duration",
     "subdomain": "Cytokines",
     "finding": "Plasma cytokine abnormalities depend strongly on illness duration: a pro- and anti-inflammatory activation pattern early (under 3 years) that is absent in long-established illness.",
     "grade": "several",
     "plain": "Blood inflammation markers look disturbed in people ill for a short time and normal in people ill for years, which may be why studies disagree.",
     "detail": "Hornig 2015 (Sci Adv) used two multicentre cohorts with controls frequency-matched on season, site, age and sex: 52 cases ill under 3 years, 246 ill longer, 348 controls. Early cases showed prominent activation of both pro- and anti-inflammatory cytokines and dissociation of inter-cytokine regulatory networks; this was not present in the longer-duration group. Cytokine alterations correlated more strongly with duration than with severity measures. This duration-dependence is the single most useful explanation for the field's inconsistency, and it has been widely cited but not formally replicated with a comparably large early-illness sample; it also cannot distinguish a biological evolution from survivorship/sampling differences between recent-onset and long-ill cohorts.",
     "studies": [
      {
       "cite": "Hornig 2015, Science Advances",
       "n": "52 early vs 246 long-duration vs 348 controls",
       "design": "multicentre case-control, 51-plex immunoassay",
       "result": "Distinct early cytokine activation and network dissociation, absent later; duration a stronger correlate than severity",
       "url": "https://doi.org/10.1126/sciadv.1400121",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Corbitt 2019, BMC Neurology",
       "n": "15 included studies",
       "design": "systematic review of cytokines",
       "result": "Findings inconclusive; cytokines would not serve as reliable biomarkers",
       "url": "https://doi.org/10.1186/s12883-019-1433-0",
       "verified": true
      }
     ],
     "open_questions": [
      "Would a prospective inception cohort (post-infection) reproduce the early signature and show it fading?",
      "Does early immunomodulation change outcome - never tested"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour",
      "immune-exhaustion"
     ],
     "test_available": "research only (multiplex cytokine panels are not clinically interpretable)"
    },
    {
     "id": "immune-cytokine-severity",
     "subdomain": "Cytokines",
     "finding": "In the largest single cytokine study, 17 cytokines rose in step with disease severity although only TGF-beta (up) and resistin (down) differed between all patients and controls.",
     "grade": "several",
     "plain": "Comparing all patients with all controls shows almost nothing, but the sicker the patient, the higher a group of inflammatory signals.",
     "detail": "Montoya 2017 (PNAS) measured 51 cytokines by Luminex in 192 ME/CFS patients and 392 healthy controls, regressing each on severity with covariates for age, sex, race and non-specific binding. On average only TGF-beta was elevated (p=0.0052) and resistin lower (p=0.0052). Seventeen cytokines showed significant upward linear trends with severity (CCL11, CXCL1, CXCL10, IFN-gamma, IL-4, IL-5, IL-7, IL-12p70, IL-13, IL-17F, leptin, G-CSF, GM-CSF, LIF, NGF, SCF, TGF-alpha), 13 of them pro-inflammatory; CXCL9 correlated inversely with fatigue duration. Caveats: severity is self-reported and correlates with inactivity, BMI and sleep; multiplex platforms have substantial non-specific binding (which this paper explicitly modelled, unlike most); and the TGF-beta result is now suspect on pre-analytic grounds (see immune-tgfbeta-artefact).",
     "studies": [
      {
       "cite": "Montoya 2017, PNAS",
       "n": "192 vs 392",
       "design": "case-control, 51-plex Luminex with severity regression",
       "result": "TGF-beta up, resistin down overall; 17 cytokines rising with severity",
       "url": "https://doi.org/10.1073/pnas.1710519114",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Roerink 2017, PNAS (letter) and Roerink 2017, J Transl Med",
       "n": "50 vs 48",
       "design": "critique + independent case-control with 92-marker PEA",
       "result": "Only IL-12p40 and CSF-1 raised; TGF-beta no different between patients and controls",
       "url": "https://doi.org/10.1186/s12967-017-1371-9",
       "verified": true
      }
     ],
     "open_questions": [
      "Are severity-cytokine gradients mediated by deconditioning, adiposity and sleep loss rather than disease?",
      "Do any of the 17 track within-person symptom change?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour",
      "mast-cell"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-cytokine-meta",
     "subdomain": "Cytokines",
     "finding": "Meta-analysis of 42 studies finds small but significant elevations of TNF, IL-2, IL-4, TGF-beta and CRP, with 12 other proteins showing no difference.",
     "grade": "several",
     "plain": "Pooling all the cytokine studies shows a little extra inflammation on average, but the effects are small and inconsistent, so no blood test comes out of it.",
     "detail": "Strawbridge 2019 (Neurosci Biobehav Rev) meta-analysed 42 studies of circulating inflammatory proteins in CFS versus unaffected controls: TNF ES 0.274 (p<0.001), IL-2 0.203 (p=0.006), IL-4 0.373 (p=0.004), TGF-beta 0.967 (p<0.001), CRP 0.622 (p=0.019); 12 proteins did not differ. The authors conclude inflammation is unlikely to be a primary feature in all patients and that subgroups may exist. Blundell 2015 reviewed 38 papers and found elevated TGF-beta in 5 of 8 studies and no other consistent cytokine, with no case-control differences after exercise. Corbitt 2019 reached an explicitly inconclusive verdict. Independent support for low-grade inflammation comes from UK Biobank: Beentjes/Ponting 2025 found inflammation, insulin-resistance and liver markers shifted in both sexes among 1,455 cases vs 131,303 controls, not explained by inactivity, with small effect sizes.",
     "studies": [
      {
       "cite": "Strawbridge 2019, Neuroscience & Biobehavioral Reviews",
       "n": "42 studies",
       "design": "systematic review and meta-analysis",
       "result": "TNF, IL-2, IL-4, TGF-beta, CRP elevated (ES 0.20-0.97); 12 proteins null",
       "url": "https://doi.org/10.1016/j.neubiorev.2019.08.011",
       "verified": true
      },
      {
       "cite": "Blundell 2015, Brain Behavior and Immunity",
       "n": "38 papers, 77 cytokines",
       "design": "systematic review",
       "result": "Only TGF-beta consistently elevated (5/8 studies); no post-exercise differences",
       "url": "https://doi.org/10.1016/j.bbi.2015.07.004",
       "verified": true
      },
      {
       "cite": "Beentjes/Ponting 2025, EMBO Molecular Medicine (via Solving ME/CFS re-analysis)",
       "n": "1,455 cases vs 131,303 controls",
       "design": "UK Biobank cross-sectional, 3,237 traits incl. 2,923 proteins",
       "result": "116 traits differ in both sexes incl. inflammation markers; effects small; not explained by inactivity",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Corbitt 2019, BMC Neurology",
       "n": "15 studies",
       "design": "systematic review",
       "result": "Inconclusive; cytokines not reliable biomarkers",
       "url": "https://doi.org/10.1186/s12883-019-1433-0",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome)",
       "n": "~150 cases/105 controls across 4 proteomic cohorts + UK Biobank",
       "design": "harmonised meta-analysis",
       "result": "Interferon proteins and most immune protein sets flat or inconsistent; no single protein replicates strongly",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Is there a genuinely inflamed subgroup identifiable prospectively, and does it respond differently to anything?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "CRP and ESR are routine but usually normal or only slightly raised; not diagnostic"
    },
    {
     "id": "immune-tgfbeta-artefact",
     "subdomain": "Cytokines",
     "finding": "The most-replicated single cytokine finding, elevated TGF-beta, is largely a blood-handling artefact driven by platelet activation during centrifugation.",
     "grade": "refuted",
     "plain": "The one cytokine that kept showing up high turned out to depend on how fast the blood was spun, because platelets release it.",
     "detail": "Roerink 2018 (Neth J Med) measured plasma TGF-beta1 in two female CFS cohorts (50 patients/48 controls; 90/29) with controls recruited by patients as age-matched acquaintances. Within each cohort there was no patient-control difference, but between cohorts values differed greatly, traced to the centrifuge g-force: the lower force (1361 g) produced more platelet activation and higher P-selectin and TGF-beta. This is the clearest documented pre-analytic trap in ME/CFS immunology and it retrospectively undercuts Blundell's 5-of-8 TGF-beta tally and Montoya 2017's overall TGF-beta elevation. Roerink 2017 (J Transl Med), using a 92-marker proximity extension assay in 50 patients vs 48 neighbourhood controls, found TGF-beta no different and only IL-12p40 and CSF-1 raised.",
     "studies": [
      {
       "cite": "Roerink 2018, Netherlands Journal of Medicine",
       "n": "50 vs 48 and 90 vs 29",
       "design": "two case-control cohorts with pre-analytic comparison",
       "result": "No within-cohort difference; between-cohort discrepancy explained by centrifuge g-force and platelet activation",
       "url": "https://pubmed.ncbi.nlm.nih.gov/30220655/",
       "verified": true
      },
      {
       "cite": "Roerink 2017, J Translational Medicine",
       "n": "50 vs 48",
       "design": "case-control, 92-marker PEA + ELISA, plus anakinra RCT samples",
       "result": "IL-12p40 and CSF-1 higher; TGF-beta not different; anakinra had no effect on circulating cytokines other than IL-1Ra",
       "url": "https://doi.org/10.1186/s12967-017-1371-9",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "How many other reported cytokine and proteomic differences are pre-analytic? (A parallel leak artefact was found in the Hoel/Fluge SomaScan serum data.)"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "microclots"
     ],
     "test_available": "Not indicated"
    },
    {
     "id": "immune-il1-blockade",
     "subdomain": "Cytokines",
     "finding": "Blocking IL-1 with anakinra for four weeks did not improve fatigue in CFS, and did not change circulating cytokines beyond IL-1Ra itself.",
     "grade": "refuted",
     "plain": "A drug that blocks one of the main inflammation signals did not help, which argues against simple ongoing inflammation driving symptoms.",
     "detail": "Roerink and colleagues ran a randomised placebo-controlled trial of daily anakinra 100 mg for four weeks in 50 severely fatigued female CFS patients (reported in Ann Intern Med 2017 and in the accompanying cytokine paper). After correction for multiple testing, anakinra had no effect on circulating cytokines other than IL-1Ra, and the trial did not produce clinical benefit. A companion endocrine analysis (Roerink 2018, Horm Behav) found a blunted cortisol awakening response in patients (4.2 vs 6.1 nmol/L, p=0.036) and a small anakinra effect on hair cortisol, indicating the drug was biologically active. The trial was short (4 weeks) and in a mostly moderate, female, Dutch cohort, so it cannot exclude benefit from longer or different immunomodulation.",
     "studies": [
      {
       "cite": "Roerink 2017, Annals of Internal Medicine / J Transl Med (cytokine arm)",
       "n": "50 randomised",
       "design": "RCT, anakinra vs placebo, 4 weeks",
       "result": "No clinical benefit; no cytokine change beyond IL-1Ra",
       "url": "https://doi.org/10.1186/s12967-017-1371-9",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would IL-6 or TNF blockade, or a longer course, do anything? No adequately powered trial exists."
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "Not indicated"
    },
    {
     "id": "immune-csf-cytokines",
     "subdomain": "Cytokines",
     "finding": "Cerebrospinal fluid cytokine profiles differ from controls with raised eotaxin (CCL11) and disturbed IL-1 signalling, but CSF studies are small and partly contradicted by later proteomics.",
     "grade": "early",
     "plain": "Spinal fluid from patients looks immunologically different from healthy people's, hinting at inflammation inside the nervous system. The studies are small.",
     "detail": "Hornig 2015 (Mol Psychiatry) analysed CSF from 32 ME/CFS cases, 40 MS patients and 19 normal subjects with a 51-plex assay: raised CCL11 (eotaxin, an eosinophil chemokine) in cases and an inverse relationship between IL-1 receptor antagonist and CSF-1, CSF-2 and IL-17F without changes in IL-1 alpha or beta, interpreted as disturbed IL-1 signalling and a shift toward a Th2/allergic pattern. Peterson/Brenu 2015 found CSF cytokine differences in 18 patients. Against: a Solving ME/CFS re-analysis of NIH CSF proteomics found IL-17 proteins null in both serum and CSF, and neuronal injury markers (NEFL, GFAP) flat. Lumbar puncture cohorts are inevitably small and self-selected, and MS is an imperfect comparator.",
     "studies": [
      {
       "cite": "Hornig 2015, Molecular Psychiatry",
       "n": "32 cases vs 40 MS vs 19 normal",
       "design": "case-control CSF 51-plex",
       "result": "CCL11 raised; IL-1Ra vs CSF-1/CSF-2/IL-17F network inversion",
       "url": "https://doi.org/10.1038/mp.2015.29",
       "verified": true
      },
      {
       "cite": "Peterson/Brenu 2015, Mediators of Inflammation",
       "n": "18 patients",
       "design": "pilot CSF cytokine study",
       "result": "CSF cytokine differences reported",
       "url": "https://doi.org/10.1155/2015/929720",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome; NIH Walitt CSF)",
       "n": "NIH CSF and serum",
       "design": "re-analysis",
       "result": "IL-17 proteins null in serum and CSF; neuronal injury markers flat",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does CSF CCL11 replicate in an independent cohort with matched non-inflammatory controls?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "mast-cell",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-innate-hyperreactivity",
     "subdomain": "Innate immunity",
     "finding": "Whole-blood immune responses to microbial antigens are exaggerated in ME/CFS and worsen after exercise, alongside complement activation and metabolic abnormalities.",
     "grade": "early",
     "plain": "When patients' blood cells are shown bits of bacteria, they over-react, and the over-reaction is worse the day after exercise.",
     "detail": "Che, Lipkin and colleagues 2025 (npj Metabolic Health and Disease; medRxiv July 2025) studied 56 ME/CFS cases and 52 healthy controls recruited in New York and California with 47 matched pairs, combining plasma metabolomics, proteomics and immune responses to microbial stimulation before and 24 h after exercise. They report exaggerated innate responses to microbial antigens, complement activation, impaired TCA/beta-oxidation/urea-cycle energy production, disrupted extracellular-matrix homeostasis with release of pro-inflammatory endogenous ligands, reduced cell-cell adhesion with gut dysbiosis, copper-dependent redox disturbance and tryptophan-kynurenine dysregulation; many abnormalities worsened after exercise in patients but not controls and correlated with symptom intensity. This is a single-group study with very many readouts and no pre-registered primary endpoint; the stimulation assay is not standardised across labs. It is nonetheless one of the few designs that captures a post-exertional immune change at all.",
     "studies": [
      {
       "cite": "Che/Lipkin 2025, npj Metabolic Health and Disease",
       "n": "56 vs 52 (47 matched pairs)",
       "design": "case-control multi-omics with exercise challenge and ex vivo microbial stimulation",
       "result": "Exaggerated innate response to microbial antigens, complement activation, energy-pathway and ECM abnormalities; worse post-exercise and correlated with symptoms",
       "url": "https://doi.org/10.1038/s44324-025-00079-w",
       "verified": true
      },
      {
       "cite": "Raijmakers 2019, Journal of Infection (Q fever fatigue syndrome)",
       "n": "20 QFS, 19 CFS, 19 seropositive controls, 25 HC",
       "design": "case-control ex vivo stimulation",
       "result": "QFS PBMCs produced more IL-6, TNF-alpha and IL-1beta on Coxiella antigen stimulation; higher circulating IL-6 and IFN-gamma",
       "url": "https://doi.org/10.1016/j.jinf.2019.01.006",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome; Germain 2-day CPET)",
       "n": "SomaScan, 2-day CPET",
       "design": "re-analysis",
       "result": "Nothing changes differently after exercise at FDR in the plasma proteome (0 FDR hits)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the stimulation hyper-response replicate in an independent lab with blinded samples?",
      "Is it a property of monocytes, of plasma factors, or of both?",
      "Can it be used as a PEM readout?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "complement-thromboinflammation",
      "gut-dysbiosis",
      "cell-danger-response"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-monocytes-platelets",
     "subdomain": "Innate immunity",
     "finding": "Single-cell transcriptomics showed classical-monocyte dysregulation at baseline and platelet-activation signatures after exercise, but a re-analysis of the same data found no reproducible composition or expression differences.",
     "grade": "contested",
     "plain": "One study found patients' monocytes looked abnormal, and platelets reacted oddly to exercise. Reworking the same data did not confirm it.",
     "detail": "Vu 2024 (Cell Rep Med) applied scRNA-seq to PBMCs from patients and controls at baseline and after exercise, reporting classical-monocyte dysregulation suggestive of inappropriate differentiation and tissue migration, with the fraction of 'diseased' monocytes correlating with severity, and post-exercise patterns indicating improper platelet activation with minimal change elsewhere. A Solving ME/CFS re-analysis of the same public dataset (GSE214283; 30 vs 28, 503,739 cells) with CellTypist majority voting and person-level pseudobulk found no composition differences at baseline (lowest q=0.41) or in the exercise change, and 0 genes at q<0.05 at baseline across 15 cell types (only NDUFA10 in non-classical monocytes in the 24 h change, q=0.037); platelet-RNA+ monocyte scores were null. There is no independent ME/CFS PBMC scRNA dataset from another group, which is the central problem. Older monocyte claims (Prieto 1989, naloxone-reversible monocyte dysfunction) were never replicated.",
     "studies": [
      {
       "cite": "Vu 2024, Cell Reports Medicine",
       "n": "case-control, paired pre/post exercise",
       "design": "scRNA-seq of PBMCs",
       "result": "Classical-monocyte dysregulation correlating with severity; post-exercise platelet activation signature",
       "url": "https://doi.org/10.1016/j.xcrm.2023.101373",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/singlecell_methyl, GSE214283)",
       "n": "30 vs 28, 503,739 PBMCs",
       "design": "independent re-analysis",
       "result": "No composition difference (lowest q=0.41); 0 DE genes at q<0.05 at baseline in 15 cell types; platelet+ monocyte score null",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does any group outside Cornell have ME/CFS single-cell blood data to test this?",
      "Are monocyte findings driven by handling time and cell isolation?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "microclots",
      "complement-thromboinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-cfrna",
     "subdomain": "Cell-free RNA",
     "finding": "Plasma cell-free RNA carries an immune-weighted ME/CFS signature that classifies cases with held-out AUC 0.81, with raised plasmacytoid-dendritic, monocyte and T-cell-derived transcripts.",
     "grade": "early",
     "plain": "Fragments of RNA floating in blood plasma, mostly from immune cells, differ enough between patients and controls to build a reasonably accurate classifier.",
     "detail": "Gardella 2025 (PNAS) sequenced plasma cfRNA in 93 ME/CFS cases and 75 healthy sedentary controls. A LASSO generalised linear model trained on condition-specific signatures reached test-set AUC 0.81 and accuracy 77%. Deconvolution showed differences in platelet-derived cfRNA and elevated plasmacytoid dendritic, monocyte and T-cell-derived cfRNA; network analysis implicated cytokine signalling and T-cell exhaustion, converging with Iu 2024. Caveats: single cohort with internal hold-out rather than external validation; sedentary controls help but do not remove deconditioning confounding; cfRNA is sensitive to pre-analytic handling. A separate LLM-benchmarking paper using the same cohort (Gaudio 2026, Nat Commun) confirms the ME/CFS cohort is the hardest of three to classify, which is a useful calibration on effect size.",
     "studies": [
      {
       "cite": "Gardella 2025, PNAS",
       "n": "93 vs 75",
       "design": "case-control cfRNA sequencing with machine learning",
       "result": "Held-out AUC 0.81, accuracy 77%; raised pDC, monocyte and T-cell cfRNA; cytokine-signalling and exhaustion networks",
       "url": "https://doi.org/10.1073/pnas.2507345122",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (downloads)",
       "n": "5 blood RNA cohorts + cfRNA + SomaScan",
       "design": "re-analysis",
       "result": "No gene set passes FDR in blood gene expression; interferon slightly up in all 5 (meta p=0.12)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the classifier hold in an independent cohort and against other fatiguing illnesses?",
      "Does the cfRNA signature move with PEM or treatment?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "innate-hyperactivation"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-gpcr-autoantibodies",
     "subdomain": "Autoantibodies",
     "finding": "Elevated IgG against beta2-adrenergic and M3/M4 muscarinic receptors, reported in about 30% of patients by ELISA, is not reproduced by unbiased high-throughput antigen profiling.",
     "grade": "refuted",
     "plain": "A commercial antibody test suggested many patients have antibodies against nerve-signal receptors. When measured with better, unbiased methods, the difference disappears.",
     "detail": "Loebel 2015 (Brain Behav Immun) tested a Berlin cohort (n=268) plus pre/post samples from 25 rituximab-trial patients against a panel of GPCRs by ELISA versus 108 healthy controls: beta2, M3 and M4 antibodies significantly elevated, 29.5% of patients above threshold, correlating with IgG1-3 subclass levels and with activated HLA-DR+ T cells, TPO and ANA positivity; in rituximab responders with prolonged B-cell depletion beta2 and M4 antibodies fell, in non-responders they did not. Bynke 2020 partially validated this in two Swedish cohorts (M3 and M4 in both, beta1/beta2 in one; n=24+24 vs 24) with no correlation to severity and nothing detectable in CSF. The decisive negative is Germain, Jaycox, Ring and Hanson 2025 (IJMS 26:2799): 7,542 antibody-antigen interactions across 172 participants using REAP (6,183 extracellular human proteins, 225 viral antigens) and a 1,134-plex Luminex panel found no significant difference in any autoantibody reactivity, including beta-adrenergic receptors (no q below 0.68), with ADRB2, ADRA1A and GRK2 specifically not elevated. In POTS the same conclusion was reached with standard methodology: Hall 2022 (Circulation) found no difference across 11 receptors in 116 POTS vs 81 controls, with 98.3% of patients and 100% of controls above the manufacturer's alpha1 seropositivity threshold, i.e. the commercial cut-offs are not meaningful.",
     "studies": [
      {
       "cite": "Loebel 2015, Brain Behavior and Immunity",
       "n": "268 patients vs 108 controls (+25 trial patients)",
       "design": "case-control ELISA panel",
       "result": "beta2, M3, M4 IgG elevated; 29.5% above threshold; fell in rituximab responders only",
       "url": "https://doi.org/10.1016/j.bbi.2015.09.013",
       "verified": true
      },
      {
       "cite": "Bynke 2020, Brain Behav Immun Health",
       "n": "48 patients vs 24 controls (CSF in 24/6)",
       "design": "two-cohort validation, plasma and CSF ELISA",
       "result": "M3 and M4 raised in both cohorts, beta1/beta2 in one; no correlation with severity; nothing in CSF",
       "url": "https://doi.org/10.1016/j.bbih.2020.100107",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Germain, Jaycox, Ring & Hanson 2025, IJMS",
       "n": "172 participants (164 REAP, 103 Luminex)",
       "design": "case-control unbiased antigen profiling (REAP + 1,134-plex)",
       "result": "No autoantibody reactivity differed; no q below 0.68; ADRB2/ADRA1A/GRK2 not elevated; incidental higher EBV gp42 reactivity",
       "url": "https://doi.org/10.3390/ijms26062799",
       "verified": true
      },
      {
       "cite": "Hall 2022, Circulation (POTS)",
       "n": "116 POTS vs 81 HC",
       "design": "case-control, commercial ELISA, standard methodology",
       "result": "No difference for any of 11 receptors; ROC poor; manufacturer thresholds met by essentially everyone",
       "url": "https://doi.org/10.1161/CIRCULATIONAHA.122.059971",
       "verified": true
      }
     ],
     "open_questions": [
      "Could functional (bioassay) activity exist without differential binding, as the Wallukat-type cardiomyocyte assays claim? Those assays have never been blinded across labs.",
      "Is the ELISA signal an artefact of IgG concentration or non-specific binding (it correlated with total IgG1-3)?"
     ],
     "hypotheses": [
      "autoimmunity",
      "vascular-endothelial",
      "autonomic-small-fibre"
     ],
     "test_available": "CellTrend GPCR antibody panels are purchasable privately; the evidence does not support using them for diagnosis or treatment decisions"
    },
    {
     "id": "immune-immunoadsorption",
     "subdomain": "Autoantibodies",
     "finding": "Removing autoantibodies by immunoadsorption helps in open-label cohorts but fails against sham control, and antibody removal does not predict who improves.",
     "grade": "refuted",
     "plain": "Filtering antibodies out of the blood made patients feel better in studies without a comparison group, and did nothing once a fake treatment was used for comparison.",
     "detail": "Open-label positives: Scheibenbogen 2018 (10 patients, 5 sessions, beta2-selected) and Stein 2024 (Lancet Reg Health Eur; 20 post-COVID ME/CFS with elevated beta2-AR antibodies; total IgG down 79%, beta2 antibodies down 77%, mean SF-36 PF +17.75, 14/20 responders at six months). The controlled trials are negative: IAMPOCO (Stortz 2026, Lancet Reg Health Eur) randomised 40 post-COVID patients in a patient-blinded sham-controlled crossover - no difference in symptom severity (Post-COVID Functional Scale OR 1.17, 95% CI 0.41-3.36, p=0.771; MFI-20 difference 2.4, p=0.437; Chalder 0.09, p=0.970; Bell -2.6, p=0.246; MoCA -0.01, p=0.993; handgrip 1.3, p=0.234), with more adverse events on immunoadsorption (24 vs 10), and GPCR antibodies depleted by immunoadsorption but not sham - i.e. the mechanism worked and the patients did not improve. IA-PACS-CFS (Charite, NCT05710770, 65 randomised 2:1) also failed its primary endpoint per conference report; the paper is not yet published. Wossidlo 2026 (Infection; 18 patients) found significant antibody reduction, return to baseline within 3-6 months, no improvement in objective performance, only slight self-reported pain/fatigue change and no correlation between antibody change and symptoms. Anft 2025 (Mol Ther; 12 patients) found antibody and cytokine reduction with improved neuropsychological testing and handgrip but no improvement in self-reported symptoms or ME/CFS questionnaires, and rebound within a month.",
     "studies": [
      {
       "cite": "Stortz 2026 (IAMPOCO), Lancet Regional Health Europe",
       "n": "40",
       "design": "randomised patient-blinded sham-controlled crossover",
       "result": "No difference on any endpoint; antibodies depleted by IA not sham; more AEs on IA",
       "url": "https://doi.org/10.1016/j.lanepe.2026.101744",
       "verified": true
      },
      {
       "cite": "Wossidlo 2026, Infection",
       "n": "18",
       "design": "prospective exploratory, 5 sessions",
       "result": "Antibodies fell then rebounded; no improvement in objective performance; no antibody-symptom correlation",
       "url": "https://doi.org/10.1007/s15010-026-02914-8",
       "verified": true
      },
      {
       "cite": "Anft 2025, Molecular Therapy",
       "n": "12",
       "design": "observational, pre/post",
       "result": "Antibodies and pro-inflammatory cytokines fell, cognition and handgrip improved; questionnaires did not; rebound within a month",
       "url": "https://doi.org/10.1016/j.ymthe.2025.01.007",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Stein 2024, Lancet Regional Health Europe",
       "n": "20 (open-label)",
       "design": "prospective pre-post cohort",
       "result": "70% responders; SF-36 PF +17.75 sustained to 6 months",
       "url": "https://doi.org/10.1016/j.lanepe.2024.101161",
       "verified": true
      },
      {
       "cite": "Scheibenbogen 2018, PLOS ONE",
       "n": "10 (open-label)",
       "design": "uncontrolled pilot",
       "result": "Symptom improvement after 5 days of IgG-binding-column IA",
       "url": "https://doi.org/10.1371/journal.pone.0193672",
       "verified": true
      }
     ],
     "open_questions": [
      "Is any benefit in open-label apheresis due to volume/haemodynamic effects rather than antibody removal?",
      "Would a longer or repeated-cycle regimen beat sham (IAMPOCO was a single crossover)?"
     ],
     "hypotheses": [
      "autoimmunity",
      "low-blood-volume-preload"
     ],
     "test_available": "Offered privately in some clinics; controlled evidence is negative"
    },
    {
     "id": "immune-bc007",
     "subdomain": "Autoantibodies",
     "finding": "BC007 (rovunaptabin), an aptamer that neutralises GPCR autoantibodies, showed secondary-endpoint fatigue improvement in a 30-patient exploratory trial but failed in the larger company-run phase II.",
     "grade": "contested",
     "plain": "A drug designed to mop up the suspect antibodies looked promising in a tiny trial and failed in the bigger one. The company ran out of money.",
     "detail": "reCOVer (Hohberger 2025, eClinicalMedicine; EudraCT 2022-001781-35) was a prospective exploratory placebo-controlled double-blind randomised phase IIa trial with crossover at Erlangen: 30 post-COVID patients with GPCR functional autoantibodies and fatigue as major symptom, 1350 mg rovunaptabin or placebo at day 0 and day 48. Primary endpoint was safety (9 vs 5 treatment-emergent adverse events, p=0.13). Secondary endpoints improved: FACIT-Fatigue effect size 2.10 (p=0.0378), Bell score 3.64 (p=0.0004), Fatigue Severity Scale -2.66 (p=0.0088), and 4 of 8 quality-of-life items, with confirmed neutralisation of the autoantibodies. However the separately run Berlin Cures phase II (BLOC, NCT05911009; about 114 participants across 19 European centres, results announced November 2024) reported the drug well tolerated but with no superiority over placebo; the company subsequently halted work for lack of funds and the full data are not published. A 30-patient crossover trial with safety as its primary endpoint and multiple positive secondary endpoints is weak evidence against a failed larger trial.",
     "studies": [
      {
       "cite": "Hohberger 2025 (reCOVer), eClinicalMedicine",
       "n": "30 randomised",
       "design": "exploratory placebo-controlled double-blind RCT with crossover",
       "result": "Safety primary endpoint met; FACIT-F, Bell, FSS improved as secondary endpoints; antibodies neutralised",
       "url": "https://doi.org/10.1016/j.eclinm.2025.103358",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Berlin Cures BLOC phase II (NCT05911009), company announcement November 2024",
       "n": "~114 across 19 centres",
       "design": "double-blind placebo-controlled phase II",
       "result": "Well tolerated, no superiority over placebo; data unpublished; company halted development",
       "url": "https://clinicaltrials.gov/study/NCT05911009",
       "verified": false
      }
     ],
     "open_questions": [
      "Will the BLOC data ever be published, and was the placebo response inflated?",
      "Does any patient subgroup defined by functional (not binding) autoantibody activity respond?"
     ],
     "hypotheses": [
      "autoimmunity",
      "vascular-endothelial"
     ],
     "test_available": "Not available (development halted)"
    },
    {
     "id": "immune-igg-transfer-mice",
     "subdomain": "Autoantibodies",
     "finding": "IgG from long COVID patients transfers pain, fatigue-like behaviour and small-fibre nerve damage to mice in three independent 2026 studies; no equivalent published ME/CFS transfer experiment exists.",
     "grade": "several",
     "plain": "Inject antibodies from long COVID patients into mice and the mice become more pain-sensitive and less active, which is strong evidence those antibodies do something. Nobody has published the same experiment with ME/CFS antibodies.",
     "detail": "Chen, Appelman and colleagues 2026 (Cell Rep Med) transferred pooled total IgG from 34 WHO-defined long COVID patients (31 analysed; 15 post-infection asymptomatic controls) into C57BL/6 mice at 260 mg/kg (about one third of the mouse's circulating IgG), 8 mice per group, stratified into three subgroups by plasma GFAP, NFL and interferon-beta: pronounced and persistent mechanical hypersensitivity, reproduced by IgG drawn from the same still-symptomatic patients two years later, with subgroup-linked autoreactivities validated by independent assays. De Sa, Iwasaki and colleagues 2026 (Cell) found CNS- and PNS-reactive autoantibodies correlating with neurological symptoms, IgG reacting with human locus coeruleus, thalamus, adrenal and thyroid and cross-reacting with mouse sciatic nerve and meninges, and passive transfer inducing fatigue-like behaviour, loss of balance, thermal hyperalgesia, small-fibre nerve damage and increased pain-related neuronal activity. Mignolet 2026 (Acta Neuropathologica) transferred IgG from neurological long COVID patients into mice: transient mechanical allodynia and thermal hypersensitivity in week 1 with no anxiety, depression or memory effect; abolished by IgG-depleted serum or papain-digested IgG; IgG accumulated in lumbar dorsal root ganglia colocalising with proprioceptive and nociceptive neurons without local neuroinflammation. These are the template experiments from the fibromyalgia field (Goebel 2021, JCI). The honest caveats: pooled IgG at high dose, pain readouts dominate, the fatigue-like readouts are indirect, and none of this is ME/CFS.",
     "studies": [
      {
       "cite": "Chen 2026, Cell Reports Medicine",
       "n": "31 LC patients, 15 HC; 8 mice/group",
       "design": "passive IgG transfer to mice + proteome-wide autoantibody profiling",
       "result": "Persistent mechanical hypersensitivity; reproduced with IgG taken 2 years later",
       "url": "https://doi.org/10.1016/j.xcrm.2026.102693",
       "verified": true
      },
      {
       "cite": "de Sa / Iwasaki 2026, Cell",
       "n": "LC cohort + mouse transfer",
       "design": "autoantibody mapping + passive transfer",
       "result": "Fatigue-like behaviour, balance loss, thermal hyperalgesia, small-fibre damage; IgG binds locus coeruleus, thalamus, sciatic nerve, meninges",
       "url": "https://doi.org/10.1016/j.cell.2026.04.042",
       "verified": true
      },
      {
       "cite": "Mignolet 2026, Acta Neuropathologica",
       "n": "neurological LC vs asymptomatic matched controls; C57Bl6/J mice",
       "design": "passive IgG transfer, 4 daily i.p. injections",
       "result": "Transient allodynia and thermal hypersensitivity; abolished by IgG depletion or papain digestion; IgG in lumbar DRG",
       "url": "https://doi.org/10.1007/s00401-026-03019-0",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Mignolet 2026, Acta Neuropathologica",
       "n": "as above",
       "design": "passive transfer",
       "result": "No effect on anxiety, depression or spatial memory, and no local neuroinflammation - so the 'brain fog' part does not transfer",
       "url": "https://doi.org/10.1007/s00401-026-03019-0",
       "verified": true
      },
      {
       "cite": "Stortz 2026 (IAMPOCO), Lancet Reg Health Eur",
       "n": "40",
       "design": "sham-controlled crossover",
       "result": "Removing IgG from patients did not improve symptoms - hard to reconcile with a sufficient pathogenic-IgG model",
       "url": "https://doi.org/10.1016/j.lanepe.2026.101744",
       "verified": true
      }
     ],
     "open_questions": [
      "Does ME/CFS IgG (non-COVID onset) transfer anything to mice? Presented at conferences, not published.",
      "Why do mouse transfer experiments work while antibody removal in patients does not? Tissue-bound antibody, long-lived plasma cells, or irreversible downstream damage?",
      "Which antigens matter - the studies disagree on targets."
     ],
     "hypotheses": [
      "autoimmunity",
      "autonomic-small-fibre",
      "serum-factor"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-igg-endothelial",
     "subdomain": "Autoantibodies",
     "finding": "Purified IgG from ME/CFS patients fragments mitochondria and shifts energetics in human endothelial cells, an effect absent with multiple-sclerosis and healthy IgG and lost on Fab/Fc cleavage.",
     "grade": "early",
     "plain": "Patient antibodies damage the energy machinery of blood-vessel cells in a dish; antibodies from healthy people and from MS patients do not.",
     "detail": "Liu, Hollmann, Prusty and colleagues (medRxiv Aug 2025; Brain Behav Immun Health Feb 2026, 101187) isolated immunoglobulin complexes by protein G from 106 individuals: 39 ME/CFS, 15 post-COVID ME/CFS, 20 MS disease controls and 41 healthy controls, applied them to HUVEC endothelial cells, fibroblasts and PBMCs, and assayed mitochondrial morphology by confocal microscopy and energetics by Seahorse. Endothelial mitochondrial fragmentation occurred with female ME/CFS IgG (p=0.0018 vs controls) with increased spare respiratory capacity and raised IL-1beta (p=0.046), and no effect from MS or healthy IgG; the effect was lost on cleaving IgG into Fab and Fc, with the Fab fragment reproducing the energetic change and Fc alone inducing a hypometabolic phenotype; mass spectrometry found excess extracellular-matrix proteins and SPEG in the immune complexes. Authors' own limits: large inter-plate Seahorse variability forcing split experiments, IgM not captured, unexplained sex difference, autoantigen array arm only 12 vs 3, and nominal p-values across many readouts. Single lab, no independent replication. The same group previously reported ME/CFS serum fragmenting mitochondria in cell lines (Schreiner 2020, ImmunoHorizons, n=10). Context matters: the headline serum-factor claim in muscle cells failed a well-powered replication (Ryback 2026, 67 vs 53, maximal respiration +2.50 pmol/min, 95% CI -1.62 to 6.62, p=0.23).",
     "studies": [
      {
       "cite": "Liu / Prusty 2026, Brain Behavior & Immunity - Health",
       "n": "39 ME/CFS, 15 PCS-CFS, 20 MS, 41 HC",
       "design": "in vitro IgG application to endothelial cells with disease control",
       "result": "Mitochondrial fragmentation with female ME/CFS IgG (p=0.0018); IL-1beta up; effect lost on Fab/Fc cleavage",
       "url": "https://doi.org/10.1016/j.bbih.2026.101187",
       "verified": true
      },
      {
       "cite": "Hackel / Sotzny 2025, medRxiv",
       "n": "post-COVID with and without ME/CFS",
       "design": "IgG fractions applied to monocyte cell lines",
       "result": "Distinct patient-IgG-induced cytokine responses, differing between post-COVID with and without ME/CFS",
       "url": "https://doi.org/10.1101/2025.01.09.25320264",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Ryback 2026, PLOS ONE (via Solving ME/CFS re-analysis serum-check)",
       "n": "67 ME vs 53 HC",
       "design": "preregistered blinded serum-exposure replication in myoblasts",
       "result": "Maximal respiration +2.50 pmol/min (95% CI -1.62 to 6.62), p=0.23; all secondary endpoints null; CI excludes Fluge's d=1.32",
       "url": "https://doi.org/10.1371/journal.pone.0341334",
       "verified": true
      }
     ],
     "open_questions": [
      "Can another lab reproduce the IgG endothelial assay blind, with a detection-floor spike-in control?",
      "Is the signal driven by IgG specificity or by glycosylation/immune-complex composition?",
      "Does the effect size track patient severity or PEM state?"
     ],
     "hypotheses": [
      "autoimmunity",
      "serum-factor",
      "vascular-endothelial",
      "mitochondrial-energy"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-igg-subclass",
     "subdomain": "Humoral deficiency",
     "finding": "A sizeable minority of ME/CFS patients have IgG subclass or mannose-binding-lectin deficiency, with IgG3 and IgG4 the commonest phenotypes, and registry data show strong enrichment of coded antibody deficiency.",
     "grade": "several",
     "plain": "Some patients are mildly short of particular antibody types, which fits a history of frequent infections. Others have the opposite, with raised antibody levels.",
     "detail": "Guenther 2015 (Hum Immunol) retrospectively analysed 300 CFS patients: 25% had decreased levels of at least one antibody class or subclass, IgG3 and IgG4 deficiency commonest, while another 25% had elevated immunoglobulins with excess IgM and IgG2; MBL deficiency in 15% versus 6% in a historical control group; similar frequencies in a second cohort of 168. Lutz 2021 (Biomolecules) found in 262 Austrian patients that 64.9% had a reduction or deficiency in at least one immune parameter and 26.3% showed activation or inflammation, with 17.6% having unclassified antibody deficiency (IgG3/IgG4 most common) and MBL reduced in 32%, deficient in 7%. Finnish national registry data (FinnGen/FinRegistry via Risteys, as used in the Solving ME/CFS re-analysis) show postviral fatigue syndrome enriched for coded IgG subclass antibody deficiency by 20-90x observed/expected, alongside EDS/hypermobility (about 100x) - but healthcare-use bias inflates such registry enrichments, and the published literature on immunoglobulins in ME/CFS is explicitly contradictory (12 studies for, 7 against in that review tally). Both clinic series lack contemporaneous matched controls.",
     "studies": [
      {
       "cite": "Guenther 2015, Human Immunology",
       "n": "300 + 168 patients",
       "design": "retrospective clinic series vs historical controls",
       "result": "25% with reduced class/subclass (IgG3, IgG4 commonest); 25% elevated; MBL deficiency 15% vs 6%",
       "url": "https://doi.org/10.1016/j.humimm.2015.09.028",
       "verified": true
      },
      {
       "cite": "Lutz 2021, Biomolecules",
       "n": "262 patients",
       "design": "retrospective clinic series",
       "result": "64.9% with at least one reduced immune parameter; 17.6% unclassified antibody deficiency; MBL reduced in 32%",
       "url": "https://doi.org/10.3390/biom11091359",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2/epi_literature, FinnGen/FinRegistry)",
       "n": "~1,622 FinRegistry / 348 FinnGen postviral fatigue cases",
       "design": "registry enrichment",
       "result": "IgG subclass antibody deficiency O/E 44 [87]; crude and healthcare-use inflated; literature contradictory",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/epi_literature literature tally)",
       "n": "19 studies",
       "design": "literature contradiction count",
       "result": "Immunoglobulins/IgG subclasses contradictory across studies (12 for / 7 against)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the deficient subgroup differ clinically (infection frequency, severity, onset)?",
      "Is MBL deficiency anything more than a common population variant here?",
      "Does immunoglobulin replacement help the deficient subgroup? Old IVIG trials were small and conflicting; iSTAND in autoimmune POTS (n=30) was negative with both arms improving."
     ],
     "hypotheses": [
      "autoimmunity",
      "viral-persistence",
      "connective-tissue"
     ],
     "test_available": "Clinical: total IgG/IgA/IgM plus IgG1-4 subclasses; MBL is available in some centres. Cheap and interpretable by an immunologist."
    },
    {
     "id": "immune-mast-cell",
     "subdomain": "Mast cells",
     "finding": "Clinically relevant mast cell activation is reported in up to a quarter of ME/CFS patients, associates with orthostatic intolerance and POTS, and those patients report more benefit from mast-cell-directed treatment.",
     "grade": "early",
     "plain": "A quarter of patients have symptoms of over-reactive mast cells (flushing, rashes, food and smell sensitivity), and those patients more often have the fast-heart-rate-on-standing problem.",
     "detail": "Rohrhofer 2025 (Diagnostics) analysed an Austrian patient-centred survey (687 completed) plus a retrospective clinical cohort (383 patients): mast cell activation prevalence rose over the disease course, up to 25.3% meeting criteria for clinically relevant MCA, and patients with MCAS plus orthostatic intolerance reported symptom alleviation after mast-cell-targeted treatment significantly more often than those without MCAS, while beta-blocker response did not differ (p=0.637); MCA involvement was significantly associated with orthostatic intolerance, especially POTS. Laboratory-level work is thin and small: Nguyen 2017 and Balinas 2018 (both Marshall-Gradisnik group, n=11-18) characterised mast cell progenitor phenotypes and TLR3 responses in PBMCs. There is no replicated serum tryptase or urinary mediator finding in ME/CFS, criteria for MCAS are themselves contested, and self-reported treatment response is not a controlled outcome. One small retrospective case series of continuous high-dose oral cromolyn in ME/CFS with MCAS (5 patients) reported benefit.",
     "studies": [
      {
       "cite": "Rohrhofer 2025, Diagnostics",
       "n": "687 survey + 383 retrospective",
       "design": "survey plus retrospective clinical cohort",
       "result": "Up to 25.3% clinically relevant MCA; associated with OI/POTS; better reported response to mast-cell-directed treatment",
       "url": "https://doi.org/10.3390/diagnostics15222828",
       "verified": true
      },
      {
       "cite": "Nguyen 2017, Asian Pac J Allergy Immunol",
       "n": "18 patients vs 13 HC",
       "design": "flow cytometry of PBMC mast cell phenotypes",
       "result": "Altered mast cell phenotypes and RAGE/HMGB1 expression",
       "url": "https://doi.org/10.12932/AP0771",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome)",
       "n": "4 blood protein cohorts + UK Biobank",
       "design": "harmonised proteomic meta-analysis",
       "result": "No replicated mast-cell or allergic protein signature; immune protein sets flat or inconsistent",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Do objective mediator measures (serum tryptase, urinary N-methylhistamine, prostaglandin D2) differ in the MCA subgroup?",
      "Is MCA cause, consequence, or a parallel diagnosis of the same autonomic phenotype?",
      "Does a controlled trial of mast-cell stabilisers help ME/CFS with OI?"
     ],
     "hypotheses": [
      "mast-cell",
      "autonomic-small-fibre",
      "innate-hyperactivation"
     ],
     "test_available": "Clinical: serum tryptase (baseline and during a flare), urinary mediators in specialist centres; antihistamine/cromolyn trials are low-risk"
    },
    {
     "id": "immune-interferon-alpha-model",
     "subdomain": "Sickness behaviour",
     "finding": "In the interferon-alpha fatigue model, higher baseline IL-6 and IL-10 and a larger early cytokine and fatigue surge predict fatigue that persists after the immune trigger is gone.",
     "grade": "early",
     "plain": "Giving people interferon as hepatitis treatment causes fatigue; in a third it does not go away afterwards. Those people had a more reactive immune system before treatment started.",
     "detail": "Russell 2019 (Psychoneuroendocrinology) followed 55 patients on interferon-alpha for chronic hepatitis C at baseline, during 6-12 months of treatment, and six months after, comparing with 54 CFS patients and 57 healthy volunteers. Eighteen (33%) had persistent fatigue (higher six months post-treatment than baseline). Those who went on to persistent fatigue had a greater fatigue increase in the first four weeks (7.1 +/- 1.5 vs 4.0 +/- 0.8, p=0.046), a trend to higher baseline IL-6, significantly higher baseline IL-10, and larger treatment-induced rises in both (more than doubled by week 4 relative to the others: p=0.011 for IL-6, p=0.001 for IL-10). This is the only human design that measures the immune system before the trigger. Limits: iatrogenic cytokine exposure is not a natural infection, HCV patients differ in many ways, n=18 in the key group, and the model's cytokine differences did not persist to follow-up. It sits alongside the acute sickness-behaviour literature (Capuron 2002: neurovegetative symptoms including fatigue appear within two weeks of interferon-alpha and are less paroxetine-responsive than mood symptoms).",
     "studies": [
      {
       "cite": "Russell 2019, Psychoneuroendocrinology",
       "n": "55 HCV patients (18 persistent fatigue) + 54 CFS + 57 HV",
       "design": "prospective pre/during/post immune-trigger cohort",
       "result": "Higher baseline IL-10 (and trend IL-6), larger week-4 cytokine and fatigue surge in those with persistent fatigue",
       "url": "https://doi.org/10.1016/j.psyneuen.2018.11.032",
       "verified": true
      },
      {
       "cite": "Capuron 2002, Neuropsychopharmacology",
       "n": "40 melanoma patients",
       "design": "randomised paroxetine vs placebo during IFN-alpha",
       "result": "Neurovegetative symptoms (anorexia, fatigue, pain) appear within 2 weeks; mood/cognitive symptoms appear later and are the paroxetine-responsive ones",
       "url": "https://doi.org/10.1016/S0893-133X(01)00407-9",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does a pre-trigger immune profile predict ME/CFS after natural infection (needs a large inception cohort with stored baseline samples)?",
      "Is the persistent-fatigue state after interferon biologically the same as ME/CFS, or only phenotypically similar?"
     ],
     "hypotheses": [
      "sickness-behaviour",
      "innate-hyperactivation",
      "metabolic-trap-ido"
     ],
     "test_available": "research only (prospective design)"
    },
    {
     "id": "immune-sickness-behaviour",
     "subdomain": "Sickness behaviour",
     "finding": "Acute-infection symptom severity, and the pro-inflammatory cytokine response and genotypes behind it, predict who develops post-infective fatigue, while microbiological and psychological factors do not.",
     "grade": "replicated",
     "plain": "How severe your initial infection is, not which germ you had or your personality, predicts whether you stay ill. Cytokines track how ill you feel at the time.",
     "detail": "The Dubbo prospective cohort (Hickie 2006, BMJ) followed 253 patients from acute EBV, Coxiella burnetii or Ross River virus infection: 29 (12%) had a disabling fatigue-pain-cognitive-mood illness at six months and 28 (11%) met CFS criteria, with a stereotyped phenotype at similar incidence after each infection, predicted largely by acute illness severity and not by demographic, psychological or microbiological factors. Vollmer-Conna 2004 (Psychol Med) showed in 21 acute Q fever and 48 acute RRV/EBV patients that spontaneously released IL-1beta and IL-6 from PBMC cultures correlated consistently with fever, malaise, pain, fatigue, mood and concentration, i.e. cytokines index the acute sickness response in humans. Vollmer-Conna 2008 (Clin Infect Dis) found IFN-gamma +874T/A and IL-10 -592C/A polymorphisms acted synergistically on illness severity, cytokine levels and duration across all three infections. This is the most solidly replicated immune-related causal structure in the field, but it explains the onset and early course rather than why illness persists for years, and the same group's later work found no persisting cytokine abnormality.",
     "studies": [
      {
       "cite": "Hickie 2006, BMJ",
       "n": "253 followed 12 months",
       "design": "prospective post-infection inception cohort",
       "result": "11% met CFS criteria at 6 months; stereotyped phenotype, equal incidence across three pathogens; predicted by acute severity",
       "url": "https://doi.org/10.1136/bmj.38933.585764.AE",
       "verified": true
      },
      {
       "cite": "Vollmer-Conna 2004, Psychological Medicine",
       "n": "21 + 48 acute infections",
       "design": "prospective symptom-cytokine correlation",
       "result": "PBMC IL-1beta and IL-6 release correlated with fever, malaise, pain, fatigue, mood, concentration",
       "url": "https://doi.org/10.1017/S0033291704001953",
       "verified": true
      },
      {
       "cite": "Vollmer-Conna 2008, Clinical Infectious Diseases",
       "n": "Dubbo cohort",
       "design": "genotype-phenotype analysis",
       "result": "IFN-gamma +874T/A and IL-10 -592C/A synergistically affected severity, cytokine levels and illness duration",
       "url": "https://doi.org/10.1086/592967",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (downloads; DecodeME)",
       "n": "15,579 cases vs 259,909 controls",
       "design": "GWAS",
       "result": "Infection-onset and other-onset ME/CFS are genetically the same (rg 0.98) and no immune cell type is enriched, so inherited immune reactivity is not the main risk axis",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "What converts a severe acute sickness response into years of illness?",
      "Do the cytokine-gene effects replicate in modern post-COVID inception cohorts?"
     ],
     "hypotheses": [
      "sickness-behaviour",
      "innate-hyperactivation",
      "neuroinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "immune-longcovid-overlap",
     "subdomain": "Long COVID",
     "finding": "Long COVID immune profiling finds exhausted T cells, myeloid shifts, raised antibodies to non-SARS-CoV-2 viruses especially EBV, and low cortisol, giving a reference phenotype for post-infectious illness.",
     "grade": "several",
     "plain": "Long COVID studies, which are larger and better funded, find a similar picture to ME/CFS: worn-out T cells, signs of old viruses waking up, and low cortisol.",
     "detail": "Klein 2023 (Nature) enrolled 275 individuals with or without long COVID for multidimensional immune phenotyping with unbiased machine learning: differences in circulating myeloid and lymphocyte populations, exaggerated humoral responses to SARS-CoV-2, higher antibody responses to non-SARS-CoV-2 pathogens particularly EBV, and lower cortisol in long COVID, with cortisol among the strongest discriminating features. Yin 2024 (Nat Immunol) found systemic inflammation, sex-specific perturbation of cytolytic subsets, more CD4+ T cells poised to migrate to inflamed tissue, exhausted SARS-CoV-2-specific CD8+ T cells and mis-coordination between T and B cell responses. These converge with the ME/CFS exhaustion and herpesvirus-reactivation strands; complement dysregulation in active long COVID (Cervia-Hasler 2024, Science) is, unusually for this space, independently replicated. The caution is that long COVID cohorts are heterogeneous, most participants do not meet ME/CFS criteria, and the low-cortisol finding has not replicated consistently in ME/CFS.",
     "studies": [
      {
       "cite": "Klein 2023, Nature",
       "n": "275",
       "design": "cross-sectional immune phenotyping with ML",
       "result": "Myeloid/lymphocyte shifts, higher anti-EBV and other non-SARS-CoV-2 antibodies, lower cortisol",
       "url": "https://doi.org/10.1038/s41586-023-06651-y",
       "verified": true
      },
      {
       "cite": "Yin 2024, Nature Immunology",
       "n": "LC vs non-LC, 8 months post-infection",
       "design": "multi-omic + serology case-control",
       "result": "Exhausted SARS-CoV-2-specific CD8+ T cells; inflammation; uncoordinated adaptive response",
       "url": "https://doi.org/10.1038/s41590-023-01724-6",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome; long COVID Olink sets)",
       "n": "UK Explore 51 vs 51; IRIS 36 vs 9; Bansal 12 vs 44",
       "design": "re-analysis of long COVID proteomics",
       "result": "Only lower vascular mural/matrix proteins overlap with ME/CFS; interferon and immune-activation protein sets do not line up",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "How much of the long COVID immune phenotype is present in non-COVID-onset ME/CFS when measured side by side?",
      "Is the EBV antibody signal reactivation, bystander activation, or cross-reactivity?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "herpes-reactivation",
      "complement-thromboinflammation",
      "hpa-endocrine"
     ],
     "test_available": "EBV and CMV serology and PCR are clinically available; interpretation is limited (IgG shows past infection only)"
    },
    {
     "id": "immune-genetics-null",
     "subdomain": "Genetics vs immunity",
     "finding": "Common-variant genetics shows no enrichment of ME/CFS risk in any blood immune cell type, implying most immune findings are downstream consequences rather than inherited causes.",
     "grade": "replicated",
     "plain": "The largest genetic study points at brain cells, not immune cells. So the immune abnormalities are probably effects of the illness rather than its root cause.",
     "detail": "DecodeME (15,579 PEM-required cases vs 259,909 controls, SNP heritability about 9.5% on the liability scale) shows enrichment in brain tissue and neurons with no enrichment in blood immune cell types; the Solving ME/CFS re-analysis reproduced all 8 loci exactly and found MAGMA enrichment in 11 brain regions and pituitary and none of 29 immune cell types (Th17 p=0.52), with S-LDSC enrichment only in CNS tissues and chromatin and no signal in ImmGen or blood-cell ATAC-seq. HLA-DQA1*05:01 is protective (p=1.4e-10) but the earlier ME/CFS HLA candidates (C*07:04, DQB1*03:03 from Lande 2020) are not among DecodeME's hits, and the TRA locus (Ueland 2022) did not replicate. Infection-onset and other-onset cases are genetically indistinguishable (rg 0.98). The qualifier: a GWAS of common variants in 15,000 people can miss rare-variant immune contributions, and the one NK-relevant gene near a lead locus (RAB27A, needed for cytotoxic granule release) is not the authors' preferred candidate but is as plausible as their pick.",
     "studies": [
      {
       "cite": "DecodeME 2025 + Solving ME/CFS re-analysis (genetics, downloads)",
       "n": "15,579 cases vs 259,909 controls",
       "design": "GWAS with MAGMA and S-LDSC enrichment",
       "result": "Brain/neuron and pituitary enrichment; no enrichment in 29 immune cell types; locus-level tissue test null after FDR",
       "url": "",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2/genetics_mr)",
       "n": "DecodeME + Million Veteran Program",
       "design": "Mendelian randomisation and replication",
       "result": "Genetically higher EBV DNA load suggestive (OR 1.59 in MVP); overall DecodeME signal replicates (rg 0.73) while individual loci mostly do not",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Zhang 2025 (HEAL2), medRxiv",
       "n": "multi-site whole genomes",
       "design": "deep-learning rare-variant analysis",
       "result": "115 candidate risk genes with reduced expression in patients' B and T cells, especially cytotoxic CD4 T cells - rare-variant immune involvement not excluded by common-variant results",
       "url": "https://doi.org/10.1101/2025.04.15.25325899",
       "verified": true
      }
     ],
     "open_questions": [
      "Do rare immune variants (e.g. perforin-pathway, immunodeficiency panels) contribute in a subgroup?",
      "Could a genuinely causal immune mechanism be environmental (infection timing, dose) and therefore invisible to GWAS?"
     ],
     "hypotheses": [
      "autoimmunity",
      "immune-exhaustion",
      "genetic-neuronal",
      "herpes-reactivation"
     ],
     "test_available": "Clinical genetic testing is not indicated for ME/CFS; an immunodeficiency/HLH panel only if red flags (abnormal counts, recurrent severe infection)"
    },
    {
     "id": "immune-il11-mmp9",
     "subdomain": "Cytokines",
     "finding": "Newly reported elevations of serum IL-11 and MMP-9 in female ME/CFS patients are unreplicated single-cohort findings.",
     "grade": "early",
     "plain": "A 2026 study found two new inflammation-related proteins raised in patients. Nobody has checked it yet.",
     "detail": "Chinnappan 2026 (Front Immunol) quantified IL-11 and matrix metalloproteinase-9 in serum from 40 female ME/CFS patients and controls (Nova Southeastern/Institute for Neuro-Immune Medicine), reporting both elevated, with an EBV framing. IL-11 is a fibrosis- and inflammation-associated cytokine and MMP-9 a matrix-remodelling enzyme, so the finding is thematically compatible with the extracellular-matrix disturbance in Che/Lipkin 2025 and the lower vascular mural/matrix proteins found across four proteomic cohorts. But single-cohort, single-lab, two-analyte studies in this field have a poor replication record, and MMP-9 in particular is sensitive to sample handling and neutrophil contamination. Listed here as the kind of new lead a living review should carry with an explicit 'unreplicated' label.",
     "studies": [
      {
       "cite": "Chinnappan 2026, Frontiers in Immunology",
       "n": "40 female patients vs controls",
       "design": "case-control serum immunoassay",
       "result": "IL-11 and MMP-9 elevated",
       "url": "https://doi.org/10.3389/fimmu.2026.1827700",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2/proteome)",
       "n": "4 blood protein cohorts",
       "design": "meta-analysis",
       "result": "No single protein replicates strongly; effects that repeat are d 0.25-0.5",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does it replicate in an independent cohort with handling controls?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "vascular-endothelial"
     ],
     "test_available": "research only"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "autoimmunity",
     "for": "Infection-triggered onset; female predominance; open-label responses to B-cell depletion (early phase II), cyclophosphamide and especially daratumumab (6/10, 5 sustained, serum IgG fell more in responders); patient IgG does things control and MS IgG do not in endothelial cells (Liu/Prusty 2026, 39 ME vs 20 MS vs 41 HC); long COVID IgG transfers pain, fatigue-like behaviour and small-fibre damage to mice in three independent 2026 studies; IgG subclass abnormalities common.",
     "against": "Rituximab failed in a 151-patient RCT (26% vs 35% placebo); immunoadsorption removed GPCR antibodies without clinical benefit against sham (IAMPOCO n=40, all endpoints null; IA-PACS-CFS n=65 failed); BC007's larger phase II failed; unbiased profiling of 7,542 antibody-antigen interactions in 172 people found no differential reactivity (no q below 0.68), including beta-adrenergic receptors; BCR repertoires show no clonality, no somatic-hypermutation shift and no autoimmunity signature; no ME/CFS-specific autoantigen has been identified; common-variant genetics shows no immune cell enrichment and HLA associations do not fit an autoimmune pattern."
    },
    {
     "id": "immune-exhaustion",
     "for": "CD8+ effector-memory exhaustion transcription factors, chromatin and metabolic reprogramming (Iu 2024); independent cfRNA exhaustion and cytokine-signalling signatures (Gardella 2025); reduced CD8+ mitochondrial membrane potential and glycolysis (Mandarano 2020); exhausted virus-specific CD8+ T cells in long COVID (Yin 2024); early-immunosenescence CD28-CD57- cells in mild/moderate patients (Lee 2025).",
     "against": "Re-analysis of the same public scRNA data found exhausted-CD8 gating null and no differentially expressed genes at FDR in any of 15 cell types; TCR repertoires show no clonal expansion, which is hard to square with chronic antigen-driven exhaustion; no immune cell type is genetically enriched; no antigen has been identified."
    },
    {
     "id": "innate-hyperactivation",
     "for": "Exaggerated whole-blood responses to microbial antigens, worse 24 h after exercise and correlated with symptom intensity (Che/Lipkin 2025, 56 vs 52); Q fever fatigue syndrome PBMCs over-produce IL-6, TNF and IL-1beta on antigen stimulation; monocyte dysregulation in scRNA (Vu 2024); meta-analytic small elevations of TNF, IL-2, IL-4, CRP; 17 cytokines rising with severity (Montoya 2017); UK Biobank inflammation and liver/insulin markers shifted in both sexes.",
     "against": "IL-1 blockade with anakinra gave no clinical benefit and changed no cytokine but IL-1Ra; the best-replicated single cytokine (TGF-beta) is a platelet/centrifugation artefact; re-analysis of five blood RNA cohorts found no gene set passing FDR and interferon only nominally up (meta p=0.12); monocyte scRNA findings do not survive independent re-analysis of the same data; no immune cell type is genetically enriched."
    },
    {
     "id": "sickness-behaviour",
     "for": "Dubbo cohort: 11% met CFS criteria six months after three different pathogens with a stereotyped phenotype predicted by acute illness severity, not by pathogen or psychological factors; PBMC IL-1beta and IL-6 release tracks acute fever, malaise, pain, fatigue, mood and concentration; IFN-gamma and IL-10 polymorphisms synergistically set severity and duration; interferon-alpha reliably produces neurovegetative fatigue within two weeks (Capuron 2002) and persistent fatigue in a third of patients, predicted by higher baseline IL-10 and a bigger early surge (Russell 2019).",
     "against": "The model explains onset far better than persistence: cytokines are not persistently abnormal in long-duration illness (Hornig 2015), cytokine inhibition does not help, and the acute cytokine signal fades while the illness does not."
    },
    {
     "id": "herpes-reactivation",
     "for": "Higher anti-EBV antibody responses in long COVID (Klein 2023); incidental elevated EBV gp42 reactivity in the otherwise negative antigen-profiling study; IgG to arginine-rich EBNA4/EBNA6 sequences and homologous human peptides (Hoheisel 2025); genetically higher EBV DNA load suggestive in MVP (OR 1.59); exploratory re-analysis signals - fewer CMV-driven adaptive NK expansions in patients (1/30 vs 9/28, post-hoc p=0.005), more KLRG1+CX3CR1+ effector CD8 (d +0.60), higher EBV-specific TCR-beta share in severe patients' CD8 (0.88% vs 0.45%, p=0.06).",
     "against": "Cliff 2019 found no difference in seroprevalence for six human herpesviruses in 251 patients; the re-analysis signals are exploratory and do not survive multiple-testing correction (q 0.28); valganciclovir evidence rests on one 30-patient trial selected on antibody titres, which are a poor marker of active infection."
    },
    {
     "id": "serum-factor",
     "for": "Patient IgG fragments endothelial mitochondria and alters energetics with no effect from MS or healthy IgG, lost on Fab/Fc cleavage (Liu/Prusty 2026, 106 individuals); long COVID IgG transfer reproduces symptoms in mice; daratumumab responses track falling serum IgG.",
     "against": "The flagship serum-factor result (patient serum raising myoblast respiration) failed a preregistered, blinded, well-powered replication (67 vs 53, maximal respiration estimate 2.50 pmol/min, 95% CI -1.62 to 6.62, p=0.23, CI excluding the original d=1.32); no serum-to-cell assay has separated patients from controls in more than one laboratory; removing circulating IgG from patients by immunoadsorption produced no clinical benefit against sham."
    },
    {
     "id": "mast-cell",
     "for": "Up to 25.3% of ME/CFS patients meet criteria for clinically relevant mast cell activation, with prevalence rising over the disease course and a significant association with orthostatic intolerance and POTS; those patients report more benefit from mast-cell-directed treatment; small flow-cytometry studies find altered mast cell progenitor phenotypes; CSF eotaxin (an eosinophil/allergic chemokine) elevated.",
     "against": "Almost all the human evidence is self-reported or retrospective clinic data with no controls; MCAS criteria are themselves contested; no replicated objective mediator (tryptase, urinary metabolites) finding exists in ME/CFS; blood proteomics across four cohorts shows no allergic or mast-cell signature; no controlled trial of mast-cell stabilisers in ME/CFS."
    },
    {
     "id": "viral-persistence",
     "for": "Chronic antigenic stimulation interpretation of the naive/switched-memory B-cell shift (Walitt 2024); exhaustion programmes in CD8+ T cells; early-immunosenescence phenotype in mild/moderate patients 'indicative of persistent viral infection' (Lee 2025); exaggerated response to microbial antigens.",
     "against": "No persisting pathogen has been identified in ME/CFS blood; TCR and BCR repertoires show no clonal expansion or antigen-driven maturation; herpesvirus seroprevalence is normal; immune cell types are not genetically enriched."
    },
    {
     "id": "complement-thromboinflammation",
     "for": "Complement activation reported in the Che/Lipkin 2025 multi-omics with worsening after exercise; complement dysregulation in active long COVID (Cervia-Hasler 2024) is one of the few independently replicated post-viral blood abnormalities; a CDC study reports higher C3 in ME/CFS.",
     "against": "The strong complement evidence is long COVID rather than ME/CFS; one failed replication after age/BMI adjustment; platelet/microclot markers were flat in a harmonised four-cohort proteomic re-analysis."
    }
   ],
   "glossary": [
    {
     "term": "Natural killer (NK) cell",
     "plain": "A white blood cell that kills virus-infected and abnormal cells without needing to be trained first."
    },
    {
     "term": "Cytotoxicity assay",
     "plain": "A lab test that mixes immune cells with target cells and measures how many targets they kill. It needs fresh blood, which is why results vary between labs."
    },
    {
     "term": "T-cell exhaustion",
     "plain": "A worn-out state that killer T cells enter after long periods of fighting an infection or tumour; they stay present but respond poorly."
    },
    {
     "term": "Switched-memory B cell",
     "plain": "A B cell that has already met an antigen and changed the class of antibody it makes. Fewer of them, with more untrained (naive) cells, can suggest an unusual pattern of immune stimulation."
    },
    {
     "term": "Autoantibody",
     "plain": "An antibody that targets the body's own tissue instead of a germ."
    },
    {
     "term": "GPCR autoantibody (beta2, M3, M4)",
     "plain": "Antibodies said to target receptors that nerves use to control blood vessels, heart rate and glands. Commercial tests exist; better methods have not confirmed differences between patients and controls."
    },
    {
     "term": "Immunoadsorption / apheresis",
     "plain": "Filtering antibodies out of the blood through a column, like a short-term dialysis for antibodies."
    },
    {
     "term": "Passive transfer",
     "plain": "Injecting antibodies from a patient into an animal to see whether the symptoms travel with the antibodies. It is the strongest available test of whether antibodies cause something."
    },
    {
     "term": "Cytokine",
     "plain": "A small signalling protein immune cells use to talk to each other; also what makes you feel ill during an infection."
    },
    {
     "term": "Cell-free RNA (cfRNA)",
     "plain": "RNA fragments floating free in blood plasma, released when cells die or turn over. They give a readout of what many tissues and cells are doing."
    },
    {
     "term": "MAIT cell",
     "plain": "An unconventional T cell that patrols mucosal surfaces such as the gut and responds to bacterial metabolites."
    },
    {
     "term": "IgG subclass deficiency",
     "plain": "Being low in one of the four flavours of the main blood antibody, which can make some infections more frequent."
    },
    {
     "term": "Mannose-binding lectin (MBL)",
     "plain": "A blood protein of the innate immune system that tags microbes for destruction; low levels are a common inherited variation."
    },
    {
     "term": "Mast cell activation",
     "plain": "Over-release of histamine and other mediators by tissue immune cells, causing flushing, itching, gut symptoms and sensitivity to foods, smells and drugs."
    },
    {
     "term": "TRPM3",
     "plain": "A calcium channel in cell membranes. One group reports it works poorly in patients' NK cells."
    },
    {
     "term": "Sham-controlled trial",
     "plain": "A trial where the comparison group gets a convincing fake procedure, which matters enormously here because 35% improved on placebo in the rituximab trial."
    },
    {
     "term": "Pre-analytic artefact",
     "plain": "A difference created by how blood was collected, spun or stored rather than by the illness. TGF-beta in ME/CFS turned out to be one."
    }
   ],
   "key_reviews": [
    {
     "cite": "Baraniuk, Eaton-Fitch & Marshall-Gradisnik 2024, Frontiers in Immunology",
     "url": "https://doi.org/10.3389/fimmu.2024.1440643",
     "why": "The definitive pooled estimate for the NK cytotoxicity literature (28 papers, g 0.96) - read alongside Querec 2023 for why it has not become a test."
    },
    {
     "cite": "Strawbridge 2019, Neuroscience & Biobehavioral Reviews",
     "url": "https://doi.org/10.1016/j.neubiorev.2019.08.011",
     "why": "The only quantitative meta-analysis of circulating inflammatory proteins (42 studies); gives the honest effect sizes."
    },
    {
     "cite": "Corbitt 2019, BMC Neurology",
     "url": "https://doi.org/10.1186/s12883-019-1433-0",
     "why": "Systematic review concluding cytokines are not reliable biomarkers - the counterweight to single-cohort cytokine papers."
    },
    {
     "cite": "Mountford 2024, Pain Reports (international workshop position statement)",
     "url": "https://doi.org/10.1097/PR9.0000000000001167",
     "why": "Sets out how passive-transfer evidence should be read in symptom-based disorders, which is the key methodological frame for the 2026 IgG mouse studies."
    },
    {
     "cite": "Fehrer 2026, Autoimmunity Reviews (3rd Charite Fatigue Center conference)",
     "url": "https://doi.org/10.1016/j.autrev.2026.104043",
     "why": "Current expert survey of the autoimmunity/post-COVID strand including unpublished trial results."
    },
    {
     "cite": "Wirth & Scheibenbogen 2020, Autoimmunity Reviews",
     "url": "https://doi.org/10.1016/j.autrev.2020.102527",
     "why": "The most-cited mechanistic version of the beta2-adrenergic autoantibody hypothesis; useful to read now that its testable predictions have mostly failed."
    },
    {
     "cite": "Solving ME/CFS re-analysis, deep2/singlecell_methyl and deep2/proteome SUMMARY.md",
     "url": "",
     "why": "Systematic re-analysis of public immune-cell, methylation, repertoire and protein data; documents which published immune claims fail, and two specific artefacts (granulocyte contamination, cell-leak) that mislead naive users of these datasets."
    }
   ],
   "gaps": [
    "No immune measurement during PEM: there is no paired rest / post-exertional-crash study of NK function, T-cell phenotype or autoantibodies, despite PEM being the defining feature.",
    "No published passive-transfer experiment with ME/CFS IgG (as opposed to long COVID or fibromyalgia IgG), even though that is now a standard, affordable design.",
    "No independent laboratory has attempted the TRPM3 patch-clamp or the IgG-endothelial-mitochondria assay; both rest entirely on one group each.",
    "No independent ME/CFS single-cell blood dataset exists outside one research group, so single-cell claims cannot be externally replicated.",
    "No prospective inception cohort with pre-infection immune samples large enough to test whether a 'primed' immune profile predicts ME/CFS after natural infection (the interferon-alpha model is the only proxy).",
    "Severe and very severe patients are almost absent from immune studies that require travel; the few in-home studies are small, and the one well-powered serum replication included only 6 severe patients.",
    "No standardised, multi-site-validated functional immune assay exists at all - NK cytotoxicity failed that test, and nothing has replaced it.",
    "Why antibody-removal trials fail while antibody-transfer experiments work is unexplained and untested (tissue-bound IgG, long-lived plasma cells, irreversible downstream damage are all untested explanations).",
    "The daratumumab signal has no randomised test yet, and no second study of its proposed NK-count predictor.",
    "Mast cell activation in ME/CFS has no objective mediator study with controls and no controlled treatment trial, despite being common and treatable in principle.",
    "Pre-analytic standardisation is still not routine: after the TGF-beta centrifugation artefact and the SomaScan cell-leak artefact, immune studies in this field still rarely report or adjust for handling variables.",
    "Tissue immunology is almost untouched: muscle, gut, dorsal root ganglia and brainstem immune cells have barely been examined in ME/CFS, while the long COVID IgG studies point precisely at peripheral sensory ganglia."
   ]
  },
  {
   "domain": "infection",
   "title": "Infection and viruses",
   "overview": "The most solid finding in this area is about onset: about 1 in 10 people meet ME/CFS criteria six months after several unrelated infections (EBV, Q fever, Ross River virus, Giardia, SARS-CoV-2), and how severe the first illness was predicts this better than psychological factors do. Post-COVID cohorts confirm that infection raises the rate of new ME/CFS several-fold. Whether any virus is still active in people who already have ME/CFS is far less clear. Blood antibody and PCR studies of herpesviruses give mixed results. Tissue findings for enterovirus, HHV-6 and SARS-CoV-2 are small or unreplicated, though some of them are striking. SARS-CoV-2 antigen and RNA do persist in a minority of people for months, but three randomised trials of nirmatrelvir in established long COVID (about 1,200 people) were negative, and antiviral trials in ME/CFS are tiny and unreplicated. The XMRV episode, a retrovirus finding that turned out to be lab contamination, is a reminder that one striking virus finding needs blinded multi-lab replication.",
   "findings": [
    {
     "id": "infection-dubbo-stereotyped-pifs",
     "subdomain": "Post-infectious onset",
     "finding": "About 11-12% of people with confirmed EBV, Q fever or Ross River virus infection meet CFS criteria at 6 months, at a similar rate for all three pathogens, and the severity of the acute illness predicts it.",
     "grade": "replicated",
     "plain": "Three very different germs (a herpesvirus, a bacterium and a mosquito-borne virus) left about 1 in 10 people with the same long-lasting fatigue illness. The sicker someone was at the start, the more likely they were to stay ill.",
     "detail": "The Dubbo prospective cohort (n=253, rural Australia) followed people from laboratory-confirmed acute infection: 29/253 (12%) had a post-infective fatigue syndrome at 6 months and 28 (11%) met CFS criteria. The phenotype was 'stereotyped' across pathogens, and acute severity predicted it, whereas demographic, psychological and microbiological factors did not. Because the result does not depend on which pathogen caused the infection, it points to the host response rather than to the particular microbe. The cohort was small, with few cases per pathogen, and was followed to 12 months.",
     "studies": [
      {
       "cite": "Hickie 2006, BMJ",
       "n": "253",
       "design": "prospective cohort from acute infection",
       "result": "12% prolonged illness at 6 months, 11% CFS criteria; similar across EBV, Q fever, RRV; predicted by acute severity",
       "url": "https://doi.org/10.1136/bmj.38933.585764.AE",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What immune or metabolic feature of severe acute illness carries forward into the chronic state?",
      "Does early intervention during the acute phase change the 6-month rate?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour",
      "cell-danger-response"
     ],
     "test_available": "No predictive test; documentation of the acute infection (serology/PCR at onset) is useful for subtyping."
    },
    {
     "id": "infection-katz-post-mono-adolescents",
     "subdomain": "Post-infectious onset",
     "finding": "After infectious mononucleosis in adolescents, 13%, 7% and 4% met paediatric CFS criteria at 6, 12 and 24 months; all 13 cases still ill at 24 months were female.",
     "grade": "replicated",
     "plain": "After glandular fever, many teenagers are still unwell at six months, but most recover over two years. Those still ill at two years were all girls.",
     "detail": "Katz 2009 (n=301 adolescents aged 12-18, Chicago) screened for non-recovery and made diagnoses by blind review. Greater fatigue severity at 12 months predicted persistence, and acute-phase steroids did not raise risk. Jason and colleagues' follow-up of a larger college-student mono cohort (about 238 cases) found that baseline GI symptoms and low IL-13/IL-5 predicted non-recovery, per a secondary summary in the Solving ME/CFS re-analysis. Together with Dubbo, these data show the spontaneous recovery rates any treatment trial in recent-onset illness has to beat.",
     "studies": [
      {
       "cite": "Katz 2009, Pediatrics",
       "n": "301",
       "design": "prospective cohort after IM",
       "result": "CFS 13% / 7% / 4% at 6 / 12 / 24 months; all 24-month cases female",
       "url": "https://doi.org/10.1542/peds.2008-1879",
       "verified": true
      },
      {
       "cite": "Jason 2022 (via Solving ME/CFS re-analysis epi summary)",
       "n": "238 mono cases",
       "design": "prospective cohort, pre-illness baseline",
       "result": "baseline GI symptoms and low IL-13/IL-5 predicted non-recovery",
       "url": "",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Why are late persisters predominantly female?",
      "Does EBV load or lytic activity during IM predict persistence?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "immune-exhaustion"
     ],
     "test_available": "EBV serology (VCA IgM/IgG, EBNA IgG) confirms the IM trigger; no predictive test."
    },
    {
     "id": "infection-giardia-bergen",
     "subdomain": "Post-infectious onset",
     "finding": "A waterborne Giardia outbreak in Bergen (2004) left chronic fatigue in 26% of exposed people vs 11% of matched controls 10 years later (adjusted OR 3.0), and more than half of those referred with lasting fatigue met CFS criteria.",
     "grade": "several",
     "plain": "A gut parasite outbreak in Norway, which was not a virus, also left a lasting excess of chronic fatigue and IBS a decade later.",
     "detail": "Litleskare 2018 followed 1,252 laboratory-confirmed cases and 2,504 age- and sex-matched controls: at 10 years, chronic fatigue was 26% vs 11% (aOR 3.01, 95% CI 2.22-4.08) and IBS 43% vs 14% (aOR 4.74). Response rates at 10 years were around 46% of cases, so selection bias is possible. 'Chronic fatigue' here is a questionnaire measure, not a CFS diagnosis. Naess 2012 clinically evaluated 96 referred patients with persistent post-Giardia fatigue and found 58 (60%) met CDC CFS criteria. A gut-confined protozoan producing the same syndrome is strong evidence against any single-virus explanation.",
     "studies": [
      {
       "cite": "Litleskare 2018, Clin Gastroenterol Hepatol",
       "n": "1,252 exposed vs 2,504 controls",
       "design": "prospective matched cohort, 10 years",
       "result": "chronic fatigue 26% vs 11%, aOR 3.01; IBS aOR 4.74",
       "url": "https://doi.org/10.1016/j.cgh.2018.01.022",
       "verified": true
      },
      {
       "cite": "Naess 2012, BMC Gastroenterol",
       "n": "96 referred",
       "design": "clinical case series",
       "result": "58/96 (60%) met CDC CFS criteria; 57% reported a progressive course",
       "url": "https://doi.org/10.1186/1471-230X-12-13",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is persisting gut barrier/microbiome disturbance the bridge to systemic symptoms?",
      "How many of the 10-year 'chronic fatigue' cases would meet modern ME/CFS criteria with PEM?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-q-fever-qfs",
     "subdomain": "Post-infectious onset",
     "finding": "Q fever fatigue syndrome (QFS) follows roughly 20% of acute Q fever and persists more than 10 years after infection in many patients, almost all of whom report post-exertional malaise; doxycycline did not help.",
     "grade": "several",
     "plain": "After the large Dutch Q fever outbreak (2007-2010), many people developed a lasting ME-like illness. Antibiotics aimed at the bacterium did not improve it.",
     "detail": "Spronk 2023 surveyed 368 QFS patients 10 or more years after infection: median EQ-5D index 0.63, and 98.9% reported PEM complaints. Younger and middle-aged patients did worse. The Qure RCT (n=154) found that doxycycline was no better than placebo, and the initial CBT benefit had faded by 1-year follow-up (Raijmakers 2019). The failure of an antimicrobial against the original pathogen is consistent with a post-infectious host process rather than ongoing Coxiella infection. The ~20% figure comes from the Reukers 2019 introduction, a secondary source.",
     "studies": [
      {
       "cite": "Spronk 2023, Epidemiol Infect",
       "n": "368",
       "design": "cross-sectional, ≥10 years post-infection",
       "result": "median EQ-5D 0.63; PEM complaints 98.9%",
       "url": "https://doi.org/10.1017/S0950268823001401",
       "verified": true
      },
      {
       "cite": "Raijmakers 2019, J Psychosom Res (Qure follow-up)",
       "n": "154",
       "design": "RCT follow-up (CBT vs doxycycline vs placebo)",
       "result": "neither doxycycline nor CBT differed from placebo at 1 year post-treatment",
       "url": "https://doi.org/10.1016/j.jpsychores.2018.11.007",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do QFS cohorts show the same immune/metabolic signatures as EBV- or COVID-onset ME/CFS?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "sickness-behaviour"
     ],
     "test_available": "Coxiella serology documents the trigger; no test for QFS itself."
    },
    {
     "id": "infection-post-covid-mecfs-incidence",
     "subdomain": "Post-infectious onset",
     "finding": "In RECOVER-Adult, new ME/CFS (IOM criteria) developed at 2.66 per 100 person-years after SARS-CoV-2 infection vs 0.93 in uninfected participants (HR 4.93), and 4.5% of infected participants met criteria.",
     "grade": "replicated",
     "plain": "COVID-19 roughly multiplies the chance of developing new ME/CFS by four to five. About 1 in 20 infected adults in a large US study met the diagnostic criteria.",
     "detail": "Vernon 2025 (RECOVER-Adult, n=11,785 infected vs 1,439 uninfected) found 531/11,785 (4.5%) vs 9/1,439 (0.6%), and 88.7% of post-COVID ME/CFS cases also met RECOVER long COVID criteria. PEM (24%) was the most common ME/CFS symptom. The criteria were applied from self-report. A meta-analysis of 13 studies (1,973 long COVID patients) found 51% met ME/CFS criteria, but heterogeneity was high and the populations were selected clinic cohorts (Dehlia 2024). Registry signals of the same direction exist for H1N1 influenza (HR 2.04, Norway, Magnus 2015) and for COVID (HR ~1.6, Chen 2024 via the Solving ME/CFS re-analysis). Healthcare-seeking bias inflates some registry estimates (af Geijerstam 2026, via re-analysis), and the INSPIRE cohort found no excess after a COVID-positive vs COVID-negative test (Unger 2024, via re-analysis).",
     "studies": [
      {
       "cite": "Vernon 2025, J Gen Intern Med",
       "n": "11,785 infected vs 1,439 uninfected",
       "design": "prospective observational cohort",
       "result": "incidence 2.66 vs 0.93 per 100 PY; HR 4.93 (3.62-6.71)",
       "url": "https://doi.org/10.1007/s11606-024-09290-9",
       "verified": true
      },
      {
       "cite": "Dehlia 2024, J Infect",
       "n": "1,973 (13 studies)",
       "design": "meta-analysis",
       "result": "51% (42-60%) of long COVID patients met ME/CFS criteria",
       "url": "https://doi.org/10.1016/j.jinf.2024.106297",
       "verified": true
      },
      {
       "cite": "Magnus 2015, Vaccine",
       "n": "whole Norwegian population",
       "design": "registry cohort",
       "result": "CFS/ME HR 2.04 after pandemic influenza; 0.97 after adjuvanted vaccine",
       "url": "https://doi.org/10.1016/j.vaccine.2015.10.018",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Unger 2024 (INSPIRE), via Solving ME/CFS re-analysis",
       "n": "~4,500",
       "design": "prospective cohort, test-positive vs test-negative",
       "result": "no difference in ME/CFS-like illness between COVID+ and COVID- symptomatic participants",
       "url": "",
       "verified": false
      }
     ],
     "open_questions": [
      "How much of the registry excess is healthcare-seeking bias?",
      "Does vaccination or variant change the per-infection ME/CFS risk?",
      "Do post-COVID and post-EBV ME/CFS share biology?"
     ],
     "hypotheses": [
      "viral-persistence",
      "innate-hyperactivation",
      "autoimmunity"
     ],
     "test_available": "No test; diagnosis is clinical (IOM 2015 criteria)."
    },
    {
     "id": "infection-ebv-serology-not-different",
     "subdomain": "EBV",
     "finding": "In large well-characterised cohorts, standard EBV and other herpesvirus seroprevalence and overall IgG reactivity do not differ between ME/CFS patients and controls.",
     "grade": "several",
     "plain": "Ordinary EBV antibody tests look the same in people with ME/CFS as in healthy people, so a 'high EBV titre' on a routine panel does not diagnose anything.",
     "detail": "UK ME/CFS Biobank (Cliff 2019; 251 ME/CFS including 54 severe, 107 healthy, 46 MS): no seroprevalence differences for six herpesviruses, whereas EBV seroprevalence was higher in MS. Blomberg 2019 (Swedish Canada-criteria cohorts vs blood donors, multiplex assay) found no significant overall differences, only minor relative differences for some antigens. Loebel 2017 peptide array (92 CFS vs 50 HC, 3,054 EBV peptides) found profiles 'quite similar' to controls. The exception was raised IgG to an EBNA-6 repeat, which has homology to human proteins. A meta-analytic tally in the Solving ME/CFS re-analysis lists 10 positive vs 2 negative EBV-antibody studies, so smaller older studies were more positive than the larger recent ones, a pattern typical of publication bias.",
     "studies": [
      {
       "cite": "Cliff 2019, Front Immunol",
       "n": "251 vs 107 (+46 MS)",
       "design": "case-control biobank",
       "result": "no difference in seroprevalence for 6 herpesviruses",
       "url": "https://doi.org/10.3389/fimmu.2019.00796",
       "verified": true
      },
      {
       "cite": "Blomberg 2019, Front Immunol",
       "n": "several Swedish cohorts vs blood donors",
       "design": "case-control multiplex serology",
       "result": "no significant overall IgG differences to herpesviral antigens",
       "url": "https://doi.org/10.3389/fimmu.2019.01946",
       "verified": true
      },
      {
       "cite": "Loebel 2017, PLoS One",
       "n": "92 vs 50",
       "design": "peptide microarray case-control",
       "result": "similar profiles; higher IgG to EBNA-6 repeat peptides",
       "url": "https://doi.org/10.1371/journal.pone.0179124",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Shikova 2020, J Med Virol",
       "n": "58 vs 50",
       "design": "case-control serology + nested PCR",
       "result": "IgG/IgM no different, but plasma EBV DNA (active infection) 24.1% vs 4% (p=0.0027)",
       "url": "https://doi.org/10.1002/jmv.25744",
       "verified": true
      }
     ],
     "open_questions": [
      "Is there a signal restricted to infection-onset or recent-onset patients?",
      "Would quantitative titres timed to crashes differ when single snapshots don't?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "autoimmunity"
     ],
     "test_available": "EBV VCA IgM/IgG, EBNA IgG, EA-D IgG (clinical); uninformative as an ME/CFS test."
    },
    {
     "id": "infection-ebv-deficient-memory",
     "subdomain": "EBV",
     "finding": "Charité studies report a deficient EBV-specific memory B- and T-cell response in CFS (absent or low EBNA-1/VCA antibody-secreting memory B cells in up to 76% of patients, fewer polyfunctional EBNA-1-specific T cells), while CMV responses were normal.",
     "grade": "early",
     "plain": "Instead of fighting EBV too hard, some patients' immune memory for EBV seems weak, which could let the virus reactivate more easily.",
     "detail": "Loebel 2014 (PLoS One): EBNA-IgG low or absent in 10% of patients, and memory B-cell ELISpot showed diminished EBNA-1/VCA antibody-secreting cells in up to 76%. Ex vivo EBV-induced TNF-α/IFN-γ was lower, and triple-cytokine EBNA-1-specific CD4/CD8 cells were reduced, with no CMV difference. EBER-DNA but not BZLF-1 RNA was more frequent in blood cells, suggesting increased latent, not lytic, load. Sepúlveda 2022 re-analysed the 2017 array and found classification only when restricted to infection-onset patients (two candidate antigens, needing confirmation). The Solving ME/CFS single-cell re-analysis found a non-significant trend toward more EBV-specific TCRβ in severe patients' CD8 (0.88% vs 0.45%, p=0.06), which points the other way, to expansion rather than deficiency. Newer long COVID tetramer work (Ma 2026, Cell Rep Med) finds SARS-CoV-2-, CMV- and EBV-specific CD8 T cells skewed to terminally differentiated, exhausted, cytolytic states in long COVID.",
     "studies": [
      {
       "cite": "Loebel 2014, PLoS One",
       "n": "CFS vs HC (tens per group)",
       "design": "case-control cellular immunology",
       "result": "reduced EBV memory B/T responses; normal CMV responses",
       "url": "https://doi.org/10.1371/journal.pone.0085387",
       "verified": true
      },
      {
       "cite": "Sepúlveda 2022, Front Med",
       "n": "92 vs 50",
       "design": "re-analysis of peptide array",
       "result": "no discrimination overall; two antigens discriminated infection-onset patients",
       "url": "https://doi.org/10.3389/fmed.2022.921101",
       "verified": true
      },
      {
       "cite": "Ma 2026, Cell Rep Med",
       "n": "long COVID vs recovered (tetramer cohort)",
       "design": "case-control CD8 tetramer profiling",
       "result": "EBV/CMV/SARS-CoV-2-specific CD8 terminally differentiated, exhausted, cytolytic in LC",
       "url": "https://doi.org/10.1016/j.xcrm.2026.102946",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (TCR, PRJNA940635)",
       "n": "~38 per group per cell type",
       "design": "re-analysis of public TCR repertoire",
       "result": "EBV-matched CD8 clonotypes 0.88% vs 0.45% in severe (p=0.06, q>0.6)",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication of memory B-cell ELISpot deficits outside Charité?",
      "HLA-typed tetramer studies against lytic antigens (BZLF1, BMLF1) in ME/CFS?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "immune-exhaustion"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-ebv-dna-load-blood",
     "subdomain": "EBV",
     "finding": "Detectable cell-free EBV DNA in plasma has been reported more often in ME/CFS (24% vs 4% in one Bulgarian study), but EBV DNA in blood cells is equally common in patients and controls.",
     "grade": "early",
     "plain": "Almost everyone carries EBV in their blood cells. Some small studies find free-floating viral DNA, a sign of activity, more often in patients, but this has not been firmly confirmed.",
     "detail": "Shikova 2020 (58 vs 50): PBMC EBV DNA 81% vs 84% (no difference), plasma EBV DNA 24.1% vs 4% (p=0.0027). Loebel 2014 found more EBER-DNA but not BZLF-1 RNA in blood cells. In long COVID, EBV viremia at acute infection predicted later PASC (Su 2022, n=309), but EBV viremia at a median of 4 months did not associate with long COVID in Peluso 2023 (n=280), while serologic markers (EA-D IgG) did. Persistently detectable EBV DNA months after IM warrants exclusion of chronic active EBV, which is rare but treatable.",
     "studies": [
      {
       "cite": "Shikova 2020, J Med Virol",
       "n": "58 vs 50",
       "design": "case-control nested PCR",
       "result": "plasma EBV DNA 24.1% vs 4%; PBMC DNA no difference",
       "url": "https://doi.org/10.1002/jmv.25744",
       "verified": true
      },
      {
       "cite": "Su 2022, Cell",
       "n": "309",
       "design": "prospective multi-omic cohort (COVID)",
       "result": "EBV viremia at diagnosis one of four PASC-anticipating factors",
       "url": "https://doi.org/10.1016/j.cell.2022.01.014",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Peluso 2023, J Clin Invest",
       "n": "280",
       "design": "post-COVID cohort",
       "result": "LC fatigue associated with EA-D IgG (OR 2.12) and high EBNA IgG, but not with ongoing EBV viremia",
       "url": "https://doi.org/10.1172/JCI163669",
       "verified": true
      }
     ],
     "open_questions": [
      "Does plasma EBV DNA rise during PEM crashes?",
      "Large-cohort quantitative plasma EBV PCR in ME/CFS has not been done."
     ],
     "hypotheses": [
      "herpes-reactivation",
      "viral-persistence"
     ],
     "test_available": "EBV DNA PCR (whole blood/plasma) is clinically available; mainly to exclude chronic active EBV."
    },
    {
     "id": "infection-ebv-genetic-load-mr",
     "subdomain": "EBV",
     "finding": "Genetic instruments for higher EBV DNA load in blood (Kamitaki 2026) are associated with ME/CFS risk in the Million Veteran Program (OR 1.59, p=0.012) but not significantly in DecodeME (OR 1.13, p=0.25).",
     "grade": "early",
     "plain": "People whose genes make them carry more EBV appear somewhat more likely to get ME/CFS in one large dataset but not clearly in another. This is a weak hint that EBV control matters.",
     "detail": "A Solving ME/CFS Mendelian randomisation re-analysis used EBV load instruments from Kamitaki 2026 (n≈655k). It gave a combined OR of about 1.23 (p≈0.02), and HLA instruments at r²<0.01 held (p=0.012, 0.043). HLA patterns that raise antibodies to the lytic EBV proteins ZEBRA and EA-D also align with higher risk, but negative-control serologies (VZV, HHV-6B) align similarly, so the HLA signal is not EBV-specific. Genetic correlation of ME/CFS with infectious mononucleosis was 0.11 ± 0.14, and with EBV load 0.05 ± 0.04, both non-significant. Reverse MR was underpowered. Not yet replicated by an independent group.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (genetics_mr), using Kamitaki 2026 instruments, DecodeME and MVP",
       "n": "DecodeME ~15k cases; MVP",
       "design": "two-sample Mendelian randomisation",
       "result": "EBV load OR 1.13 (DecodeME, ns), 1.59 (MVP, p=0.012)",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis, negative-control serologies",
       "n": "same",
       "design": "MR specificity check",
       "result": "VZV and HHV-6B HLA instruments also align, undermining EBV specificity",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does the signal hold with full Kamitaki summary statistics and the larger DecodeME release?",
      "Is it the MHC driving everything?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "genetic-neuronal",
      "autoimmunity"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-herpes-dutpase-antibodies",
     "subdomain": "EBV",
     "finding": "Antibodies against herpesvirus dUTPases (EBV, HHV-6, VZV), proposed as a marker of lytic or abortive-lytic reactivation, are higher in ME/CFS in studies from one lab, including a 2026 longitudinal study (873 serum samples, 40 patients vs 16 controls) where levels tracked fatigue severity.",
     "grade": "early",
     "plain": "One research group measures antibodies to a viral enzyme made when herpesviruses partly wake up. They repeatedly find more of these antibodies in ME/CFS, but other labs have not yet checked.",
     "detail": "Halpin 2017: 30.9-52.7% of ME/CFS patients had antibodies to multiple herpesviral and/or human dUTPases vs 17.2% of controls, and anti-EBV dUTPase was higher in ME/CFS. Palomo 2026 (J Med Virol): longitudinal sampling, with EBV/HHV-6/VZV dUTPase IgG significantly higher (p<0.001). 72.5% of patients co-expressed antibodies to multiple herpesvirus and HERV-K dUTPases vs 31% of controls, and EBV and HHV-6 dUTPase IgG correlated with fatigue. The controls were few (n=16). In healthy cohorts positivity ranges from 40-84% and rises with age (Alharshawi 2022), so baseline rates are high. The assays are not commercial, and all data come from the Ariza lab.",
     "studies": [
      {
       "cite": "Halpin 2017, J Med Virol",
       "n": "ME/CFS and GWI cohorts vs controls",
       "design": "case-control serology",
       "result": "multiple dUTPase antibodies 31-53% vs 17%; anti-EBV dUTPase higher",
       "url": "https://doi.org/10.1002/jmv.24810",
       "verified": true
      },
      {
       "cite": "Palomo 2026, J Med Virol",
       "n": "40 vs 16 (873 + 378 serial samples)",
       "design": "longitudinal case-control",
       "result": "EBV/HHV-6/VZV dUTPase IgG higher (p<0.001); correlated with fatigue",
       "url": "https://doi.org/10.1002/jmv.70769",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Alharshawi 2022, J Med Virol",
       "n": "255 healthy",
       "design": "cross-sectional healthy cohorts",
       "result": "40-84% of asymptomatic people have anti-dUTPase antibodies; higher with age",
       "url": "https://doi.org/10.1002/jmv.27728",
       "verified": true
      }
     ],
     "open_questions": [
      "Independent replication with blinded samples?",
      "Does dUTPase protein itself (an immune modulator via TLR2) circulate during crashes?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "innate-hyperactivation"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-ebv-reactivation-long-covid",
     "subdomain": "EBV",
     "finding": "Serologic evidence of recent EBV reactivation (EA-D IgG) is associated with long COVID fatigue (OR 2.12) in a 280-person cohort, consistent with an earlier small report.",
     "grade": "several",
     "plain": "People with long COVID more often have antibody signs that EBV recently reactivated, which may be one route by which COVID leads to lasting illness.",
     "detail": "Peluso 2023 (JCI, n=280): LC fatigue and neurocognitive symptoms were associated with EA-D IgG positivity or high EBNA IgG, independent of covariates, but not with EBV viremia. Prior CMV infection was associated with lower LC risk. Gold 2021 (n=68) reported 66.7% of LC vs 10% of controls positive for EA-D IgG or VCA IgM, a small study with a convenience control group. Cervia-Hasler 2024 found classical complement activation linked to higher herpesvirus antibody titres in active LC. Serology cannot distinguish a cause of LC from a consequence of a general immune disturbance.",
     "studies": [
      {
       "cite": "Peluso 2023, J Clin Invest",
       "n": "280",
       "design": "post-COVID cohort, adjusted regression",
       "result": "EA-D IgG associated with fatigue (OR 2.12); no association with viremia; CMV seropositivity protective",
       "url": "https://doi.org/10.1172/JCI163669",
       "verified": true
      },
      {
       "cite": "Gold 2021, Pathogens",
       "n": "30 LC vs 20 controls (+18)",
       "design": "cross-sectional",
       "result": "EBV reactivation serology 66.7% vs 10%",
       "url": "https://doi.org/10.3390/pathogens10060763",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do people with ME/CFS (non-COVID) show the same EA-D pattern at onset?",
      "Is reactivation sustained or a one-off after acute COVID?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "complement-thromboinflammation"
     ],
     "test_available": "EBV EA-D IgG is clinically available; interpretation is non-specific."
    },
    {
     "id": "infection-herpes-complement-link",
     "subdomain": "Herpes-complement",
     "finding": "In active long COVID, classical complement pathway activation tracked raised antibody titres against several herpesviruses, alongside markers of haemolysis, platelet activation and monocyte-platelet aggregates.",
     "grade": "early",
     "plain": "In long COVID, the blood's complement defence system stays switched on, and this goes along with higher antibodies to herpesviruses. One explanation is that immune complexes against reactivated viruses keep complement firing.",
     "detail": "Cervia-Hasler 2024 (Science) ran proteomics on >6,500 proteins in 268 longitudinal samples from Zurich/Basel cohorts followed to 12 months. Active LC showed terminal complement dysregulation and ongoing alternative and classical pathway activation, and the classical arm was associated with increased anti-herpesvirus (e.g., EBV, CMV) titres. Machine learning put complement and thromboinflammation proteins at the top. The cohort was mostly post-hospital-era COVID. No ME/CFS (non-COVID) replication yet. Complement activation fits several mechanisms, and herpes reactivation is only one of them.",
     "studies": [
      {
       "cite": "Cervia-Hasler 2024, Science",
       "n": "268 samples (~113 COVID patients + controls)",
       "design": "longitudinal proteomics cohort",
       "result": "complement dysregulation in active LC; classical activation associated with herpesvirus antibody titres",
       "url": "https://doi.org/10.1126/science.adg7942",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does the herpes-complement association appear in classic ME/CFS?",
      "Would antivirals reduce complement activation in LC?"
     ],
     "hypotheses": [
      "complement-thromboinflammation",
      "herpes-reactivation",
      "microclots"
     ],
     "test_available": "Complement panels (C3, C4, CH50, sC5b-9) are clinically available but not validated for LC/ME/CFS."
    },
    {
     "id": "infection-hhv6-tissue-mirna",
     "subdomain": "HHV-6",
     "finding": "HHV-6 microRNA (miR-aU14) was detected by in situ hybridisation in brain and spinal cord tissue from 3 ME/CFS post-mortem donors but not in 24 controls (Kasimir 2022).",
     "grade": "early",
     "plain": "A small autopsy study found signs of active HHV-6 in the brain and spinal cord of three people with ME/CFS and none in controls. This needs repeating in many more brains.",
     "detail": "Kasimir 2022 (Front Mol Biosci) used FISH against an HHV-6 miRNA in frontal lobe, basal ganglia and spinal cord from 3 ME/CFS donors, 3 disease controls and 21 Cambridge Brain Bank controls. The authors also report evidence of EBV activity in tissue. Limitations: n=3 cases, no blinding is described in the abstract, miRNA FISH specificity in post-mortem tissue, and no quantitative PCR or sequencing confirmation. The authors argue that blood negativity in many studies reflects localised tissue reactivation. In vitro, HHV-6 reactivation from integrated genomes fragments mitochondria and induces an antiviral state, and patient serum transferred a similar effect (Schreiner 2020, Immunohorizons).",
     "studies": [
      {
       "cite": "Kasimir 2022, Front Mol Biosci",
       "n": "3 ME/CFS vs 24 controls",
       "design": "post-mortem tissue FISH",
       "result": "HHV-6 miR-aU14 in brain/spinal cord only in ME/CFS",
       "url": "https://doi.org/10.3389/fmolb.2022.1044964",
       "verified": true
      },
      {
       "cite": "Schreiner 2020, Immunohorizons",
       "n": "cell lines + 10 patient sera",
       "design": "in vitro proteomics / serum transfer",
       "result": "HHV-6 reactivation fragments mitochondria, inhibits PDH and FAO; induces antiviral state",
       "url": "https://doi.org/10.4049/immunohorizons.2000006",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Cliff 2019, Front Immunol",
       "n": "251 vs 107",
       "design": "serology",
       "result": "no HHV-6 seroprevalence difference",
       "url": "https://doi.org/10.3389/fimmu.2019.00796",
       "verified": true
      },
      {
       "cite": "Shikova 2020, J Med Virol",
       "n": "58 vs 50",
       "design": "PCR",
       "result": "HHV-6 DNA in PBMC 82.8% vs 82%; plasma 1.7% vs 0%",
       "url": "https://doi.org/10.1002/jmv.25744",
       "verified": true
      }
     ],
     "open_questions": [
      "Blinded multi-site replication in brain banks (e.g., with ME/CFS tissue now being collected)?",
      "Is reactivated HHV-6 cause or consequence of illness?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "neuroinflammation",
      "mitochondrial-energy",
      "cell-danger-response"
     ],
     "test_available": "research only (blood HHV-6 PCR is available but generally uninformative)"
    },
    {
     "id": "infection-cihhv6",
     "subdomain": "HHV-6",
     "finding": "Inherited chromosomally integrated HHV-6 (iciHHV-6, ~1% of people) has been reported at roughly twice the population rate in a neurological/fatigue clinic cohort, but this has not been confirmed in a large ME/CFS sample.",
     "grade": "early",
     "plain": "About 1 in 100 people are born with HHV-6 built into one of their chromosomes. One small study suggested this is more common in ME/CFS, and the large UK DecodeME study is checking.",
     "detail": "A 2013 University of South Florida study reported iciHHV-6 in >2% of adults with CFS and neurological symptoms vs ~0.8% population estimates. It was a small, selected clinic cohort, and the information here comes from secondary reports only. UK prevalence is about 1.4%, rising to ~3% in parts of Scotland and Ireland, so ancestry confounding matters. DecodeME has an approved sub-study to measure iciHHV-6 in ~4,500 participants, comparing infection-onset vs other onsets. No published result had been found as of this review. iciHHV-6 can produce false 'high HHV-6 DNA' results on commercial PCR panels, a common source of misinterpretation.",
     "studies": [
      {
       "cite": "USF 2013 report (secondary source: press/HHV-6 Foundation)",
       "n": "small clinic cohort",
       "design": "cross-sectional",
       "result": ">2% iciHHV-6 vs ~0.8% population",
       "url": "https://hhv-6foundation.org/cihhv-6/cfs-patients-with-cihhv-6-may-benefit-from-antiviral-treatment",
       "verified": false
      },
      {
       "cite": "DecodeME iciHHV-6 sub-study (protocol)",
       "n": "~4,500 planned",
       "design": "genetic cohort sub-study",
       "result": "pending",
       "url": "https://www.decodeme.org.uk/?p=2590",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is iciHHV-6 enriched in DecodeME, and especially in infection-onset cases?",
      "Does integrated-genome reactivation occur in patients' tissues?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "genetic-neuronal"
     ],
     "test_available": "Hair-follicle or nail HHV-6 PCR / ddPCR ratio of viral to cellular genomes can confirm iciHHV-6 (specialist labs)."
    },
    {
     "id": "infection-enterovirus-stomach-chia",
     "subdomain": "Enterovirus",
     "finding": "Enteroviral VP1 protein was found in stomach biopsies from 82% of 165 CFS patients vs 20% of controls, with EV RNA in 37% vs 5%, but this has not been independently replicated with blinded methods.",
     "grade": "contested",
     "plain": "One clinician-researcher found enterovirus protein in the stomach lining of most patients he biopsied. Other labs have not confirmed it, and the antibody stain used may cross-react.",
     "detail": "Chia 2008 (J Clin Pathol): 135/165 consecutive CFS patients' antrum biopsies stained positive for VP1 in parietal cells vs 7/34 controls (p≤0.001), and EV RNA was found in 9/24 vs 1/21 paraffin samples. Paired samples taken years apart stayed positive. The patients were the author's own referral population. The controls were biopsies taken for other gastric conditions, and the 5D8/1 VP1 antibody has known cross-reactivity concerns. Earlier muscle work found EV RNA in 41/158 postviral fatigue vs 2/152 controls (Bowles 1993), but later muscle studies were inconsistent. O'Neal & Hanson 2021 concluded the methods used were inadequate either to confirm or to rule out persistent EV infection, and a Cornell EV-targeted RNA-seq thesis (2022) detected no EVs in patients or controls.",
     "studies": [
      {
       "cite": "Chia 2008, J Clin Pathol",
       "n": "165 vs 34",
       "design": "case series with tissue controls",
       "result": "VP1 82% vs 20%; EV RNA 37% vs 5%",
       "url": "https://doi.org/10.1136/jcp.2007.050054",
       "verified": true
      },
      {
       "cite": "Bowles 1993, J Med",
       "n": "158 PFS vs 152 controls",
       "design": "muscle biopsy hybridisation",
       "result": "EV RNA 41/158 vs 2/152",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Bowles+1993+enterovirus+muscle+postviral+fatigue",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "O'Neal & Hanson 2021, Front Med (critical review)",
       "n": "review",
       "design": "critical review",
       "result": "prior methods inadequate; evidence neither confirms nor excludes persistent EV",
       "url": "https://doi.org/10.3389/fmed.2021.688486",
       "verified": true
      },
      {
       "cite": "Cornell thesis 2022 (Hanson lab)",
       "n": "small",
       "design": "EV-targeted RNA-seq",
       "result": "no EV detected in ME/CFS or controls",
       "url": "https://ecommons.cornell.edu/entities/publication/b33e3546-5489-437f-bf76-34c38575b15b",
       "verified": false
      }
     ],
     "open_questions": [
      "Blinded, multi-lab testing of gastric/intestinal tissue with sequencing-based detection of non-cytolytic (5' terminally deleted) EV RNA?",
      "Do historical ME outbreaks (e.g., Royal Free 1955) fit EV epidemiology?"
     ],
     "hypotheses": [
      "viral-persistence",
      "gut-dysbiosis"
     ],
     "test_available": "research only (stomach VP1 staining available from one private practice; not validated)"
    },
    {
     "id": "infection-sarscov2-plasma-antigen",
     "subdomain": "SARS-CoV-2 persistence",
     "finding": "SARS-CoV-2 spike, S1 or nucleocapsid antigen is detectable in plasma more often after COVID than in pre-pandemic samples (excess +11% at 3-6 months, +5.4% at 10-14 months), with more antigenemia after more severe acute illness.",
     "grade": "several",
     "plain": "In about 1 in 10 people, bits of the virus can still be found in the blood months after COVID, which suggests a hidden reservoir. Whether this causes long COVID is less certain.",
     "detail": "Peluso 2024 (Lancet Infect Dis; 171 post-COVID vs 250 pre-pandemic controls, Simoa) found 2% assay positivity in pre-pandemic controls. Antigenemia was associated with hospitalisation and worse acute illness. The association with LC symptoms was modest and not consistent across all symptom groups (detail from secondary reports). Swank 2022/23 (n=63) reported spike in a majority of PASC patients up to 12 months, a small sample later tempered by larger work. In PAX LC, circulating S1/spike levels did not change after 15 days of nirmatrelvir (Bhattacharjee 2026 preprint).",
     "studies": [
      {
       "cite": "Peluso 2024, Lancet Infect Dis",
       "n": "171 vs 250 pre-pandemic",
       "design": "cohort with true-negative comparator",
       "result": "excess antigen prevalence +11%, +8.7%, +5.4% at 3-6, 6-10, 10-14 months",
       "url": "https://doi.org/10.1016/S1473-3099(24)00211-1",
       "verified": true
      },
      {
       "cite": "Swank 2022, medRxiv (later published in Clin Infect Dis)",
       "n": "63",
       "design": "case-control plasma antigen",
       "result": "spike detected in most PASC patients up to 12 months",
       "url": "https://doi.org/10.1101/2022.06.14.22276401",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Bhattacharjee 2026, medRxiv (PAX LC exploratory)",
       "n": "82",
       "design": "RCT exploratory biomarker analysis",
       "result": "circulating S1/spike unchanged after nirmatrelvir",
       "url": "https://doi.org/10.64898/2026.02.24.26347001",
       "verified": true
      }
     ],
     "open_questions": [
      "Is antigenemia from replicating virus, translated persistent RNA, or slow-clearing protein?",
      "Does antigen positivity identify an antiviral-responsive LC subgroup?"
     ],
     "hypotheses": [
      "viral-persistence",
      "complement-thromboinflammation",
      "microclots"
     ],
     "test_available": "research only (Simoa spike/nucleocapsid assays)"
    },
    {
     "id": "infection-sarscov2-tissue-gut",
     "subdomain": "SARS-CoV-2 persistence",
     "finding": "SARS-CoV-2 RNA and protein persist in gut and other tissues for months to two years in a minority of people, and in a Beijing tissue study, viral RNA in tissue was associated with long COVID symptoms (OR 5.17).",
     "grade": "several",
     "plain": "Viral genetic material can hide in the gut and other organs long after the nose test is negative. Several groups have now seen this, and people with long COVID seem to have it more often.",
     "detail": "Zollner 2022 (n=46 IBD patients, ~7 months): gut mucosal RNA in 32/46 and nucleocapsid in 24/46, associated with post-acute symptoms. Natarajan 2022 (n=113): fecal RNA in 12.7% at 4 months and 3.8% at 7 months, linked to GI symptoms. Zuo 2024 (Lancet ID; 317 samples from 225 patients): RNA in 30%/27%/11% of solid tissues at 1/2/4 months, with tissue RNA associated with LC symptoms (OR 5.17). Peluso 2024 (Sci Transl Med; n=24 PET) found T-cell activation (F-AraG) in gut wall and spinal cord linked to LC, and RNA in rectosigmoid tissue up to 676 days in some participants. These are mostly convenience tissue samples, and some people without LC also carry RNA.",
     "studies": [
      {
       "cite": "Zollner 2022, Gastroenterology",
       "n": "46",
       "design": "endoscopy cohort (IBD)",
       "result": "gut RNA 32/46, nucleocapsid 24/46 at ~7 months",
       "url": "https://doi.org/10.1053/j.gastro.2022.04.037",
       "verified": true
      },
      {
       "cite": "Natarajan 2022, Med",
       "n": "113",
       "design": "longitudinal stool cohort",
       "result": "fecal RNA 12.7% at 4 months, 3.8% at 7 months; linked to GI symptoms",
       "url": "https://doi.org/10.1016/j.medj.2022.04.001",
       "verified": true
      },
      {
       "cite": "Zuo 2024, Lancet Infect Dis",
       "n": "225 patients, 317 samples",
       "design": "cross-sectional tissue cohort",
       "result": "tissue RNA 30%/27%/11%; associated with LC (OR 5.17)",
       "url": "https://doi.org/10.1016/S1473-3099(24)00171-3",
       "verified": true
      },
      {
       "cite": "Peluso 2024, Sci Transl Med",
       "n": "24 (PET) + 5 biopsies",
       "design": "imaging cohort + tissue ISH",
       "result": "T-cell activation in gut/spinal cord with LC; gut RNA up to ~2 years",
       "url": "https://doi.org/10.1126/scitranslmed.adk3295",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do tissue reservoirs exist in non-COVID ME/CFS for other viruses?",
      "Why do antivirals not help if reservoirs exist (wrong drug, too short, too late, or reservoir not causal)?"
     ],
     "hypotheses": [
      "viral-persistence",
      "gut-dysbiosis",
      "neuroinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-paxlovid-lc-null",
     "subdomain": "Antiviral trials",
     "finding": "Three randomised placebo-controlled trials of 15-25 days of nirmatrelvir-ritonavir in established long COVID (STOP-PASC n=155, PAX LC n=100, RECOVER-VITAL n=959; about 1,214 people in total) found no benefit on primary or secondary outcomes.",
     "grade": "replicated",
     "plain": "Paxlovid, the COVID antiviral, has been tested several times in people who already have long COVID, and it did not help.",
     "detail": "STOP-PASC (Geng 2024, JAMA IM): 15 days, 2:1 randomisation, no difference in pooled severity of 6 symptoms at 10 weeks; mostly vaccinated participants, mean 17.5 months after infection. PAX LC (Sawano 2025, Lancet ID): adjusted PROMIS-29 physical difference -0.55 (p=0.54). RECOVER-VITAL (Baden 2026, Lancet ID; 69 sites): 15- or 25-day regimens, no benefit in cognitive, autonomic or exercise phenotypes. All were safe apart from dysgeusia. Caveats: participants were not selected for antigenemia or reservoir evidence, illness was long-standing, and the drug's tissue penetration is uncertain. Ensitrelvir was also null per a secondary summary.",
     "studies": [
      {
       "cite": "Geng 2024, JAMA Intern Med (STOP-PASC)",
       "n": "155",
       "design": "RCT",
       "result": "no difference in core symptom severity at 10 weeks",
       "url": "https://doi.org/10.1001/jamainternmed.2024.2007",
       "verified": true
      },
      {
       "cite": "Sawano 2025, Lancet Infect Dis (PAX LC)",
       "n": "100",
       "design": "decentralised RCT",
       "result": "PROMIS-29 PHSS difference -0.55 (95% CI -2.32 to 1.21)",
       "url": "https://doi.org/10.1016/S1473-3099(25)00073-8",
       "verified": true
      },
      {
       "cite": "Baden 2026, Lancet Infect Dis (RECOVER-VITAL)",
       "n": "959",
       "design": "RCT, 3 arms (15d, 25d, placebo)",
       "result": "no benefit in any phenotype",
       "url": "https://doi.org/10.1016/S1473-3099(26)00406-8",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would trials enriched for antigen-positive participants, or using longer/combination antivirals or monoclonal antibodies, differ?"
     ],
     "hypotheses": [
      "viral-persistence"
     ],
     "test_available": "n/a"
    },
    {
     "id": "infection-acute-antiviral-prevention",
     "subdomain": "Antiviral trials",
     "finding": "Treating acute COVID-19 with nirmatrelvir may reduce later long COVID: a small terminated Norwegian RCT found 26% vs 43% at 3 months (RR 0.60), echoing observational VA data, but larger prospective evidence is lacking.",
     "grade": "early",
     "plain": "Taking the antiviral early, while COVID is still active, might lower the chance of developing long COVID, but the only trial so far was small and stopped early.",
     "detail": "PANORAMIC Norway (Oppegaard 2026, Lancet ID): enrolled 144 of a planned 2,000 before stopping for poor recruitment, and found long COVID in 17/66 vs 33/77 (RR 0.60, 95% CI 0.37-0.98, p=0.039). An unblinding effect via dysgeusia (86% vs 18%) is possible because the outcomes were self-reported. VA cohort (Xie 2023, JAMA IM; 35,717 treated vs 246,076) reported lower risk, while Fung 2023 (older Medicare patients) found no reduction. Metformin during acute COVID (COVID-OUT) also reduced later LC, per the Solving ME/CFS treatments summary. Together this suggests the early acute phase may be a window for intervention, which fits the Dubbo finding that acute severity predicts later illness.",
     "studies": [
      {
       "cite": "Oppegaard 2026, Lancet Infect Dis (PANORAMIC Norway)",
       "n": "144",
       "design": "RCT (terminated early)",
       "result": "LC 26% vs 43%, RR 0.60",
       "url": "https://doi.org/10.1016/S1473-3099(26)00244-6",
       "verified": true
      },
      {
       "cite": "Xie 2023, JAMA Intern Med",
       "n": "35,717 vs 246,076",
       "design": "observational VA cohort",
       "result": "nirmatrelvir associated with reduced PCC risk",
       "url": "https://doi.org/10.1001/jamainternmed.2023.0743",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Fung 2023, JAMA Intern Med",
       "n": "Medicare older adults",
       "design": "observational cohort",
       "result": "no reduction in PCC with nirmatrelvir or molnupiravir",
       "url": "https://doi.org/10.1001/jamainternmed.2023.5099",
       "verified": false
      }
     ],
     "open_questions": [
      "Would early antivirals for EBV (e.g., during IM) prevent post-IM ME/CFS? No trial exists."
     ],
     "hypotheses": [
      "viral-persistence",
      "innate-hyperactivation"
     ],
     "test_available": "n/a"
    },
    {
     "id": "infection-valganciclovir-montoya",
     "subdomain": "Antiviral trials",
     "finding": "In a 30-patient RCT of 6 months of valganciclovir in CFS patients selected for high HHV-6 and EBV IgG, the primary MFI-20 outcome was not significant, though some secondary fatigue and cognitive trajectories favoured the drug.",
     "grade": "early",
     "plain": "A strong antiviral helped some tiredness and thinking measures in a small trial, but missed its main goal. It has never been repeated, and it can lower white blood cell counts.",
     "detail": "Montoya 2013 (J Med Virol): 2:1 randomisation. The MFI-20 difference at 9 months was non-significant. Trajectories for MFI-20 mental fatigue (p=0.039), FSS (p=0.006) and cognition (p=0.025) were significant, and responder status was 7.4x more likely (p=0.029). Monocytes fell and neutrophils rose. These came from multiple secondary endpoints with no multiplicity correction. Preceding uncontrolled data: Kogelnik 2006 (n=12, 9 responders) and Watt 2012 chart review (n=61, 52% responders, baseline titres did not predict response). Lerner's valacyclovir 'EBV subset' series had small controlled phases and are largely uncontrolled. Antibody titre selection is a weak marker of active infection.",
     "studies": [
      {
       "cite": "Montoya 2013, J Med Virol",
       "n": "30 (20 vs 10)",
       "design": "double-blind RCT",
       "result": "primary MFI-20 ns; secondary FSS, mental fatigue, cognition significant",
       "url": "https://doi.org/10.1002/jmv.23713",
       "verified": true
      },
      {
       "cite": "Watt 2012, J Med Virol",
       "n": "61",
       "design": "retrospective uncontrolled chart review",
       "result": "52% responders; titres did not predict response",
       "url": "https://doi.org/10.1002/jmv.23411",
       "verified": true
      },
      {
       "cite": "Lerner 2007, In Vivo",
       "n": "small",
       "design": "small RCT phase + 36-month open follow-up",
       "result": "Energy Index improved more on valacyclovir",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Lerner+2007+valacyclovir+Epstein-Barr+subset+chronic+fatigue",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "No adequately powered replication in 13 years",
      "Would selection on lytic markers (plasma DNA, dUTPase, EA-D) rather than IgG identify responders?"
     ],
     "hypotheses": [
      "herpes-reactivation"
     ],
     "test_available": "n/a (off-label; requires blood count monitoring)"
    },
    {
     "id": "infection-herv-expression",
     "subdomain": "Endogenous retroviruses",
     "finding": "Human endogenous retrovirus (HERV) expression in blood cells differs between ME/CFS, fibromyalgia and controls in small studies (HERV-K raised in moderate ME/CFS; HERV microarray profiles clustered n=43 participants perfectly by diagnosis).",
     "grade": "early",
     "plain": "Old viral DNA built into our genome can switch back on. Small studies hint that its activity pattern differs in ME/CFS, but nothing is confirmed.",
     "detail": "Rodrigues 2019: qPCR found HERV-K overexpressed only in moderately affected patients, with no HERV-W difference. Giménez-Orenga 2025 (eLife; HERV-V3 microarrays, >350k HERV elements, PBMC, n=43 women): hierarchical clustering separated ME/CFS, FM, co-diagnosed and controls 'perfectly'. That sample is very small with very many features, so overfitting is likely without an independent validation set. HERV-K dUTPase antibodies co-occur with herpesvirus dUTPase antibodies in the Ariza lab data (Palomo 2026). Herpesviruses such as EBV can transactivate HERVs, which links this to the herpes hypothesis.",
     "studies": [
      {
       "cite": "Rodrigues 2019, Auto Immun Highlights",
       "n": "small (moderate + severe vs controls)",
       "design": "case-control qPCR",
       "result": "HERV-K up in moderate ME/CFS; HERV-W no difference",
       "url": "https://doi.org/10.1186/s13317-019-0122-8",
       "verified": true
      },
      {
       "cite": "Giménez-Orenga 2025, eLife",
       "n": "43",
       "design": "case-control microarray",
       "result": "HERV profiles clustered ME/CFS, FM, both and HC separately",
       "url": "https://doi.org/10.7554/eLife.104441",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication in an independent, larger, blinded cohort",
      "Are HERV proteins (Env) present in tissues or serum?"
     ],
     "hypotheses": [
      "herv-activation",
      "herpes-reactivation",
      "autoimmunity"
     ],
     "test_available": "research only"
    },
    {
     "id": "infection-temelimab-null",
     "subdomain": "Endogenous retroviruses",
     "finding": "Temelimab, an antibody against HERV-W envelope protein, did not reduce fatigue versus placebo in a 203-person RCT of HERV-W-positive post-COVID patients.",
     "grade": "refuted",
     "plain": "A drug designed to block an endogenous retrovirus protein was tested in long COVID patients who had that protein, and it worked no better than placebo.",
     "detail": "Nehme 2026 (Brain Behav Immun; GeNeuro-funded, NCT05497089): 24 weeks of monthly IV 54 mg/kg in participants selected for HERV-W ENV positivity. The PROMIS fatigue change was -3.2 vs -3.8 points, with no secondary benefit. Note that temelimab targets HERV-W, not HERV-K, so it does not test the HERV-K findings in ME/CFS. No temelimab trial in non-COVID ME/CFS exists.",
     "studies": [
      {
       "cite": "Nehme 2026, Brain Behav Immun",
       "n": "203",
       "design": "double-blind RCT",
       "result": "no difference in fatigue at 24 weeks",
       "url": "https://doi.org/10.1016/j.bbi.2026.106892",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is HERV-K (rather than HERV-W) a better target in ME/CFS?"
     ],
     "hypotheses": [
      "herv-activation"
     ],
     "test_available": "n/a"
    },
    {
     "id": "infection-xmrv-cautionary",
     "subdomain": "Retroviruses (XMRV)",
     "finding": "The 2009 report of the mouse retrovirus XMRV in 67% of CFS patients was a laboratory contamination artefact: blinded multi-lab and original-investigator studies found no XMRV/pMLV, and the Science paper was retracted.",
     "grade": "refuted",
     "plain": "In 2009 a mouse-derived virus was reported in most CFS patients. Careful blinded testing, including by the original scientists, showed it was contamination, and the paper was withdrawn.",
     "detail": "Lombardi 2009 (Science) was partially retracted in 2011 and fully retracted later that year. Simmons 2011 (Blood XMRV Scientific Research Working Group): coded replicate samples sent to 9 labs, and reactivity was similar in CFS and controls. Alter 2012 (mBio; Lipkin-led, 147 CFS vs 146 controls, including the original investigators' labs) found no XMRV or pMLV. Lessons for this field: sensitive PCR contamination (mouse DNA in reagents and cell lines), unblinded testing, and premature clinical use (antiretrovirals were taken by some patients). The episode also set back trust and funding for virus research in ME/CFS.",
     "studies": [
      {
       "cite": "Simmons 2011, Science",
       "n": "15 positive vs 15 negative (coded replicates)",
       "design": "blinded multi-laboratory",
       "result": "assays did not reproducibly detect XMRV/MLV; CFS = controls",
       "url": "https://doi.org/10.1126/science.1213841",
       "verified": true
      },
      {
       "cite": "Alter 2012, mBio",
       "n": "147 vs 146",
       "design": "blinded multicenter",
       "result": "no XMRV or pMLV in any group",
       "url": "https://doi.org/10.1128/mBio.00266-12",
       "verified": true
      },
      {
       "cite": "Silverman 2011, Science (partial retraction)",
       "n": "-",
       "design": "retraction notice",
       "result": "contamination acknowledged in part of the data",
       "url": "https://doi.org/10.1126/science.1212182",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [],
     "hypotheses": [
      "viral-persistence"
     ],
     "test_available": "n/a (no valid XMRV test; commercial tests sold in 2010-11 were not meaningful)"
    },
    {
     "id": "infection-viral-vs-bacterial-registry",
     "subdomain": "Post-infectious onset",
     "finding": "In Finnish registry data, ME/CFS (G93.3) is enriched 3-6-fold for prior viral infections (HSV, EBV, influenza, COVID, viral CNS infection) but not for bacterial pneumonia or erysipelas.",
     "grade": "early",
     "plain": "In national health records, people with ME/CFS more often had earlier viral illnesses, but not more bacterial ones, which suggests viruses play a particular role.",
     "detail": "Solving ME/CFS re-analysis of FinnGen R12 and FinRegistry (Risteys) found observed/expected ratios of 2-6 for viral infection codes vs ≤1 for bacterial codes, and protozoal intestinal infection was also enriched (18x). Limitations: FinnGen and FinRegistry overlap, so they are not independent. Coding and healthcare-seeking bias can inflate associations, and the timing of infection relative to onset is not always resolved. Infection-onset ME/CFS shares genetic risk with other onsets (DecodeME). The Giardia and Q fever outbreaks show that non-viral triggers also occur, so the viral excess is not absolute.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (epi_literature; FinnGen R12 / FinRegistry)",
       "n": "1,622 (FinRegistry), 348 (FinnGen) G93.3 cases",
       "design": "registry co-occurrence (O/E, Fisher, BH-FDR)",
       "result": "viral infections O/E 3-6; bacterial pneumonia/erysipelas not enriched",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Age/sex-matched time-to-event hazard ratios by infection class in UK Biobank or Danish registries"
     ],
     "hypotheses": [
      "viral-persistence",
      "herpes-reactivation",
      "innate-hyperactivation"
     ],
     "test_available": "n/a"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "viral-persistence",
     "for": "SARS-CoV-2 antigen/RNA persists in plasma and tissues of a minority for months to years, and tissue RNA associated with LC (Zuo 2024 OR 5.17). Enterovirus protein in stomach biopsies (Chia 2008). Early nirmatrelvir may prevent LC (small RCT).",
     "against": "Three RCTs of nirmatrelvir in established LC (about 1,200 people) were null, and spike levels did not fall with treatment. Enterovirus findings are unreplicated with blinded methods. XMRV was contamination. The Giardia and Q fever triggers and failed doxycycline in QFS fit a 'hit-and-run' rather than persistent infection."
    },
    {
     "id": "herpes-reactivation",
     "for": "Lytic-marker serology (EA-D) associates with LC fatigue (Peluso 2023). dUTPase antibodies higher in ME/CFS (one lab, repeated). HHV-6 miRNA in ME/CFS brain (n=3). Genetic EBV-load MR suggestive (MVP OR 1.59). Plasma EBV DNA more frequent in one study. Herpes titres track complement activation in LC.",
     "against": "Large serology studies (UK Biobank, Swedish cohorts) show no difference. PBMC viral DNA is equally common. The valganciclovir RCT missed its primary outcome and was never replicated. MR signal is not EBV-specific on negative controls. Most positive findings come from single labs."
    },
    {
     "id": "herv-activation",
     "for": "HERV-K overexpression in moderate ME/CFS (Rodrigues 2019). HERV transcript profiles separate ME/CFS from FM (n=43). HERV-K dUTPase antibodies co-occur with herpes dUTPase antibodies. Definition: reactivation of human endogenous retroviral elements, possibly transactivated by herpesviruses, driving immune dysregulation.",
     "against": "All studies are small and unreplicated. The anti-HERV-W antibody temelimab failed in HERV-W-positive post-COVID patients (n=203)."
    },
    {
     "id": "immune-exhaustion",
     "for": "Deficient EBV-specific memory B/T responses (Loebel 2014). Exhausted, terminally differentiated virus-specific CD8 T cells in LC (Ma 2026).",
     "against": "Single-cell re-analysis found no exhausted CD8 signature in ME/CFS, and EBV-specific clonotypes trended higher, not lower, in severe cases."
    },
    {
     "id": "complement-thromboinflammation",
     "for": "Classical complement activation associated with herpesvirus antibody titres in active LC (Cervia-Hasler 2024).",
     "against": "Not yet replicated in non-COVID ME/CFS, and causal direction unknown."
    },
    {
     "id": "innate-hyperactivation",
     "for": "Severity of acute illness, not pathogen type, predicts post-infective fatigue (Dubbo). The stereotyped syndrome after diverse pathogens suggests a common host response.",
     "against": "Few studies measure the acute immune response prospectively, and baseline cytokines have predicted outcome inconsistently."
    },
    {
     "id": "sickness-behaviour",
     "for": "Stereotyped phenotype across pathogens, with no ongoing pathogen required (Dubbo, QFS doxycycline failure).",
     "against": "Does not explain why only ~10% persist, or tissue-specific viral findings."
    },
    {
     "id": "gut-dysbiosis",
     "for": "Giardia outbreak produced lasting fatigue + IBS. SARS-CoV-2 gut persistence. Enterovirus in stomach (contested).",
     "against": "Gut reservoir data are mostly in LC and convenience samples."
    }
   ],
   "glossary": [
    {
     "term": "Post-infective fatigue syndrome (PIFS)",
     "plain": "A lasting illness of fatigue, pain, poor concentration and unrefreshing sleep that begins after a documented infection."
    },
    {
     "term": "Lytic vs latent",
     "plain": "Herpesviruses hide quietly in cells for life (latent). When they 'wake up' and make new virus or viral proteins, that is lytic activity, or reactivation."
    },
    {
     "term": "EA-D, VCA, EBNA",
     "plain": "Parts of Epstein-Barr virus that antibodies are measured against. EA-D antibodies suggest recent reactivation, VCA IgM suggests a new infection, and EBNA IgG shows an old past infection."
    },
    {
     "term": "dUTPase",
     "plain": "An enzyme some herpesviruses make when partly reactivated. It can itself stir up inflammation, and antibodies to it are used as a sign of reactivation."
    },
    {
     "term": "iciHHV-6",
     "plain": "Inherited chromosomally integrated HHV-6: about 1 in 100 people carry HHV-6's genome in every cell, inherited from a parent. It can make blood HHV-6 tests look falsely 'high'."
    },
    {
     "term": "Enterovirus VP1",
     "plain": "A coat protein of enteroviruses (the family of polio, coxsackie and echoviruses) used to stain tissue for evidence of infection."
    },
    {
     "term": "Antigenemia",
     "plain": "Viral protein detectable in the blood."
    },
    {
     "term": "HERV",
     "plain": "Human endogenous retroviruses: remnants of ancient viral infections that make up about 8% of our DNA and can sometimes be switched back on."
    },
    {
     "term": "XMRV",
     "plain": "A mouse-derived retrovirus once reported in CFS. The finding was later shown to be lab contamination and retracted."
    },
    {
     "term": "Mendelian randomisation (MR)",
     "plain": "Using genetic variants as a natural experiment to test whether a trait (like EBV load) plausibly causes a disease."
    }
   ],
   "key_reviews": [
    {
     "cite": "O'Neal & Hanson 2021, Front Med",
     "url": "https://doi.org/10.3389/fmed.2021.688486",
     "why": "Critical review of enterovirus methods in ME/CFS: why past studies neither proved nor excluded persistent infection."
    },
    {
     "cite": "Proal et al. 2023, Nat Immunol",
     "url": "https://doi.org/10.1038/s41590-023-01601-2",
     "why": "Review of evidence for a SARS-CoV-2 tissue reservoir in long COVID and research priorities for antiviral trials."
    },
    {
     "cite": "Hickie et al. 2006, BMJ",
     "url": "https://doi.org/10.1136/bmj.38933.585764.AE",
     "why": "The foundational prospective cohort showing a stereotyped post-infective syndrome across pathogens."
    },
    {
     "cite": "Alter et al. 2012, mBio",
     "url": "https://doi.org/10.1128/mBio.00266-12",
     "why": "The definitive blinded study closing the XMRV episode, and a model for testing a pathogen claim."
    },
    {
     "cite": "Blomberg et al. 2019, Front Immunol",
     "url": "https://doi.org/10.3389/fimmu.2019.01946",
     "why": "Careful multiplex herpesvirus serology in Canada-criteria ME/CFS with a discussion of why serology is limited."
    }
   ],
   "gaps": [
    "No adequately powered, replicated antiviral RCT in ME/CFS (valganciclovir n=30 in 2013 is still the best evidence).",
    "No LC antiviral trial has enrolled people selected by biomarker evidence of persistence (antigenemia or tissue RNA).",
    "No prospective trial of early antiviral treatment during infectious mononucleosis to prevent post-IM ME/CFS.",
    "Blinded, multi-lab, sequencing-based tissue studies (gut, muscle, brain) for enterovirus, herpesvirus and SARS-CoV-2 in non-COVID ME/CFS are missing.",
    "Longitudinal viral sampling timed to PEM crashes (plasma EBV/HHV-6 DNA, dUTPase, EA-D) has barely been done.",
    "DecodeME iciHHV-6 prevalence results are not yet published.",
    "Independent replication of dUTPase antibody findings outside the originating lab.",
    "Whether post-COVID, post-EBV, post-Giardia and post-Q-fever ME/CFS share the same downstream biology."
   ]
  },
  {
   "domain": "omics",
   "title": "Metabolism and blood omics",
   "overview": "Blood metabolomics and proteomics consistently find that ME/CFS patients differ from controls on average, but the differences are small (mostly 0.2-0.5 SD), partly cohort-specific, and confounded by medication, caffeine, diet and sample handling. The better-supported signals are modest: lower plasmalogens and some phosphatidylcholines, slightly lower carnitines, lower gut-derived IPA, lower aromatic lactates (a new cross-cohort re-analysis finding), higher methionine sulfoxide, and lower muscle-derived proteins in blood that are not explained by activity. Bigger claims, such as a uniform hypometabolic 'cell danger' state, an IDO metabolic trap, divergent metabolic or proteomic responses to exercise, reproducible methylation signatures, and diagnostic classifiers with near-perfect accuracy, have not held up in independent data or remain untested outside the original lab. No omics test is ready for diagnosis. The largest gaps are severe patients, sampling during the 48-72 h PEM window, disease controls, and pairing omics with fresh-cell function.",
   "findings": [
    {
     "id": "omics-metabolome-small-effects",
     "subdomain": "Metabolomics: overall",
     "finding": "Blood metabolome differences between ME/CFS and controls are real at group level but small (mostly 0.2-0.5 SD) and partly cohort-specific; early near-perfect classification did not carry over to new cohorts.",
     "grade": "replicated",
     "plain": "Patients' blood chemistry differs from healthy people's on average, but only slightly, and the specific list of chemicals changes from study to study.",
     "detail": "Single-cohort studies reported AUCs of 0.82-0.96 (Naviaux 2016, Nagy-Szakal 2018, Che 2022), but these were estimated within the training data. A harmonised re-analysis of 10 public cohorts found 74 compounds at q<0.05 with pooled effects mostly 0.2-0.5 SD; two Cornell cohorts agreed only weakly (rho 0.16) and cross-cohort transfer AUCs were 0.56-0.83. Medication exposure (paracetamol, gabapentin, psychoactives, acid suppressants detected in blood) and caffeine are major confounders that most original papers did not adjust for.",
     "studies": [
      {
       "cite": "Naviaux 2016, PNAS",
       "n": "45 vs 39",
       "design": "case-control, targeted LC-MS/MS (612 metabolites)",
       "result": "20 pathways abnormal; 80% of discriminating metabolites lower; AUC 0.94 (men, 8 metabolites) and 0.96 (women, 13) within the same data",
       "url": "https://doi.org/10.1073/pnas.1607571113",
       "verified": true
      },
      {
       "cite": "Nagy-Szakal 2018, Sci Rep",
       "n": "50 vs 50",
       "design": "case-control, untargeted plasma metabolomics + stool metagenomics",
       "result": "altered choline, carnitine and complex lipids; ceramide higher in the ME/CFS+IBS subgroup; cross-validated AUC 0.82 (metabolome), 0.84 combined with stool",
       "url": "https://doi.org/10.1038/s41598-018-28477-9",
       "verified": true
      },
      {
       "cite": "Che 2022, Int J Mol Sci",
       "n": "106 vs 91",
       "design": "case-control, targeted + untargeted plasma metabolomics",
       "result": "lower plasmalogens/ether phospholipids, phosphatidylcholines and sphingomyelins (p<0.001); higher dicarboxylic acids; ML AUC up to 0.873",
       "url": "https://doi.org/10.3390/ijms23147906",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide meta-analysis)",
       "n": "10 cohorts, ~600 cases / ~430 controls",
       "design": "harmonised meta-analysis of public per-person data (Metabolon, Fiehn, LC-MS/MS, NMR)",
       "result": "74 compounds q<0.05; effects mostly 0.2-0.5 SD; panel trained on BioMapAI transfers with AUC 0.60-0.83",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets)",
       "n": "two Cornell cohorts, 575 shared compounds",
       "design": "cross-cohort concordance",
       "result": "patient-control differences agree only faintly (rho 0.16); model trained on one scores AUC 0.56 on the other; a public GitHub copy of Germain 2020 has case/control labels swapped",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Xiong 2025 (BioMapAI), Nat Med",
       "n": "249 participants, 4-year longitudinal",
       "design": "deep neural net on stool metagenome, plasma metabolome, immune profiling, labs, symptoms",
       "result": "10-fold CV AUC 0.915 (all omics); external cohorts AUC 0.59-0.77 (Guo, Raijmakers stool; Germain metabolome 0.68, Che 0.61) with 19-79% feature overlap",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Which differences persist in drug-free, fasting, rapidly processed samples?",
      "Does any metabolite change track severity or PEM within a person?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "cell-danger-response",
      "gut-dysbiosis"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-cdr-hypometabolic",
     "subdomain": "Metabolomics: cell danger response",
     "finding": "Naviaux 2016's picture of a homogeneous 'hypometabolic' dauer-like state (80% of discriminating metabolites decreased) has not been confirmed as a uniform pattern in later independent cohorts.",
     "grade": "contested",
     "plain": "One influential 2016 study said patients' cells were in a protective 'low-power' mode, with most chemicals reduced. Later studies found more mixed and smaller changes.",
     "detail": "Naviaux's 45/39 cohort with targeted LC-MS/MS found lower sphingolipids, phospholipids, purines and other pathways and proposed the cell danger response (CDR). An Open Medicine Foundation validation cohort was reported informally (2018) to reproduce 23 of 30 pathway abnormalities, but no peer-reviewed replication paper was found. Hoel 2021 found heterogeneous metabotypes rather than one state; Che 2022 and Xiong 2023 partly echo the lipid reductions. In the cross-cohort re-analysis, the Naviaux data still carry some replicated signals (e.g. low aromatic lactates on a non-Metabolon platform), but there is no global hypometabolic shift across cohorts.",
     "studies": [
      {
       "cite": "Naviaux 2016, PNAS",
       "n": "45 vs 39",
       "design": "case-control, targeted LC-MS/MS (612 metabolites)",
       "result": "20 pathways abnormal; 80% of discriminating metabolites lower; AUC 0.94 (men, 8 metabolites) and 0.96 (women, 13) within the same data",
       "url": "https://doi.org/10.1073/pnas.1607571113",
       "verified": true
      },
      {
       "cite": "OMF metabolomics validation update (MEAction 2018)",
       "n": "not stated",
       "design": "unpublished replication cohort summary",
       "result": "23/30 pathway abnormalities reported as replicated",
       "url": "https://www.meaction.net/2018/02/10/update-on-the-metabolomics-validation-study-by-omf/",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Hoel 2021, JCI Insight",
       "n": "83 vs 35",
       "design": "case-control, serum metabolomics, lipidomics, hormones",
       "result": "common changes consistent with energy strain and altered fatty-acid/amino-acid fuel use; 3 patient subsets (metabotypes), 2 with distinct energy-metabolism patterns",
       "url": "https://doi.org/10.1172/jci.insight.149217",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide meta-analysis)",
       "n": "10 cohorts, ~600 cases / ~430 controls",
       "design": "harmonised meta-analysis of public per-person data (Metabolon, Fiehn, LC-MS/MS, NMR)",
       "result": "74 compounds q<0.05; effects mostly 0.2-0.5 SD; panel trained on BioMapAI transfers with AUC 0.60-0.83",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Has the OMF validation cohort been published with per-person data?",
      "Is any CDR signature (e.g. purinergic signalling) measurable in tissue rather than plasma?"
     ],
     "hypotheses": [
      "cell-danger-response",
      "mitochondrial-energy"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-lipids-plasmalogens",
     "subdomain": "Metabolomics: lipids and sphingolipids",
     "finding": "Lower plasmalogens, some phosphatidylcholines and sphingomyelins are reported across several cohorts and platforms, with modest effect sizes.",
     "grade": "several",
     "plain": "Some fats that form cell membranes, especially a type called plasmalogens, are a little lower in patients' blood in several studies.",
     "detail": "Che 2022 (106/91) reported lower plasmalogens/ether phospholipids, PCs and sphingomyelins (p<0.001) and proposed peroxisomal dysfunction. Naviaux 2016 found lower sphingolipids; Xiong 2023 lower sphingomyelins; Nagy-Szakal 2018 higher ceramides only in the IBS subgroup. In the cross-cohort re-analysis PC(14:0/16:0) was -0.42 SD (4 cohorts, 3 non-Metabolon) and plasmalogen P-16:0/18:2 -0.38 SD; dihydroceramide d18:0/16:0 was +0.28. Diet (dairy, fish) and BMI are not well controlled.",
     "studies": [
      {
       "cite": "Che 2022, Int J Mol Sci",
       "n": "106 vs 91",
       "design": "case-control, targeted + untargeted plasma metabolomics",
       "result": "lower plasmalogens/ether phospholipids, phosphatidylcholines and sphingomyelins (p<0.001); higher dicarboxylic acids; ML AUC up to 0.873",
       "url": "https://doi.org/10.3390/ijms23147906",
       "verified": true
      },
      {
       "cite": "Naviaux 2016, PNAS",
       "n": "45 vs 39",
       "design": "case-control, targeted LC-MS/MS (612 metabolites)",
       "result": "20 pathways abnormal; 80% of discriminating metabolites lower; AUC 0.94 (men, 8 metabolites) and 0.96 (women, 13) within the same data",
       "url": "https://doi.org/10.1073/pnas.1607571113",
       "verified": true
      },
      {
       "cite": "Xiong 2023, Cell Host Microbe",
       "n": "short-term 75, long-term 79 vs 79 controls",
       "design": "case-control, stool metagenomics + plasma metabolomics",
       "result": "plasma butyrate, bile acids, benzoate lower; sphingomyelins depleted; dysbiosis mostly in short-term patients",
       "url": "https://doi.org/10.1016/j.chom.2023.01.001",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "up to 10 cohorts",
       "design": "meta-analysis",
       "result": "PC(14:0/16:0) -0.42 SD; plasmalogen P-16:0/18:2 -0.38 SD; dihydroceramide +0.28 SD",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Nagy-Szakal 2018, Sci Rep",
       "n": "50 vs 50",
       "design": "case-control, untargeted plasma metabolomics + stool metagenomics",
       "result": "altered choline, carnitine and complex lipids; ceramide higher in the ME/CFS+IBS subgroup; cross-validated AUC 0.82 (metabolome), 0.84 combined with stool",
       "url": "https://doi.org/10.1038/s41598-018-28477-9",
       "verified": true
      }
     ],
     "open_questions": [
      "Are plasmalogens low in tissue (muscle, red cells) or only plasma?",
      "Do they relate to peroxisomal enzyme activity or to diet?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "cell-danger-response"
     ],
     "test_available": "research only (plasmalogen panels exist in some specialist labs)"
    },
    {
     "id": "omics-aromatic-lactates",
     "subdomain": "Metabolomics: aromatic lactates",
     "finding": "Four aromatic 2-hydroxy acids (phenyllactate, 4-hydroxyphenyllactate, indolelactate, imidazole lactate) are lower in ME/CFS in 5 of 5 cohorts (combined -0.53 SD, I²=0), robust to drug, caffeine, diet and kidney-function adjustment.",
     "grade": "several",
     "plain": "A family of small chemicals made from amino acids is consistently lower in patients across five separate studies. It is a group-level difference, not a test.",
     "detail": "Solving ME/CFS re-analysis: p=4e-11, holds on Naviaux's targeted LC-MS/MS platform (-0.64), -0.64 in people with no prescription drug detected, -0.73 in men. The precursor keto acids are not lower, so either the reduction step or a dietary source (fermented foods) is implicated. In BioMapAI paired stool, gut species do not predict these compounds (CV R²≈0), unlike IPA. They do not track severity (|r|<0.2). Not yet reported by original study authors; needs targeted, fasting confirmation and D- vs L-isomer separation (bacterial D vs host L).",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "5 cohorts (BioMapAI, Germain 2018/2020/2022, Naviaux)",
       "design": "harmonised meta-analysis with confounder models",
       "result": "combined -0.53 SD, 5/5 cohorts lower, I²=0; not stool-predicted; not severity-linked",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is this host lactate-dehydrogenase-type reduction, renal handling, or fermented-food intake?",
      "D- vs L-phenyllactate ratio?",
      "Does it differ from other fatigue conditions?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "gut-dysbiosis"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-ipa",
     "subdomain": "Metabolomics: tryptophan / gut metabolites",
     "finding": "Indole-3-propionate (IPA), a gut-bacterial tryptophan product, is lower in ME/CFS plasma in 4 of 4 cohorts (-0.33 SD) and tracks butyrate-producing gut species.",
     "grade": "several",
     "plain": "A chemical made only by certain gut bacteria is a bit lower in patients, which fits the gut-bacteria differences found in several studies.",
     "detail": "Pooled -0.33 SD (95% CI -0.49 to -0.16), p=0.0001; individual AUC only 0.57-0.60. In BioMapAI paired stool, IPA correlates with F. prausnitzii and Bifidobacterium and inversely with R. gnavus; adjusting for IPA-linked species reduces the difference from -0.40 to -0.09 (optimistic same-data selection). Xiong 2023 and BioMapAI report reduced microbial butyrate and altered tryptophan metabolism. Interpreted as a gut-ecology marker rather than a host defect.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets / deep2)",
       "n": "4 plasma cohorts",
       "design": "meta-analysis",
       "result": "IPA -0.33 SD, p=0.0001; AUC 0.57-0.60",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Xiong 2025 (BioMapAI), Nat Med",
       "n": "249 participants, 4-year longitudinal",
       "design": "deep neural net on stool metagenome, plasma metabolome, immune profiling, labs, symptoms",
       "result": "10-fold CV AUC 0.915 (all omics); external cohorts AUC 0.59-0.77 (Guo, Raijmakers stool; Germain metabolome 0.68, Che 0.61) with 19-79% feature overlap",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      },
      {
       "cite": "Xiong 2023, Cell Host Microbe",
       "n": "short-term 75, long-term 79 vs 79 controls",
       "design": "case-control, stool metagenomics + plasma metabolomics",
       "result": "plasma butyrate, bile acids, benzoate lower; sphingomyelins depleted; dysbiosis mostly in short-term patients",
       "url": "https://doi.org/10.1016/j.chom.2023.01.001",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does raising IPA (fibre, polyphenols) change anything clinically?",
      "Is low IPA a consequence of diet changes caused by illness?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-kynurenine-ido",
     "subdomain": "Metabolomics: kynurenine pathway / IDO trap",
     "finding": "The IDO metabolic-trap model predicts tryptophan-kynurenine pathway disruption, but blood data are inconsistent: one cohort found higher kynurenine, others found no difference or slightly lower kynurenine.",
     "grade": "contested",
     "plain": "A mathematical theory proposed that a tryptophan-processing enzyme gets stuck in ME/CFS. Blood measurements of that pathway point in different directions in different studies.",
     "detail": "Kashi, Davis and Phair 2019 modelled IDO1 substrate inhibition plus common non-functional IDO2 variants producing a bistable 'trap'; it is a hypothesis paper with no patient data. Kavyani 2024 reported higher kynurenine correlating with severity and lower downstream 3-HK, 3-HAA, QUIN, picolinate. Groven 2021 (49 CFS/57 FM/54 HC women) found differences mainly in FM, not CFS vs controls. The cross-cohort re-analysis found kynurenine -0.27 SD (lower), kyn/trp ratio -0.19 (q=0.06), lower kynurenine with >4 years illness, and no change in IDO1/KYNU proteins. Plasma kynurenine says little about intracellular IDO kinetics, which is what the model is about.",
     "studies": [
      {
       "cite": "Kashi, Davis, Phair 2019, Diagnostics",
       "n": "none (model)",
       "design": "mathematical model + variant database search",
       "result": "IDO1 substrate inhibition + IDO2 loss + LAT1 asymmetry gives two steady states",
       "url": "https://doi.org/10.3390/diagnostics9030082",
       "verified": true
      },
      {
       "cite": "Kavyani 2024, Mol Neurobiol",
       "n": "not in abstract",
       "design": "case-control, plasma KP metabolites + cytokines + proteomics",
       "result": "higher kynurenine linked to severity; lower 3-HK, 3-HAA, QUIN, PIC; hepcidin about halved",
       "url": "https://doi.org/10.1007/s12035-023-03784-z",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Groven 2021, Psychoneuroendocrinology",
       "n": "49 CFS, 57 FM, 54 HC (women)",
       "design": "case-control, plasma KP metabolites",
       "result": "main differences FM vs HC; quinolinic acid differs CFS vs FM; no clear CFS vs HC kynurenine shift",
       "url": "https://ntnuopen.ntnu.no/ntnu-xmlui/handle/11250/2826213",
       "verified": false
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide meta-analysis)",
       "n": "10 cohorts, ~600 cases / ~430 controls",
       "design": "harmonised meta-analysis of public per-person data (Metabolon, Fiehn, LC-MS/MS, NMR)",
       "result": "kynurenine -0.27 SD (lower); kyn/trp -0.19, q=0.06; lower in >4 y illness; IDO1/KYNU protein null",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Abujrais 2024, ACS Chem Neurosci",
       "n": "38 vs 24",
       "design": "case-control, targeted LC-MS",
       "result": "3-hydroxyanthranilic acid and 3-hydroxykynurenine differ; no headline kynurenine rise",
       "url": "https://doi.org/10.1021/acschemneuro.4c00444",
       "verified": true
      }
     ],
     "open_questions": [
      "Do IDO2 variants differ in frequency in large genetic data (DecodeME)?",
      "Intracellular tryptophan/kynurenine flux in patient cells under stress?"
     ],
     "hypotheses": [
      "metabolic-trap-ido",
      "innate-hyperactivation"
     ],
     "test_available": "Plasma tryptophan/kynurenine available in some commercial labs; not diagnostic"
    },
    {
     "id": "omics-carnitines",
     "subdomain": "Metabolomics: acylcarnitines and fatty acids",
     "finding": "Free carnitine and long-chain acylcarnitines are slightly lower in ME/CFS across cohorts, contrary to the earlier idea of excess circulating fatty acids.",
     "grade": "several",
     "plain": "Carriers that move fats into the cell's power stations are a little lower in patients' blood.",
     "detail": "Reported as altered/low in Naviaux 2016, Nagy-Szakal 2018, Germain 2017 and Germain 2022 (carnitines low). Cross-cohort re-analysis: carnitine -0.21 SD, long-chain acylcarnitines -0.22 SD, long-chain fatty acids slightly lower in all 4 cohorts examined; acetylcarnitine/carnitine ratio null. Germain 2020 reported lower acyl cholines. Effect sizes are small and diet/fasting status is rarely recorded. Older acetylcarnitine findings (Reuter and Evans 2011) recalled from memory.",
     "studies": [
      {
       "cite": "Nagy-Szakal 2018, Sci Rep",
       "n": "50 vs 50",
       "design": "case-control, untargeted plasma metabolomics + stool metagenomics",
       "result": "altered choline, carnitine and complex lipids; ceramide higher in the ME/CFS+IBS subgroup; cross-validated AUC 0.82 (metabolome), 0.84 combined with stool",
       "url": "https://doi.org/10.1038/s41598-018-28477-9",
       "verified": true
      },
      {
       "cite": "Germain 2017, Mol BioSyst",
       "n": "17 vs 15",
       "design": "pilot case-control, plasma metabolomics",
       "result": "74/361 metabolites p<0.05, 35 at q<0.15; fatty-acid and lipid pathways",
       "url": "https://doi.org/10.1039/c6mb00600k",
       "verified": true
      },
      {
       "cite": "Germain 2020, Metabolites",
       "n": "26 vs 26 (women)",
       "design": "case-control, Metabolon + complex lipids (~1,750 compounds)",
       "result": "acyl cholines lower in two cohorts; steroid changes",
       "url": "https://doi.org/10.3390/metabo10010034",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "multiple cohorts",
       "design": "meta-analysis",
       "result": "carnitine -0.21 SD; long-chain acylcarnitines -0.22 SD",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Reuter & Evans 2011, J Intern Med",
       "n": "44 patients",
       "design": "case-control serum carnitine profile",
       "result": "lower acetylcarnitine / altered acylcarnitines",
       "url": "https://doi.org/10.1111/j.1365-2796.2011.02405.x",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets)",
       "n": "4 cohorts",
       "design": "meta-analysis",
       "result": "hypothesis of higher fatty acids in patients reversed: long-chain fatty acids and acylcarnitines slightly lower",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Muscle carnitine and fatty-acid oxidation flux in vivo?",
      "Effect of fasting and dietary restriction on these values?"
     ],
     "hypotheses": [
      "mitochondrial-energy"
     ],
     "test_available": "Plasma acylcarnitine profile (clinical metabolic labs); not diagnostic for ME/CFS"
    },
    {
     "id": "omics-energy-fuel-pdh",
     "subdomain": "Metabolomics: energy metabolism",
     "finding": "Several studies infer impaired carbohydrate oxidation (pyruvate dehydrogenase restraint, TCA/urea cycle shifts), but simple blood ratios (lactate/pyruvate, citrulline/arginine) do not differ across pooled cohorts.",
     "grade": "contested",
     "plain": "Some studies suggest patients' cells burn sugar less efficiently. Blood measures that should show this are mostly normal when many studies are pooled.",
     "detail": "Fluge 2016 (200/102) found low TCA-fuelling amino acids in women and higher PDK1/2/4 mRNA in PBMCs; Hoel 2021 described energy strain with altered fuel use in subsets; Yamano 2016 found TCA/urea-cycle ratios (validation AUC 0.75). Armstrong 2015 (NMR, 34/25 women) reported lower blood lactate and glutamate and higher glucose. In the pooled re-analysis, lactate/pyruvate (-0.10), citrulline/arginine, ornithine/arginine and hypoxanthine/xanthine were null; creatine/creatinine +0.19. These are inferences from plasma about tissue metabolism; direct tissue flux measurements are scarce.",
     "studies": [
      {
       "cite": "Fluge 2016, JCI Insight",
       "n": "200 vs 102",
       "design": "case-control, serum amino acids + PBMC mRNA",
       "result": "lower TCA-fuelling amino acids mainly in women; PDK1/2/4, SIRT4, PPARδ mRNA higher; suggests impaired pyruvate dehydrogenase",
       "url": "https://doi.org/10.1172/jci.insight.89376",
       "verified": true
      },
      {
       "cite": "Hoel 2021, JCI Insight",
       "n": "83 vs 35",
       "design": "case-control, serum metabolomics, lipidomics, hormones",
       "result": "common changes consistent with energy strain and altered fatty-acid/amino-acid fuel use; 3 patient subsets (metabotypes), 2 with distinct energy-metabolism patterns",
       "url": "https://doi.org/10.1172/jci.insight.149217",
       "verified": true
      },
      {
       "cite": "Yamano 2016, Sci Rep",
       "n": "133 samples (93 train / 40 validation)",
       "design": "case-control, CE-MS plasma",
       "result": "TCA and urea-cycle intermediates differ; ornithine/citrulline + pyruvate/isocitrate AUC 0.80 train, 0.75 validation",
       "url": "https://doi.org/10.1038/srep34990",
       "verified": true
      },
      {
       "cite": "Armstrong 2015, Metabolomics",
       "n": "34 vs 25 (women)",
       "design": "case-control, 1H NMR blood and urine",
       "result": "lower lactate, acetate, glutamate; higher glucose (via review table)",
       "url": "https://doi.org/10.1007/s11306-015-0816-5",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "multiple cohorts",
       "design": "pre-specified ratio meta-analysis",
       "result": "lactate/pyruvate, NO/urea-cycle and purine ratios null; no support for an arginine/NO substrate problem",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Does PDH activity measured in muscle biopsies differ?",
      "Are Hoel's metabotypes stable over time in the same person?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "serum-factor"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-ketones",
     "subdomain": "Metabolomics: ketones",
     "finding": "Ketone bodies are associated with ME/CFS in UK Biobank NMR data, but 3-hydroxybutyrate shows no consistent difference in pooled case-control cohorts.",
     "grade": "contested",
     "plain": "One very large data set links ketones (fuel made from fat) to ME/CFS; smaller studies pooled together do not.",
     "detail": "Huang 2024 (1,194 ME/CFS in UK Biobank, NMR) reported lipoprotein and ketone-body associations; their ML model (19 baseline characteristics + 9 NMR biomarkers) reached AUC 0.83, with much of the signal from non-omic features. Fasting time and diet differ by group and strongly affect ketones. Pooled re-analysis: 3-hydroxybutyrate null; ketones used as a covariate did not explain other signals.",
     "studies": [
      {
       "cite": "Huang 2024, Commun Med",
       "n": "1,194 ME/CFS vs 53,009 controls + comorbid groups",
       "design": "UK Biobank NMR metabolomics, association + ML",
       "result": "168 biomarker associations incl. surface lipids and ketone bodies; AUC 0.83 with 19 baseline characteristics + 9 NMR markers",
       "url": "https://doi.org/10.1038/s43856-024-00669-7",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets)",
       "n": "4 cohorts",
       "design": "meta-analysis",
       "result": "3-hydroxybutyrate null",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Ketones under controlled fasting with orthostatic measures?"
     ],
     "hypotheses": [
      "mitochondrial-energy"
     ],
     "test_available": "Blood beta-hydroxybutyrate (routine); not diagnostic"
    },
    {
     "id": "omics-exercise-metabolome",
     "subdomain": "Metabolomics: exercise response",
     "finding": "Germain 2022 reported growing metabolic divergence after a 2-day CPET; within-person re-analysis finds the exercise response itself barely differs (blunted lactate and glucose rise aside).",
     "grade": "contested",
     "plain": "A study took blood before and after two exercise tests and saw bigger differences after. Re-analysed person by person, the changes caused by exercise look mostly the same in patients and controls.",
     "detail": "Germain 2022 (60/45, 1,157 metabolites, 4 time points) found female significant counts rising from 6 to 54 across time points. Re-analysis: day-1 and day-2 case-control differences correlate 0.85 and are only ~6% larger on day 2; within-person change scores are at chance (permutation p=0.12-0.61). Surviving signals: smaller lactate rise (likely lower workload), no post-exercise glucose rise in patients, oxidised cys-gly rising only in patients. Five of 8 people with missing post-exercise samples were severely affected men.",
     "studies": [
      {
       "cite": "Germain 2022, JCI Insight",
       "n": "60 vs 45",
       "design": "2-day CPET, 4 time points, Metabolon (1,157 metabolites)",
       "result": "baseline differences; authors report growing patient-control divergence after exercise in lipid and energy pathways",
       "url": "https://doi.org/10.1172/jci.insight.157621",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (PEM)",
       "n": "60 vs 45, 404 samples",
       "design": "within-person change, mixed models, permutation",
       "result": "exercise response at chance; 3 modest signals; no reproducible response subgroups (ARI 0.01)",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Samples at 48-72 h, when PEM peaks?",
      "Workload-matched comparisons?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "small-vessel-capillary"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-collagen-metso",
     "subdomain": "Metabolomics: collagen turnover",
     "finding": "Collagen breakdown products (hydroxyproline, galactosyl-hydroxylysine and related) are higher in ME/CFS plasma (+0.52 SD, 4/4 Metabolon cohorts; hydroxyproline also up on 3 other platforms).",
     "grade": "early",
     "plain": "Fragments released when collagen (the body's connective-tissue protein) is broken down are a bit higher in patients.",
     "detail": "Solving ME/CFS re-analysis only: p=2e-8 for the module; hydroxyproline +0.31 SD across 8 cohorts. Galactosyl-hydroxylysine weakens after drug adjustment. Collagen/gelatin supplements and diet are not excluded. Fits loosely with lower ECM/fibroblast and vascular mural proteins in the proteome re-analysis, but the direction (more breakdown, less matrix protein) needs direct markers (CTX-I, PIIINP).",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "4 Metabolon + 4 other cohorts",
       "design": "meta-analysis",
       "result": "collagen module +0.52 SD; hydroxyproline +0.31 SD",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Link to hypermobility / POTS subgroups?",
      "Supplement and diet history?"
     ],
     "hypotheses": [
      "connective-tissue",
      "small-vessel-capillary"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-redox",
     "subdomain": "Redox / oxidative stress",
     "finding": "Markers of oxidative stress (methionine sulfoxide, oxidised glutathione products, lower brain glutathione) are repeatedly reported, but they are non-specific and also seen in depression.",
     "grade": "several",
     "plain": "Signs that cells are under chemical 'rust' stress show up in several studies, but the same signs appear in other illnesses too.",
     "detail": "Methionine sulfoxide/methionine +0.35 SD in 7/7 cohorts across 3 platforms (I²=0), though methionine oxidises during storage; 5-oxoproline/glutamate -0.25 SD (Solving ME/CFS re-analysis). Germain 2018 proposed broad redox imbalance; Germain 2022 data show oxidised cys-gly rising after exercise in patients only. Shungu 2012 (MRS, 15 CFS/15 MDD/13 HC) found lower cortical glutathione and higher ventricular lactate in both CFS and depression. Antioxidant trials have not shown clear benefit.",
     "studies": [
      {
       "cite": "Germain 2018, Metabolites",
       "n": "32 vs 19 (women)",
       "design": "case-control, Metabolon (832 metabolites)",
       "result": "14 metabolites differ; authors propose broad redox imbalance",
       "url": "https://doi.org/10.3390/metabo8040090",
       "verified": true
      },
      {
       "cite": "Shungu 2012, NMR Biomed",
       "n": "15 CFS, 15 MDD, 13 HC",
       "design": "1H MRS case-control",
       "result": "higher ventricular lactate, lower cortical glutathione in CFS and MDD; no CFS-MDD difference in GSH",
       "url": "https://doi.org/10.1002/nbm.2772",
       "verified": false
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 metabolome-wide)",
       "n": "7 cohorts",
       "design": "meta-analysis",
       "result": "methionine sulfoxide ratio +0.35 SD, I²=0",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does MetSO hold in rapidly processed samples with matched storage time?",
      "Is oxidative stress cause, consequence, or shared with any chronic illness?"
     ],
     "hypotheses": [
      "oxidative-stress",
      "mitochondrial-energy"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-nad",
     "subdomain": "NAD+",
     "finding": "NAD+ depletion is a proposed mechanism but has not been directly established in ME/CFS cohorts; NAD precursor trials raise NAD+ without clear clinical benefit.",
     "grade": "early",
     "plain": "NAD+ is a molecule cells need for energy. The idea that patients lack it is mostly theory; supplements raise it but have not clearly helped.",
     "detail": "Reviews (Dehhaghi 2022) link the kynurenine pathway to NAD+ synthesis. Castro-Marrero 2015 (n=73) and 2021 (n=207) RCTs of CoQ10 200 mg + NADH 20 mg reported modest improvements in some fatigue/cognitive scores. A long-COVID RCT of nicotinamide riboside 2 g/day (n=58) raised blood NAD+ 2.6-3.1x but showed no between-group difference in cognition or fatigue (p=0.59), with high dropout. Vitamin B3 pathway changes appeared in Abujrais 2024 untargeted data.",
     "studies": [
      {
       "cite": "Dehhaghi 2022, Aging Dis",
       "n": "review",
       "design": "narrative review",
       "result": "links KP hyperactivation and NAD+ metabolism in ME/CFS",
       "url": "https://doi.org/10.14336/ad.2021.0824",
       "verified": true
      },
      {
       "cite": "Castro-Marrero 2021, Nutrients",
       "n": "207 (104 vs 103)",
       "design": "12-week RCT CoQ10 + NADH",
       "result": "reduction in cognitive fatigue and some QoL measures reported",
       "url": "https://doi.org/10.3390/nu13082658",
       "verified": true
      },
      {
       "cite": "Castro-Marrero 2015, Antioxid Redox Signal",
       "n": "73",
       "design": "8-week RCT CoQ10 + NADH",
       "result": "fatigue impact score reduced; PBMC NAD+/NADH, ATP higher",
       "url": "https://doi.org/10.1089/ars.2014.6181",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Wu 2025, eClinicalMedicine (long COVID)",
       "n": "58",
       "design": "24-week RCT nicotinamide riboside",
       "result": "NAD+ up 2.6-3.1x; no difference in cognition, fatigue, sleep",
       "url": "https://doi.org/10.1016/j.eclinm.2025.103633",
       "verified": true
      }
     ],
     "open_questions": [
      "Is intracellular NAD+/NADH measurably low in patient muscle or immune cells?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "metabolic-trap-ido"
     ],
     "test_available": "research only (some commercial NAD+ tests exist but are not validated for ME/CFS)"
    },
    {
     "id": "omics-ukb-blood-traits",
     "subdomain": "Proteomics and blood traits: UK Biobank",
     "finding": "In UK Biobank, ME/CFS cases show many small blood-trait differences (inflammation, insulin resistance, liver markers) that replicate partly in All of Us and are not explained by low physical activity.",
     "grade": "several",
     "plain": "In a huge UK database, people with an ME/CFS diagnosis have many slightly different blood results, and this is not just because they move less.",
     "detail": "Beentjes, Ponting and colleagues used semi-parametric (Super Learner, one-step) estimators with activity, walking pace and other mediators. 9 of 14 tested traits replicated in All of Us; people reporting PEM showed stronger differences. Caveats: diagnosis is from records/self-report, so case definition is loose; UK Biobank skews healthier and older; differences are small and single traits do not separate groups. Olink proteomics summary statistics show LEP, ADH4, BCHE, complement factors up and CA14, SOD3 down (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Beentjes 2025, EMBO Mol Med",
       "n": "up to 1,455 cases vs 131,303 controls (UK Biobank); All of Us replication",
       "design": "population cohort, 3,237 blood traits, mediation analysis",
       "result": "hundreds of traits differ (116 in both sexes): inflammation, insulin resistance, liver; 9/14 replicated in All of Us; activity mediates only 1/3,237; no single trait discriminates",
       "url": "https://doi.org/10.1038/s44321-025-00258-8",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do these survive stricter case definitions (e.g. DecodeME-style questionnaires)?",
      "How much reflects BMI, medication and comorbidity?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "hpa-endocrine"
     ],
     "test_available": "Routine labs (CRP, HbA1c, liver enzymes) exist but are not diagnostic"
    },
    {
     "id": "omics-proteome-muscle-low",
     "subdomain": "Proteomics: tissue-of-origin",
     "finding": "Muscle-derived intracellular proteins (myoglobin, troponins, CA3 and similar) are lower in resting ME/CFS blood across three SomaScan cohorts and UK Biobank Olink, and this is not explained by activity measures.",
     "grade": "several",
     "plain": "Proteins that normally leak from muscle into blood are a little lower in patients, perhaps meaning less muscle or slower muscle turnover, not ongoing muscle damage.",
     "detail": "Hoel 2026 reported reduced intracellular skeletal-muscle proteins. Solving ME/CFS re-analysis: muscle theme d -0.50 (95% CI -0.76 to -0.24), q=0.002, I² 0.07 across Hoel (-0.85), Germain (-0.43), Walitt (-0.34); UKB competitive shift negative; not mediated by walking pace, not related to steps, Bell score or time reclined. Lean mass was not measured, so reduced muscle mass remains a plausible explanation.",
     "studies": [
      {
       "cite": "Hoel 2026, Cell Rep Med",
       "n": "50 vs 29",
       "design": "case-control, SomaScan 7k serum proteomics",
       "result": "1,823 aptamers differ (845 FDR); secreted proteins up, intracellular (skeletal muscle) proteins down; lower activated-neutrophil secretion",
       "url": "https://doi.org/10.1016/j.xcrm.2026.102647",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "~150 vs ~105 per-person + UKB",
       "design": "random-effects meta-analysis of protein sets",
       "result": "muscle_damage set d -0.50, q=0.002",
       "url": "/#data",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does it survive adjustment for DXA lean mass?",
      "Relation to muscle biopsy fibre size and capillarisation?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "small-vessel-capillary"
     ],
     "test_available": "CK and myoglobin are routine but expected low-normal; not diagnostic"
    },
    {
     "id": "omics-proteome-hoel-leak",
     "subdomain": "Proteomics: SomaScan artefacts",
     "finding": "Most of the ~845 FDR-significant serum proteins in Hoel 2026 appear driven by a sample-handling (cell-leak) signal that is absent from other cohorts; about 13 survive adjustment.",
     "grade": "contested",
     "plain": "A large protein study found hundreds of differences, but a re-analysis suggests most came from how samples were handled rather than the illness.",
     "detail": "Solving ME/CFS re-analysis built a nuclear/cell-leak score: case-control d -1.16 in Hoel, ~0 in Germain, Walitt serum and Walitt CSF. After adjustment Hoel keeps 13 FDR hits (COMP, C6, ANTXR2, LAMA2, FAP and others). Hoel's raw statistics correlate with Germain only through intracellular proteins, and not after adjustment. Hoel authors validated a selection by antibody methods. Across ~6,000 proteins meta-analysed in 4 cohorts, only 32 reach q<0.05 and replicated effects are d 0.25-0.5 (ITGAV, NOTCH3, THBS1, LUM, ADH4, GSTA1, BAFF up).",
     "studies": [
      {
       "cite": "Hoel 2026, Cell Rep Med",
       "n": "50 vs 29",
       "design": "case-control, SomaScan 7k serum proteomics",
       "result": "1,823 aptamers differ (845 FDR); secreted proteins up, intracellular (skeletal muscle) proteins down; lower activated-neutrophil secretion",
       "url": "https://doi.org/10.1016/j.xcrm.2026.102647",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "Hoel 50/24 + Germain + Walitt + UKB",
       "design": "artefact score and leak-adjusted meta-analysis",
       "result": "leak score separates only Hoel (d -1.16); 13 hits survive",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Were case and control samples processed with different delays?",
      "Do the 13 surviving proteins replicate prospectively?"
     ],
     "hypotheses": [
      "vascular-endothelial",
      "connective-tissue"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-proteome-liver-vascular",
     "subdomain": "Proteomics: replicated small shifts",
     "finding": "Hepatocyte enzymes (ADH4, GSTA1, AGXT, BCHE) are modestly higher, and vascular mural/matrix proteins (ITGAV, NOTCH3, LUM, THBS1, TGF-beta family) modestly lower, in more than one cohort.",
     "grade": "early",
     "plain": "A few groups of blood proteins, from the liver and from the walls of small vessels, are slightly shifted in several data sets.",
     "detail": "Liver set d +0.40 (q=0.013) but defined after seeing data and tracks BMI; up in Germain and UKB, partly Hoel, not in long COVID. Mural/ECM: ITGAV -0.34 (4 cohorts), NOTCH3 -0.32, TGF-beta set -0.44 (q=0.013); a long COVID UK Olink set also shows lower ECM/endothelial proteins. Endothelial 'activation' markers go down, not up, and platelet/microclot proteins are not raised. Expected candidates (renin, ACE, endothelin, BNP, EPO, hepcidin, NEFL, GFAP, IL-6, CRP) are null.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "4 blood cohorts",
       "design": "set and single-protein meta-analysis",
       "result": "liver +0.40; TGF-beta -0.44; ITGAV -0.34; many named candidates null",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Beentjes 2025, EMBO Mol Med",
       "n": "up to 1,455 cases vs 131,303 controls (UK Biobank); All of Us replication",
       "design": "population cohort, 3,237 blood traits, mediation analysis",
       "result": "hundreds of traits differ (116 in both sexes): inflammation, insulin resistance, liver; 9/14 replicated in All of Us; activity mediates only 1/3,237; no single trait discriminates",
       "url": "https://doi.org/10.1038/s44321-025-00258-8",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do liver proteins survive adjustment for fatty-liver index and ALT/GGT?",
      "Do lower NOTCH3/ITGAV reflect pericyte loss or shedding?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "vascular-endothelial",
      "complement-thromboinflammation"
     ],
     "test_available": "Routine liver tests; otherwise research only"
    },
    {
     "id": "omics-exertion-proteome",
     "subdomain": "Proteomics: exertion",
     "finding": "Germain 2025 reported patient-specific proteomic changes during recovery after a 2-day CPET, but a within-person re-analysis of the same data finds no protein whose exercise response differs at FDR.",
     "grade": "contested",
     "plain": "One large study reported that patients' blood proteins react differently to exercise. When re-analysed person by person, no single protein clearly did.",
     "detail": "Germain 2025: 79 ME/CFS vs 53 sedentary controls, 6,361 proteins, 2 CPETs 24 h apart; pathway-level claims of suppressed T/B-cell and IL-17 signalling and raised glycolysis. Re-analysis: 0 FDR hits for differential change; no tissue score changes with exertion. Smaller EV studies after CPET (Giloteaux 2024, 18/17 women; Glass 2025, 10/12 men) report altered EV cargo responses (TCA down, complement up at 15 min; ER-stress proteins correlating with PEM), but n is small.",
     "studies": [
      {
       "cite": "Germain 2025, Mol Cell Proteomics",
       "n": "79 vs 53 (sedentary controls)",
       "design": "2-day CPET, SomaScan 7k, longitudinal",
       "result": "widespread changes after exertion, most in recovery; suppressed T/B signalling and IL-17, higher glycolysis proteins; sex differences",
       "url": "https://doi.org/10.1016/j.mcpro.2025.101467",
       "verified": true
      },
      {
       "cite": "Giloteaux 2024, J Extracell Vesicles",
       "n": "18 vs 17 (women)",
       "design": "CPET, EV TMT proteomics, 3 time points",
       "result": "EV proteome response differs; changes correlate with severity",
       "url": "https://doi.org/10.1002/jev2.12403",
       "verified": true
      },
      {
       "cite": "Glass 2025, Clin Transl Med",
       "n": "10 vs 12 (men)",
       "design": "CPET, EV proteomics",
       "result": "TCA proteins down, complement up 15 min post-exercise; ER-stress changes track PEM",
       "url": "https://doi.org/10.1002/ctm2.70346",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "Germain 79/51 per-person",
       "design": "within-person change",
       "result": "0 FDR hits for differential exercise response",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Sampling at 48-72 h?",
      "Effort-matched comparison and correction for lower workload?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "mitochondrial-energy",
      "complement-thromboinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-ev",
     "subdomain": "Extracellular vesicles",
     "finding": "Plasma extracellular vesicle (EV) counts may be higher in ME/CFS, but no EV protein or microRNA difference has survived multiple-testing correction in a replicated cohort.",
     "grade": "early",
     "plain": "Tiny particles cells release into blood may be more numerous in patients, but their contents have not shown a reliable difference yet.",
     "detail": "Rydland 2026 (49/50, SEC + MS proteomics): higher EV concentration; 11 of 424 proteins differ nominally (erythroid, hepatocyte, plasma-cell origin), none after FDR. Re-analysis of Rydland and Giloteaux EV data: no FDR hits. Eguchi 2026 EV-miRNA study had 6 ME/CFS. EV isolation methods vary widely and platelet activation during handling changes EV counts.",
     "studies": [
      {
       "cite": "Rydland 2026, Biochem Biophys Rep",
       "n": "49 vs 50",
       "design": "case-control, EV proteomics",
       "result": "more EVs; 11 nominal protein differences, none FDR",
       "url": "https://doi.org/10.1016/j.bbrep.2026.102679",
       "verified": true
      },
      {
       "cite": "Eguchi 2026, J Transl Med",
       "n": "6 ME/CFS, 6 ICF, 8 depression, 4 HC",
       "design": "EV miRNA profiling",
       "result": "candidate EV-miRNAs; very small n",
       "url": "https://doi.org/10.1186/s12967-026-08695-w",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "Rydland + Giloteaux EV sets",
       "design": "re-analysis",
       "result": "no FDR hits",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Standardised EV isolation across sites?",
      "Do EV counts change with PEM?"
     ],
     "hypotheses": [
      "complement-thromboinflammation",
      "serum-factor"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-csf-proteome",
     "subdomain": "CSF proteomics",
     "finding": "CSF proteomics in ME/CFS is limited to small cohorts and has not produced a replicated protein difference; the largest recent study (31 patients) had no controls.",
     "grade": "early",
     "plain": "Studies of the fluid around the brain and spine are few and small, and have not found a consistent difference.",
     "detail": "Bragee 2026 (31 patients, 902 proteins): within-patient associations with POTS (neutrophil degranulation, platelet activation) and severity (complement, coagulation, IGFBP); re-analysis found 0 FDR hits POTS vs no-POTS. Walitt 2024 CSF SomaScan (~17 vs 21) showed no FDR hits in re-analysis, and no rise in neuronal injury markers. Earlier CSF proteome reports (Schutzer 2011, Baraniuk 2005) are recalled from memory and small.",
     "studies": [
      {
       "cite": "Bragee 2026, Sci Rep",
       "n": "31 patients, no controls",
       "design": "CSF LC-MS proteomics",
       "result": "POTS- and severity-associated pathways; hypothesis-generating",
       "url": "https://doi.org/10.1038/s41598-026-46965-1",
       "verified": true
      },
      {
       "cite": "Schutzer 2011, PLoS One",
       "n": "~43 CFS vs controls (pooled)",
       "design": "CFS vs neurologic post-Lyme vs controls, CSF MS",
       "result": "distinct CSF proteomes reported",
       "url": "https://doi.org/10.1371/journal.pone.0017287",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "Walitt CSF + Bragee",
       "design": "re-analysis",
       "result": "0 FDR hits; NEFL/GFAP null",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Adequately powered CSF study with controls and severe patients?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "brainstem-craniocervical"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-transcriptome-sc",
     "subdomain": "Transcriptomics and single-cell",
     "finding": "Blood transcriptome and single-cell RNA studies report monocyte and platelet abnormalities, but no gene or cell subtype difference replicates across independent cohorts.",
     "grade": "contested",
     "plain": "Studies reading which genes are switched on in blood cells found possible differences, but they have not repeated in other groups of patients.",
     "detail": "Vu 2024 (scRNA, ~30/28, before and 24 h after CPET) reported classical monocyte dysregulation correlating with severity and platelet activation signatures post-exercise. Re-analysis of the same data at person-level pseudobulk: 0 genes at q<0.05 at baseline in 15 cell types; composition null. Across 4 bulk RNA cohorts, 0 genome-wide FDR genes; a plasma-cell/immunoglobulin score is lower in 2/4 (meta d -0.42, p=0.06). Higher mitochondrial RNA fraction in lymphocytes (CD8 Tem d +0.85) is a lead that did not replicate in bulk PBMC. Walitt 2024 reported sex-specific PBMC expression differences in 17/21 people.",
     "studies": [
      {
       "cite": "Vu 2024, Cell Rep Med",
       "n": "~30 vs 28 (PBMC, pre/post CPET)",
       "design": "scRNA-seq",
       "result": "monocyte dysregulation tracking severity; platelet activation post-exercise",
       "url": "https://doi.org/10.1016/j.xcrm.2023.101373",
       "verified": true
      },
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 PI-ME/CFS vs 21 HV",
       "design": "deep phenotyping incl. PBMC RNA, flow, metabolomics",
       "result": "sex-specific gene expression and metabolic differences; fewer switched-memory B cells",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 single-cell/methylation, proteome)",
       "n": "503,739 cells (58 people); 4 bulk RNA cohorts",
       "design": "person-level pseudobulk DE, cross-cohort meta",
       "result": "no replicated gene or cell-type difference",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "An independent PBMC scRNA cohort (only one lab has published so far)?",
      "Severe and housebound patients?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "innate-hyperactivation",
      "herpes-reactivation"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-methylation",
     "subdomain": "Epigenetics / DNA methylation",
     "finding": "Reported genome-wide blood DNA methylation differences have not replicated across cohorts, at least one large set is explained by cell-composition differences, and epigenetic clocks are not accelerated.",
     "grade": "contested",
     "plain": "Chemical tags on DNA were reported to differ in patients, but a closer look shows the differences mostly reflect which blood cells were in the sample.",
     "detail": "de Vega 2014/2017/2018, Trivedi 2018 (13/12 discovery, 17,296 CpGs; pyrosequencing in 33/31) and Helliwell 2020 (RRBS, 10/10) each reported many differentially methylated sites with little overlap. Re-analysis of 4 cohorts (177/107): in GSE93266, control 'PBMC' DNA contained much more granulocyte DNA (median 48% vs 14%); 44,794 FDR CpGs fall to 0 after composition adjustment; meta-analysis 0 CpGs at q<0.05. Horvath, Hannum, PhenoAge, GrimAge, DunedinPACE null in T cells and saliva. Commercial epigenetic-age tests are not supported for ME/CFS.",
     "studies": [
      {
       "cite": "Trivedi 2018, PLoS One",
       "n": "13 vs 12 (+33/31 validation)",
       "design": "EPIC array + pyrosequencing",
       "result": "17,296 CpGs in 6,368 genes; 307 promoters",
       "url": "https://doi.org/10.1371/journal.pone.0201066",
       "verified": true
      },
      {
       "cite": "Helliwell 2020, Clin Epigenetics",
       "n": "10 vs 10",
       "design": "RRBS",
       "result": "76 fragments, 394 CpGs; overlap with earlier gene lists",
       "url": "https://doi.org/10.1186/s13148-020-00960-z",
       "verified": true
      },
      {
       "cite": "de Vega 2014, PLoS One",
       "n": "PBMC case-control",
       "design": "450K array",
       "result": "differential methylation in immune genes",
       "url": "https://doi.org/10.1371/journal.pone.0104757",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 single-cell/methylation)",
       "n": "4 cohorts, 177 vs 107",
       "design": "composition-adjusted EWAS, clocks",
       "result": "0 replicated CpGs; clocks normal",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Sorted-cell methylation in a large, age-matched cohort?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "genetic-neuronal"
     ],
     "test_available": "research only (consumer epigenetic-age tests are not validated here)"
    },
    {
     "id": "omics-nanoneedle",
     "subdomain": "Diagnostics: nanoneedle impedance",
     "finding": "A nanoelectronic impedance assay separated 20 ME/CFS from 20 controls perfectly after hyperosmotic stress, but there is no published independent replication.",
     "grade": "early",
     "plain": "A chip measuring how blood cells react to salt stress separated a small group of patients from controls completely. Nobody outside the lab has confirmed it.",
     "detail": "Esfandyarpour 2019 (PNAS) used PBMCs incubated in plasma; patients' impedance rose after NaCl stress, controls' did not; plasma swap suggested a plasma factor. The only independent attempt found was a 4 vs 4 bioimpedance preprint (Fernandez 2023) with a different device, which reported higher impedance at one frequency and did not test the original protocol. No disease-control group (e.g. MS, depression) was reported in the original at scale; device-to-device variability is unaddressed in published work.",
     "studies": [
      {
       "cite": "Esfandyarpour 2019, PNAS",
       "n": "20 vs 20",
       "design": "case-control, nanoelectronics impedance",
       "result": "complete separation after hyperosmotic stress; plasma swap effect",
       "url": "https://doi.org/10.1073/pnas.1901274116",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Fernandez 2023, Authorea preprint",
       "n": "4 vs 4",
       "design": "bioimpedance spectroscopy, different device",
       "result": "higher impedance at 1.36 kHz; not a replication of the original protocol",
       "url": "https://doi.org/10.22541/au.168909663.38868952/v1",
       "verified": true
      }
     ],
     "open_questions": [
      "Blinded multi-site validation with disease controls?",
      "What molecule in plasma drives the effect?"
     ],
     "hypotheses": [
      "serum-factor",
      "sodium-calcium-overload"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-raman",
     "subdomain": "Diagnostics: Raman spectroscopy",
     "finding": "Raman spectroscopy of PBMCs or plasma separates ME/CFS from controls in single-centre studies (plasma AUC 0.83-0.85), without external validation.",
     "grade": "early",
     "plain": "A laser technique that reads the chemical 'fingerprint' of blood looked promising in two labs, but has not been tested in new patients elsewhere.",
     "detail": "Xu 2019 (single-cell Raman, PBMCs) found higher phenylalanine-associated bands in patients, similar to mtDNA-depleted rho0 cells; small n. Heidarifard 2026 (115 ME vs 45 sedentary controls, plasma at rest and 90 min after a standardised stress) reported AUC 0.85 and 0.83, specificity 73% and 69%. Models are trained and tested within one cohort; spectral features are hard to map to specific molecules.",
     "studies": [
      {
       "cite": "Xu 2019, Analyst",
       "n": "small (PBMCs from patients and controls)",
       "design": "single-cell Raman microspectroscopy",
       "result": "higher phenylalanine bands in CFS PBMCs",
       "url": "https://doi.org/10.1039/c8an01437j",
       "verified": true
      },
      {
       "cite": "Heidarifard 2026, Int J Mol Sci",
       "n": "115 vs 45",
       "design": "plasma Raman + ML, rest and post-stress",
       "result": "AUC 0.85 (rest), 0.83 (post-stress)",
       "url": "https://doi.org/10.3390/ijms27114937",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent external test set and disease controls?"
     ],
     "hypotheses": [
      "mitochondrial-energy"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-cfrna",
     "subdomain": "Diagnostics: cell-free RNA",
     "finding": "Plasma cell-free RNA classified ME/CFS vs sedentary controls with test-set AUC 0.81 and suggested platelet, pDC and monocyte-derived differences; not yet externally validated.",
     "grade": "early",
     "plain": "RNA fragments floating in blood plasma could tell patients from controls about 4 times in 5 in one study.",
     "detail": "Gardella 2025 (PNAS; 93 vs 75 sedentary controls): LASSO model, held-out test AUC 0.81, accuracy 77%; deconvolution suggested differences in platelet-derived cfRNA and higher pDC, monocyte and T-cell cfRNA; network analysis pointed to cytokine signalling and T-cell exhaustion. Held-out test within one cohort; the plasma-cell score that dips in PBMC data was null in this cfRNA set in re-analysis.",
     "studies": [
      {
       "cite": "Gardella 2025, PNAS",
       "n": "93 vs 75",
       "design": "case-control, plasma cfRNA-seq + ML",
       "result": "test AUC 0.81; platelet, pDC, monocyte cfRNA differences",
       "url": "https://doi.org/10.1073/pnas.2507345122",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Performance in a second cohort and against fibromyalgia, depression, long COVID?"
     ],
     "hypotheses": [
      "immune-exhaustion",
      "complement-thromboinflammation"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-ml-classifiers",
     "subdomain": "Diagnostics: machine-learning classifiers",
     "finding": "Multi-omic machine-learning classifiers (BioMapAI and others) perform well in cross-validation but drop to AUC ~0.6-0.77 on external cohorts, so none is usable for diagnosis.",
     "grade": "contested",
     "plain": "Computer models trained on many blood and stool measurements do well on the data they learned from but much worse on new patients.",
     "detail": "BioMapAI (Xiong 2025, 249 participants, 4-year longitudinal sampling) reached 10-fold CV AUC 0.915 with all omics; on external cohorts AUC was 0.72 and 0.59 (Guo stool), 0.61-0.77 (Raijmakers), 0.68 (Germain metabolome) and 0.61 (Che), with only 19-79% feature overlap. Repeated visits per person raise the question of whether CV folds were split by person; disease controls were not included. A small metabolite panel derived in the Solving ME/CFS re-analysis transferred with AUC 0.60-0.83. Huang 2024 UK Biobank AUC 0.83 relied heavily on 19 baseline (non-omic) characteristics. No multi-omic layer converges on a shared pathway beyond chance (re-analysis).",
     "studies": [
      {
       "cite": "Xiong 2025 (BioMapAI), Nat Med",
       "n": "249 participants, 4-year longitudinal",
       "design": "deep neural net on stool metagenome, plasma metabolome, immune profiling, labs, symptoms",
       "result": "10-fold CV AUC 0.915 (all omics); external cohorts AUC 0.59-0.77 (Guo, Raijmakers stool; Germain metabolome 0.68, Che 0.61) with 19-79% feature overlap",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      },
      {
       "cite": "Huang 2024, Commun Med",
       "n": "1,194 ME/CFS (UK Biobank)",
       "design": "NMR + baseline characteristics ML",
       "result": "AUC 0.83, recall 0.70",
       "url": "https://doi.org/10.1038/s43856-024-00669-7",
       "verified": true
      },
      {
       "cite": "Naviaux 2016, PNAS",
       "n": "45 vs 39",
       "design": "case-control, targeted LC-MS/MS (612 metabolites)",
       "result": "20 pathways abnormal; 80% of discriminating metabolites lower; AUC 0.94 (men, 8 metabolites) and 0.96 (women, 13) within the same data",
       "url": "https://doi.org/10.1073/pnas.1607571113",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets)",
       "n": "two Cornell cohorts, 575 shared compounds",
       "design": "cross-cohort concordance",
       "result": "patient-control differences agree only faintly (rho 0.16); model trained on one scores AUC 0.56 on the other; a public GitHub copy of Germain 2020 has case/control labels swapped",
       "url": "/#data",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome convergence)",
       "n": "4 omic layers",
       "design": "pathway-score correlation with permutation null",
       "result": "no protein-RNA-metabolite-genetics convergence beyond chance (one modest protein-metabolite rho 0.28)",
       "url": "/#data",
       "verified": true
      }
     ],
     "open_questions": [
      "Pre-registered external validation with disease controls (fibromyalgia, depression, MS, long COVID)?",
      "Person-level train/test splits for longitudinal data?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "immune-exhaustion"
     ],
     "test_available": "research only"
    },
    {
     "id": "omics-coverage-limits",
     "subdomain": "Most comprehensive blood studies",
     "finding": "Even the deepest blood omics studies sample mainly at rest, in mild-to-moderate patients, in plasma or serum only, and rarely test fresh-cell function or the 48-72 h PEM window.",
     "grade": "replicated",
     "plain": "The biggest blood studies so far mostly leave out the sickest patients, the days when symptoms flare after effort, and live-cell tests.",
     "detail": "Walitt 2024 (NIH, 17/21) is the deepest per person but tiny and excluded many severe patients by design. BioMapAI has 4 years of visits but resting samples only. Germain 2022/2025 and Giloteaux/Glass cover 0, 15 min and 24 h after CPET but not 48-72 h, and the most impaired cannot do CPET (5 of 8 missing post-exercise samples were severe men). UK Biobank is large but one baseline time point with loose case definition. Hoel 2021/2026 are resting serum. Fresh-cell assays (PBMC respiration, NK function, red-cell deformability) are rarely paired with omics; compartments like muscle, CSF and gut tissue are rarely sampled together.",
     "studies": [
      {
       "cite": "Walitt 2024, Nat Commun",
       "n": "17 vs 21",
       "design": "deep phenotyping, multiple compartments",
       "result": "many layers measured, small n",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Xiong 2025 (BioMapAI), Nat Med",
       "n": "249 participants, 4-year longitudinal",
       "design": "deep neural net on stool metagenome, plasma metabolome, immune profiling, labs, symptoms",
       "result": "10-fold CV AUC 0.915 (all omics); external cohorts AUC 0.59-0.77 (Guo, Raijmakers stool; Germain metabolome 0.68, Che 0.61) with 19-79% feature overlap",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      },
      {
       "cite": "Germain 2025, Mol Cell Proteomics",
       "n": "79 vs 53 (sedentary controls)",
       "design": "2-day CPET, SomaScan 7k, longitudinal",
       "result": "widespread changes after exertion, most in recovery; suppressed T/B signalling and IL-17, higher glycolysis proteins; sex differences",
       "url": "https://doi.org/10.1016/j.mcpro.2025.101467",
       "verified": true
      },
      {
       "cite": "Beentjes 2025, EMBO Mol Med",
       "n": "up to 1,455 cases vs 131,303 controls (UK Biobank); All of Us replication",
       "design": "population cohort, 3,237 blood traits, mediation analysis",
       "result": "hundreds of traits differ (116 in both sexes): inflammation, insulin resistance, liver; 9/14 replicated in All of Us; activity mediates only 1/3,237; no single trait discriminates",
       "url": "https://doi.org/10.1038/s44321-025-00258-8",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Who will run a multi-omic study in severe/housebound patients with home sampling?",
      "Dense sampling through a PEM episode?"
     ],
     "hypotheses": [],
     "test_available": "n/a"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "cell-danger-response",
     "for": "Naviaux 2016: 80% of discriminating metabolites lower; lipid/purine changes; Hoel 2021 energy strain.",
     "against": "No peer-reviewed replication of a homogeneous hypometabolic profile; pooled cohorts show small, mixed shifts."
    },
    {
     "id": "mitochondrial-energy",
     "for": "Fluge 2016 PDH pattern; lower carnitines/acylcarnitines; low muscle proteins in blood; Germain 2022 blunted glucose rise; Hoel 2021 metabotypes.",
     "against": "Lactate/pyruvate and other energy ratios null in pooled data; exercise metabolome response mostly normal within person."
    },
    {
     "id": "metabolic-trap-ido",
     "for": "Model plausibility (Kashi 2019); Kavyani 2024 higher kynurenine tracking severity.",
     "against": "Pooled data show slightly lower kynurenine; kyn/trp ratio null; IDO1/KYNU protein null; plasma cannot test intracellular kinetics."
    },
    {
     "id": "gut-dysbiosis",
     "for": "Low IPA in 4/4 cohorts tied to butyrate producers; Xiong 2023 low plasma butyrate; BioMapAI connectivity.",
     "against": "Aromatic lactates, the strongest metabolite signal, are not stool-predicted."
    },
    {
     "id": "oxidative-stress",
     "for": "Methionine sulfoxide +0.35 SD in 7/7 cohorts; Germain 2018; post-exercise oxidised cys-gly; Shungu 2012 low brain GSH.",
     "against": "Same changes in depression; storage artefacts possible; antioxidant trials unconvincing."
    },
    {
     "id": "connective-tissue",
     "for": "Higher collagen breakdown products; lower ECM/fibroblast and mural proteins.",
     "against": "Re-analysis only; diet/supplements not excluded."
    },
    {
     "id": "small-vessel-capillary",
     "for": "Lower NOTCH3, ITGAV, TGF-beta and vascular smooth-muscle protein sets.",
     "against": "Endothelial activation and platelet/microclot protein sets are lower, not higher."
    },
    {
     "id": "immune-exhaustion",
     "for": "cfRNA T-cell exhaustion signature (Gardella 2025); Germain 2025 suppressed T/B signalling after exertion; plasma-cell RNA dip.",
     "against": "No replicated single-cell or bulk transcript difference."
    },
    {
     "id": "serum-factor",
     "for": "Nanoneedle plasma-swap effect; Fluge 2016 serum effects on myoblasts.",
     "against": "No independent replication of the nanoneedle assay."
    },
    {
     "id": "complement-thromboinflammation",
     "for": "Complement up in Hoel and UKB; EV complement rise post-exercise in men.",
     "against": "Complement direction inconsistent across cohorts (down in Walitt); platelet set lower."
    }
   ],
   "glossary": [
    {
     "term": "Metabolomics",
     "plain": "Measuring hundreds to thousands of small molecules (sugars, fats, amino acids, drug traces) in a sample at once."
    },
    {
     "term": "Proteomics (SomaScan, Olink)",
     "plain": "Measuring thousands of proteins at once; SomaScan uses DNA-like aptamers, Olink uses antibody pairs. Both can be distorted by how a sample is handled."
    },
    {
     "term": "Effect size (SD, d)",
     "plain": "How far apart the group averages are, in units of normal person-to-person spread. 0.2 is small, 0.5 moderate, 0.8 large."
    },
    {
     "term": "AUC",
     "plain": "How well a test separates two groups: 0.5 is a coin flip, 1.0 is perfect. Values from the same data used to build a model are usually too optimistic."
    },
    {
     "term": "Cell danger response (CDR)",
     "plain": "Naviaux's idea that cells under threat switch into a protective, low-energy mode, which in ME/CFS fails to switch off."
    },
    {
     "term": "IDO metabolic trap",
     "plain": "A model in which the tryptophan-processing enzyme IDO1 gets stuck in a low-activity state because of substrate inhibition and inactive IDO2 variants."
    },
    {
     "term": "Plasmalogens",
     "plain": "Membrane fats made partly in peroxisomes; thought to protect against oxidation."
    },
    {
     "term": "Acylcarnitines",
     "plain": "Fats bound to carnitine for transport into mitochondria; their profile hints at how fat is burned."
    },
    {
     "term": "Extracellular vesicles (EVs)",
     "plain": "Tiny membrane bubbles released by cells into blood, carrying proteins and RNA."
    },
    {
     "term": "Cell-free RNA (cfRNA)",
     "plain": "RNA fragments in plasma released by dying or active cells, showing which tissues are busy."
    },
    {
     "term": "DNA methylation / epigenetic clock",
     "plain": "Chemical tags on DNA that change gene activity; clocks estimate biological age from them. Results depend heavily on the mix of cell types."
    },
    {
     "term": "Cross-validation vs external validation",
     "plain": "Testing a model on parts of the same data set vs on a completely separate group of people. Only the second shows real-world performance."
    }
   ],
   "key_reviews": [
    {
     "cite": "Yamano 2021, Int J Mol Sci",
     "url": "https://doi.org/10.3390/ijms22073423",
     "why": "Overview of metabolite biomarker studies in ME/CFS and animal models."
    },
    {
     "cite": "Davis 2025, Front Neurosci",
     "url": "https://doi.org/10.3389/fnins.2025.1498981",
     "why": "Review of lipid, energy and oxidative-stress omics across ME/CFS, Gulf War illness and fibromyalgia."
    },
    {
     "cite": "Kavyani 2022, Cell Mol Life Sci",
     "url": "https://doi.org/10.1007/s00018-022-04380-5",
     "why": "Review of the kynurenine pathway case in ME/CFS."
    },
    {
     "cite": "Beentjes 2025, EMBO Mol Med",
     "url": "https://doi.org/10.1038/s44321-025-00258-8",
     "why": "Largest blood-trait analysis, with explicit handling of inactivity as a confounder."
    },
    {
     "cite": "Xiong 2025 (BioMapAI), Nat Med",
     "url": "https://doi.org/10.1038/s41591-025-03788-3",
     "why": "Largest longitudinal multi-omic data set; reports external validation numbers worth reading critically."
    }
   ],
   "gaps": [
    "No multi-omic study in severe or housebound patients with home sampling.",
    "No dense sampling across a PEM episode (48-72 h and beyond); exertion studies stop at 24 h.",
    "Very few studies include disease controls (fibromyalgia, depression, MS, deconditioning, long COVID) needed to test specificity.",
    "Drug exposure, caffeine, fasting status and sample processing time are rarely recorded or adjusted, despite large effects on metabolomics and proteomics.",
    "No pre-registered, multi-site external validation of any diagnostic classifier (nanoneedle, Raman, cfRNA, BioMapAI).",
    "Targeted confirmation of low aromatic lactates (with D/L isomers) and high collagen breakdown in fasting, drug-free samples.",
    "Lean mass not measured alongside the low muscle-protein signal.",
    "Fresh-cell functional assays (PBMC respiration, NK cytotoxicity, red-cell deformability) are not paired with omics in the same people.",
    "Only one lab's PBMC single-cell RNA data exist; no independent scRNA cohort.",
    "Intracellular NAD+/NADH and tryptophan flux have not been measured directly in patient tissue."
   ],
   "comprehensive_studies": [
    {
     "name": "Walitt 2024 (NIH intramural)",
     "n": "17 PI-ME/CFS vs 21 HV",
     "compartments": "plasma, CSF, PBMC, stool, muscle physiology, brain imaging",
     "timepoints": "mostly single visit; exercise tests",
     "severity": "mild-moderate; strict entry criteria",
     "fresh_cell_function": "flow cytometry only",
     "limits": "very small n; results by sex underpowered"
    },
    {
     "name": "BioMapAI / Xiong 2025 (JAX, Bateman Horne)",
     "n": "249 participants, 4 years",
     "compartments": "plasma metabolome, stool metagenome, immune flow, routine labs",
     "timepoints": "up to 4 yearly visits at rest",
     "severity": "mixed, mostly ambulatory",
     "fresh_cell_function": "stimulated cytokine flow",
     "limits": "no disease controls; external AUC 0.59-0.77"
    },
    {
     "name": "Germain 2022 / 2025, Giloteaux 2024, Glass 2025 (Cornell)",
     "n": "60/45 metabolome; 79/53 proteome; EV subsets 18/17 F, 10/12 M",
     "compartments": "plasma metabolome, SomaScan proteome, EVs",
     "timepoints": "pre/post CPET day 1 and day 2 (15 min, 24 h)",
     "severity": "able to complete 2 CPETs",
     "fresh_cell_function": "none in these papers",
     "limits": "no 48-72 h samples; most impaired excluded or missing"
    },
    {
     "name": "Beentjes 2025 (UK Biobank, All of Us)",
     "n": "up to 1,455 cases vs 131,303",
     "compartments": "routine biochemistry, counts, NMR, Olink",
     "timepoints": "single baseline",
     "severity": "unknown; record/self-report diagnosis",
     "fresh_cell_function": "none",
     "limits": "loose case definition; healthy-volunteer bias"
    },
    {
     "name": "Hoel 2021 / Hoel 2026 (Bergen)",
     "n": "83/35 metabolome; 50/29 proteome",
     "compartments": "serum",
     "timepoints": "single resting",
     "severity": "mild to severe",
     "fresh_cell_function": "none",
     "limits": "sample-handling signal in proteome; per-person data not public"
    }
   ]
  },
  {
   "domain": "endocrine",
   "title": "Hormones",
   "overview": "The most consistent hormone finding in ME/CFS is a mildly under-active stress-hormone (HPA) axis: on average cortisol is slightly low in saliva, urine and hair, and feedback suppression is exaggerated. A 2026 meta-analysis of 46 case-control studies confirms this pattern. The shifts are small, most patients fall inside normal reference ranges, and they may be a consequence of illness, sleep disruption or inactivity rather than a cause. Low-dose hydrocortisone gave at best a small, short-lived benefit with side effects, and fludrocortisone failed in two large trials. Newer leads concern the hormones that hold water and salt in the body: low vasopressin and a 'renin-aldosterone paradox' that would fit the low blood volume seen in many patients. These come from small or uncontrolled studies, and the vasopressin finding is not matched by copeptin, which is the more reliable test. The female preponderance (about 1.5 to 3 times more women) is solid, but genetics shows no sex-specific risk variants, and the role of sex hormones, menopause and gynaecological conditions is still only descriptive.",
   "findings": [
    {
     "id": "endocrine-hpa-low-cortisol",
     "subdomain": "HPA axis",
     "finding": "On average, people with ME/CFS have mildly lower free cortisol (salivary awakening and morning cortisol, 24-h urinary cortisol and hair cortisol) than healthy controls.",
     "grade": "replicated",
     "plain": "Cortisol is the body's main stress and energy-mobilising hormone. On average it runs a little low in ME/CFS, but the gap is modest and most individual results are still normal.",
     "detail": "Woo et al. 2026 pooled 46 case-control datasets (1,388 patients, 1,349 controls, 71.9% female) and found lower salivary awakening and morning cortisol, 24-h urinary free cortisol and hair cortisol. Hair cortisol, which reflects about 3 months of exposure, was also lower in a small adolescent study (Vroegindeweij 2024). In the Solving ME/CFS literature tally, 19 groups report lower cortisol and 5 higher. In untargeted plasma metabolomics the signal is weak and inconsistent: Germain 2020 −0.64 SD, Germain 2022 −0.13, BioMapAI −0.05, with men −0.35 (Solving ME/CFS re-analysis). The meta-analysis does not separate cause from effect: sleep disruption, inactivity, illness duration and antidepressant use can all lower cortisol.",
     "studies": [
      {
       "cite": "Woo 2026, Molecular Psychiatry",
       "n": "46 studies; 1,388 vs 1,349",
       "design": "systematic review and meta-analysis",
       "result": "Lower salivary awakening/morning, 24-h urinary and hair cortisol",
       "url": "https://doi.org/10.1038/s41380-026-03608-1",
       "verified": true
      },
      {
       "cite": "Vroegindeweij 2024, Psychoneuroendocrinology",
       "n": "12 ME/CFS, 20 Q-fever fatigue, 8 post-COVID, 19 JIA vs 57 controls",
       "design": "case-control (LC-MS/MS hair cortisol)",
       "result": "Lower hair cortisol in fatigued patients than controls",
       "url": "https://doi.org/10.1016/j.psyneuen.2024.107117",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (epi_literature tally)",
       "n": "24 study groups",
       "design": "vote count of published studies",
       "result": "19 lower vs 5 higher cortisol",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis of BioMapAI plasma metabolome",
       "n": "about 236",
       "design": "case-control re-analysis",
       "result": "Plasma cortisol −0.05 SD overall (men −0.35, p=0.04)",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Is low cortisol present at onset, or does it develop with illness duration and inactivity?",
      "Does cortisol track PEM or severity within a person over time?",
      "How much do medication, sleep timing and sampling time explain?"
     ],
     "hypotheses": [
      "hpa-endocrine",
      "sickness-behaviour"
     ],
     "test_available": "Yes: 9am serum cortisol, salivary awakening cortisol, short Synacthen test (mainly to exclude true adrenal insufficiency)"
    },
    {
     "id": "endocrine-hpa-feedback",
     "subdomain": "HPA axis",
     "finding": "Challenge tests show a blunted cortisol response to ACTH and exaggerated cortisol suppression after a glucocorticoid dose, consistent with enhanced negative feedback.",
     "grade": "several",
     "plain": "When the stress-hormone system is pushed or braked in a test, it responds less to the push and more to the brake than in healthy people.",
     "detail": "Woo 2026 included 12 pharmacological challenge studies: impaired cortisol release after ACTH stimulation and greater suppression after dexamethasone or hydrocortisone. The pattern resembles the 'hypocortisolism with enhanced feedback' described in PTSD, fibromyalgia and atypical depression, so it is not specific to ME/CFS. Insulin stress tests in the Cleare 1999 trial showed the adrenal glands were not suppressed by low-dose hydrocortisone. POTS patients showed normal adrenal responses to ACTH (Laurin 2023, n=8 vs 5).",
     "studies": [
      {
       "cite": "Woo 2026, Molecular Psychiatry",
       "n": "12 challenge studies within 46",
       "design": "meta-analysis",
       "result": "Impaired ACTH-stimulated cortisol; exaggerated glucocorticoid suppression",
       "url": "https://doi.org/10.1038/s41380-026-03608-1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Laurin 2023, Autonomic Neuroscience",
       "n": "8 POTS vs 5",
       "design": "ACTH stimulation, low-sodium diet",
       "result": "Normal aldosterone and cortisol responses in POTS",
       "url": "https://doi.org/10.1016/j.autneu.2023.103105",
       "verified": true
      }
     ],
     "open_questions": [
      "Is the feedback change central (hypothalamus/pituitary) or adrenal?",
      "Does it differ between infection-onset and gradual-onset cases?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Research only (dexamethasone suppression or CRH tests); short Synacthen test is clinical"
    },
    {
     "id": "endocrine-longcovid-cortisol",
     "subdomain": "HPA axis",
     "finding": "Lower cortisol in long COVID, reported in a 2023 Nature cohort, has not held up in later clinic and cohort studies that controlled for sampling time.",
     "grade": "contested",
     "plain": "One high-profile study found low cortisol in long COVID. Later studies that timed the blood draws carefully mostly found normal levels.",
     "detail": "Klein 2023 (n=275) reported lower cortisol in long COVID among other immune features. Dalhuisen 2024 (UCSF, n=200, banked 8am–12pm samples) found no overall difference (median 8.9 vs 8.8 µg/dL). Its sub-hour analyses were inconsistent: lower at 8–9am but higher at 9–10am in the ME/CFS-like group. Quach 2025 (Stanford clinic, n=86, 5–10am) found only 1 low value; 36% were above range. Camici 2026 (Rome, n=96) found normal blood cortisol but flattened salivary rhythms. Post-COVID controls without symptoms showed the same salivary pattern, so the change may follow infection rather than long COVID itself.",
     "studies": [
      {
       "cite": "Klein 2023, Nature",
       "n": "275 total",
       "design": "cross-sectional immune profiling",
       "result": "Cortisol lower in long COVID",
       "url": "https://doi.org/10.1038/s41586-023-06651-y",
       "verified": true
      },
      {
       "cite": "Camici 2026, Front Cell Infect Microbiol",
       "n": "83 LC, 13 recovered, controls",
       "design": "case-control salivary profile",
       "result": "Reduced morning salivary cortisol, flattened rhythm; blood cortisol not different; ACTH higher",
       "url": "https://doi.org/10.3389/fcimb.2025.1690698",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Dalhuisen 2024, medRxiv",
       "n": "144 LC vs 56 recovered",
       "design": "cross-sectional, banked plasma",
       "result": "No overall difference in morning cortisol",
       "url": "https://doi.org/10.1101/2024.11.07.24316777",
       "verified": true
      },
      {
       "cite": "Quach 2025, Biomedicines",
       "n": "86",
       "design": "retrospective clinic series",
       "result": "Low morning cortisol in 1.2%; 36% elevated",
       "url": "https://doi.org/10.3390/biomedicines13112636",
       "verified": true
      }
     ],
     "open_questions": [
      "Do serial or salivary measurements in long COVID with PEM reproduce the ME/CFS pattern?",
      "Is the change post-infectious and non-specific?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Yes: timed morning cortisol; salivary diurnal profile (private labs)"
    },
    {
     "id": "endocrine-hydrocortisone-trials",
     "subdomain": "HPA axis treatment",
     "finding": "Low-dose hydrocortisone produced at most small, short-term fatigue benefits in two RCTs, at the cost of adrenal suppression and bone loss at higher doses.",
     "grade": "contested",
     "plain": "Replacing some cortisol was tried in the 1990s. The effect was small, side effects were real, and it is not recommended.",
     "detail": "McKenzie 1998 (JAMA, n=70, 25–35 mg/day for 12 weeks): global wellness responders 66.7% vs 54.3% (p=0.31). A secondary 5-point improvement was 53% vs 29% (p=0.04), but cosyntropin tests showed adrenal suppression. A sub-study (n=23) found lumbar spine bone density fell 2.0% on hydrocortisone. Cleare 1999 (Lancet, n=32 crossover, 5–10 mg for 1 month) found fatigue fell 4.5 points more than on placebo; 28% vs 9% reached normal-range fatigue. The trial was short, strictly selected (no psychiatric comorbidity) and never replicated. Blockmans 2003 (n=100 crossover, hydrocortisone 5 mg plus fludrocortisone 50 µg for 3 months) was null.",
     "studies": [
      {
       "cite": "Cleare 1999, Lancet",
       "n": "32 (crossover)",
       "design": "RCT crossover",
       "result": "Fatigue reduced vs placebo (paired difference 4.5 points, p=0.009)",
       "url": "https://doi.org/10.1016/s0140-6736(98)04074-4",
       "verified": true
      },
      {
       "cite": "McKenzie 1998, JAMA",
       "n": "70",
       "design": "RCT",
       "result": "Primary outcome ns (p=0.31); adrenal suppression",
       "url": "https://doi.org/10.1001/jama.280.12.1061",
       "verified": true
      },
      {
       "cite": "McKenzie 2000 (BMD sub-study of the 1998 trial)",
       "n": "23",
       "design": "RCT sub-study",
       "result": "Lateral spine BMD −2.0% on hydrocortisone",
       "url": "https://pubmed.ncbi.nlm.nih.gov/10990237/",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Blockmans 2003, Am J Med",
       "n": "100 (80 completed)",
       "design": "RCT crossover, hydrocortisone + fludrocortisone",
       "result": "No difference in fatigue or well-being",
       "url": "https://doi.org/10.1016/s0002-9343(03)00182-7",
       "verified": true
      }
     ],
     "open_questions": [
      "Would a subgroup with objectively low cortisol or blunted Synacthen response benefit?",
      "No modern trial has used PEM-based criteria."
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Not applicable (treatment); not recommended outside proven adrenal insufficiency"
    },
    {
     "id": "endocrine-fludrocortisone-null",
     "subdomain": "Salt-retaining hormones (treatment)",
     "finding": "Fludrocortisone, a synthetic salt-retaining steroid, did not improve CFS with neurally mediated hypotension in a 100-person RCT, and failed again in combination with hydrocortisone.",
     "grade": "refuted",
     "plain": "A drug that makes the kidneys hold salt and water was expected to help low blood pressure on standing. In proper trials it did not help overall.",
     "detail": "Rowe 2001 (JAMA, n=100, tilt-positive neurally mediated hypotension, 0.1 mg/day for 9 weeks): ≥15-point wellness improvement in 14% vs 10% (p=0.76). Blockmans 2003 (n=100) found no effect of hydrocortisone plus fludrocortisone. Small open series in POTS still report individual responders, and the trials did not select for measured hypovolaemia or low aldosterone.",
     "studies": [
      {
       "cite": "Rowe 2001, JAMA",
       "n": "100",
       "design": "RCT",
       "result": "No benefit (14% vs 10% responders)",
       "url": "https://doi.org/10.1001/jama.285.1.52",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would patients selected for low aldosterone or measured low plasma volume respond?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "hpa-endocrine"
     ],
     "test_available": "Not applicable (treatment)"
    },
    {
     "id": "endocrine-adrenal-pituitary-minority",
     "subdomain": "Adrenal and pituitary disease",
     "finding": "Diagnosed adrenal insufficiency and hypopituitarism are 7 to 13 times more common than expected among Finnish patients coded with postviral fatigue syndrome.",
     "grade": "early",
     "plain": "A small minority of people labelled ME/CFS may have a real, treatable gland failure. That makes basic hormone screening worthwhile.",
     "detail": "In Finnish registry data (FinRegistry about 1,622 G93.3 cases, FinnGen 348), the crude observed/expected ratio is 9.5–13 for adrenal insufficiency and 6.7–7.6 for hypopituitarism. Hydrocortisone and fludrocortisone prescriptions are enriched 11–62x (Solving ME/CFS re-analysis of Risteys). These figures are not adjusted for age or sex and are inflated by healthcare use. They may reflect misdiagnosis, a true subgroup, or steroid treatment causing adrenal suppression. Case reports describe isolated ACTH and GH deficiency first labelled as CFS, which improved with hydrocortisone (Tokumasu 2021).",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (FinnGen/FinRegistry Risteys)",
       "n": "about 1,622 and 348 cases",
       "design": "registry co-occurrence (O/E)",
       "result": "Adrenal insufficiency O/E 9.5–13; hypopituitarism 6.7–7.6",
       "url": "",
       "verified": true
      },
      {
       "cite": "Tokumasu 2021, BMJ Case Reports",
       "n": "1",
       "design": "case report",
       "result": "Idiopathic ACTH + GH deficiency mimicking CFS",
       "url": "https://doi.org/10.1136/bcr-2021-244861",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What fraction of a well-phenotyped ME/CFS cohort fails a Synacthen test?",
      "Is this misdiagnosis, a subgroup, or a treatment effect?"
     ],
     "hypotheses": [
      "hpa-endocrine",
      "pituitary-adh"
     ],
     "test_available": "Yes: 9am cortisol, ACTH, short Synacthen test; pituitary panel if indicated"
    },
    {
     "id": "endocrine-low-vasopressin",
     "subdomain": "Water balance (ADH/vasopressin)",
     "finding": "After overnight fluid deprivation, plasma vasopressin was undetectable in 82% of 111 Swedish ME/CFS patients despite high plasma osmolality in 57%, suggesting partial central diabetes insipidus-like physiology.",
     "grade": "early",
     "plain": "Vasopressin (ADH) is the hormone that tells the kidneys to keep water. Some patients appear to make too little of it even when their blood is concentrated, which could worsen low blood volume.",
     "detail": "Huhmar 2025 was a consecutive, uncontrolled clinic series (n=124). Urine osmolality was below 750 mOsm/kg in 66%. In the 13 patients tested with copeptin, which is a more stable marker of vasopressin secretion, all values were within the reference range, though at the low end. Brain MRI showed no hypothalamic or pituitary lesions. Miwa 2016 (18 vs 15) found ADH 2.2 vs 3.3 pg/mL (p=0.02) alongside lower aldosterone and small hearts. In open label use, desmopressin improved orthostatic symptoms in 5 of 10 patients. Vasopressin immunoassays are unreliable, and the normal copeptin results argue for caution.",
     "studies": [
      {
       "cite": "Huhmar 2025, Endocrine Practice",
       "n": "124 (111 vasopressin, 13 copeptin)",
       "design": "observational, uncontrolled",
       "result": "Vasopressin undetectable in 82%; copeptin normal-low",
       "url": "https://doi.org/10.1016/j.eprac.2025.12.020",
       "verified": true
      },
      {
       "cite": "Miwa 2016, J Cardiology",
       "n": "18 vs 15",
       "design": "case-control",
       "result": "Lower ADH (p=0.02) and aldosterone (p=0.004)",
       "url": "https://doi.org/10.1016/j.jjcc.2016.06.003",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Huhmar 2025 copeptin subset",
       "n": "13",
       "design": "observational",
       "result": "All copeptin values within reference range",
       "url": "https://doi.org/10.1016/j.eprac.2025.12.020",
       "verified": true
      }
     ],
     "open_questions": [
      "Does a controlled water-deprivation or hypertonic saline copeptin test confirm low vasopressin?",
      "Does low vasopressin predict desmopressin response?",
      "Is it central (hypothalamic) or due to altered osmoreceptor input?"
     ],
     "hypotheses": [
      "pituitary-adh",
      "low-blood-volume-preload"
     ],
     "test_available": "Yes: copeptin with paired plasma and urine osmolality (fasting); formal water deprivation or hypertonic saline test via endocrinology"
    },
    {
     "id": "endocrine-renin-aldosterone-paradox",
     "subdomain": "Renin-angiotensin-aldosterone",
     "finding": "Patients with POTS and with ME/CFS show normal or low renin and low aldosterone despite reduced blood volume (the 'renin-aldosterone paradox').",
     "grade": "several",
     "plain": "When blood volume is low, the kidneys should release renin and the adrenals aldosterone to retain salt. In POTS and ME/CFS this response seems muted.",
     "detail": "Raj 2005 (POTS 15 vs 14, iodine-131 albumin) found a plasma volume deficit of 334 vs 10 mL with unchanged renin activity and aldosterone of 190 vs 380 pmol/L (p=0.017). Miwa 2016 (ME 18 vs 15) found aldosterone 104 vs 157 pg/mL (p=0.004) and renin activity 1.6 vs 2.5 (p=0.06). The adrenal glands respond normally to ACTH in POTS (Laurin 2023), so the defect is upstream of the adrenal. In SomaScan and Olink proteomics, renin, ACE and BNP show no group difference (Solving ME/CFS re-analysis), but those assays measure protein abundance, not posture-stimulated activity.",
     "studies": [
      {
       "cite": "Raj 2005, Circulation",
       "n": "15 vs 14",
       "design": "case-control (POTS)",
       "result": "Low aldosterone, unchanged renin despite hypovolaemia",
       "url": "https://doi.org/10.1161/01.cir.0000160356.97313.5d",
       "verified": true
      },
      {
       "cite": "Miwa 2016, J Cardiology",
       "n": "18 vs 15",
       "design": "case-control (ME)",
       "result": "Lower aldosterone and borderline lower renin",
       "url": "https://doi.org/10.1016/j.jjcc.2016.06.003",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 proteome)",
       "n": "3 SomaScan cohorts + UKB",
       "design": "re-analysis",
       "result": "Renin, ACE, BNP, EPO, hepcidin not different at rest",
       "url": "",
       "verified": true
      },
      {
       "cite": "Zhang 2025, Vasc Health Risk Manag",
       "n": "65 vs 65 children (POTS)",
       "design": "case-control",
       "result": "Angiotensin II slightly higher in paediatric POTS",
       "url": "https://doi.org/10.2147/vhrm.s553129",
       "verified": true
      }
     ],
     "open_questions": [
      "Are supine/upright renin and aldosterone low in PEM-defined ME/CFS with measured blood volume?",
      "Is the defect renal, sympathetic or central?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Yes: supine and upright renin and aldosterone; blood volume (Daxor or CO-rebreathing) in specialist centres"
    },
    {
     "id": "endocrine-low-t3",
     "subdomain": "Thyroid",
     "finding": "In one Dutch case-control study, CFS patients had normal TSH but lower total T3, T4 and free T3, with a higher reverse-T3 fraction ('low T3 syndrome' pattern in 16% vs 7%).",
     "grade": "early",
     "plain": "The thyroid itself looks normal by standard tests, but the active thyroid hormone (T3) runs slightly lower, as seen in many long-term illnesses.",
     "detail": "Ruiz-Núñez 2018 (98 vs 99, age/sex matched): TT3 −12.5%, TT4 −11.9%, deiodinase activity −14.4%, rT3 fraction +13.3%. FT3 below range in 16/98 vs 7/99 (OR 2.56, 95% CI 1.00–6.54). The median difference in free T3 was tiny (0.1%), and thyroid values correlated with hsCRP. This pattern ('non-thyroidal illness') appears in many chronic and inflammatory states and is usually adaptive. It has not been independently replicated in ME/CFS. There is no trial of T3 treatment in ME/CFS.",
     "studies": [
      {
       "cite": "Ruiz-Núñez 2018, Front Endocrinol",
       "n": "98 vs 99",
       "design": "case-control",
       "result": "Lower TT3/TT4/FT3, higher %rT3, normal TSH; low-T3 OR 2.56",
       "url": "https://doi.org/10.3389/fendo.2018.00097",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does it replicate in a PEM-defined cohort?",
      "Is it a sign of reduced metabolic rate (hypometabolism) or just non-specific illness?"
     ],
     "hypotheses": [
      "hpa-endocrine",
      "mitochondrial-energy"
     ],
     "test_available": "Yes: TSH, free T4, free T3; reverse T3 (some labs)"
    },
    {
     "id": "endocrine-gh-igf1",
     "subdomain": "Growth hormone",
     "finding": "Reports of low IGF-1 and reduced growth hormone responses in CFS conflict, and the largest controlled study found an intact GH/IGF system.",
     "grade": "contested",
     "plain": "Growth hormone helps with repair and energy. Early small studies hinted it was low, but a larger careful study found it normal.",
     "detail": "Allain 1997 found lower IGF-I (214 vs 263 µg/L) and IGF-II and a reduced GH response to hypoglycaemia. Berwaerts 1998 (20 vs 20) found IGF-I SDS −0.39 vs +0.33 (p=0.02) and a trend to lower nocturnal GH (p=0.06), but normal stimulated GH peaks. Cleare 2000 (37 vs 37, medication-free) found no difference in IGF-I/IGFBPs, urinary GH or GHRH and insulin-stress responses. In a GH treatment RCT (Moorkens 1998, n=20) IGF-1 rose but quality of life did not improve.",
     "studies": [
      {
       "cite": "Allain 1997, Biol Psychiatry",
       "n": "small case-control",
       "design": "case-control with insulin tolerance test",
       "result": "Lower IGF-I/IGF-II; blunted GH response",
       "url": "https://doi.org/10.1016/s0006-3223(96)00074-1",
       "verified": true
      },
      {
       "cite": "Berwaerts 1998, Growth Horm IGF Res",
       "n": "20 vs 20",
       "design": "case-control",
       "result": "Lower IGF-I SDS; normal stimulated GH",
       "url": "https://doi.org/10.1016/s1096-6374(98)80036-1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Cleare 2000, J Clin Endocrinol Metab",
       "n": "37 vs 37",
       "design": "case-control with GHRH and insulin tests",
       "result": "No GH deficiency; IGF system intact",
       "url": "https://doi.org/10.1210/jcem.85.4.6513",
       "verified": true
      },
      {
       "cite": "Moorkens 1998, Growth Horm IGF Res",
       "n": "20",
       "design": "RCT of GH",
       "result": "IGF-1 rose; quality of life not improved",
       "url": "https://doi.org/10.1016/s1096-6374(98)80037-3",
       "verified": true
      }
     ],
     "open_questions": [
      "Is nocturnal GH reduced as a consequence of non-restorative sleep?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Yes: IGF-1 (stimulation tests only if pituitary disease suspected)"
    },
    {
     "id": "endocrine-female-preponderance",
     "subdomain": "Sex differences",
     "finding": "ME/CFS and long COVID are more common in women (ME/CFS prevalence about 1.4% vs 0.9%; DecodeME 82% female), but genetic risk variants are not sex-specific.",
     "grade": "replicated",
     "plain": "More women than men get ME/CFS and long COVID. Inherited risk looks the same in both sexes, so the difference probably lies in hormones, immunity or exposure, not in different genes.",
     "detail": "Lim 2020 meta-analysis: pooled prevalence 1.36% in women vs 0.86% in men. Clinic cohorts are typically 75–85% female. In RECOVER-Adult (n=12,276), female sex raised long COVID risk (RR 1.31, 95% CI 1.06–1.62), but not at age 18–39 (RR 1.04). At age 40–54 the RR was similar for menopausal (1.42) and non-menopausal (1.45) women. DecodeME: 12,833 female and 2,746 male cases, no sex-biased loci, and two female-discovered signals replicate in males. The mostly male Million Veteran Program replicates the polygenic signal (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Lim 2020, J Transl Med",
       "n": "46 studies, about 1.09 million",
       "design": "meta-analysis of prevalence",
       "result": "Women 1.36% vs men 0.86%",
       "url": "https://doi.org/10.1186/s12967-020-02269-0",
       "verified": true
      },
      {
       "cite": "Shah 2025, JAMA Netw Open (RECOVER)",
       "n": "12,276",
       "design": "prospective cohort",
       "result": "Female RR 1.31 for long COVID; age- and pregnancy-dependent",
       "url": "https://doi.org/10.1001/jamanetworkopen.2024.55430",
       "verified": true
      },
      {
       "cite": "DecodeME 2025, medRxiv preprint",
       "n": "15,579 cases",
       "design": "GWAS",
       "result": "No sex-biased loci; male replication of 2 signals",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Which biological factor (sex hormones, X-linked immune genes, autoimmunity propensity) drives the excess?",
      "Why is the excess absent in young adults in RECOVER?"
     ],
     "hypotheses": [
      "hpa-endocrine",
      "autoimmunity"
     ],
     "test_available": "Not applicable"
    },
    {
     "id": "endocrine-gynaecological-menopause",
     "subdomain": "Sex hormones and menopause",
     "finding": "Women with CFS report more endometriosis, heavy bleeding, pelvic pain, hysterectomy and earlier menopause than controls in US population-based case-control studies.",
     "grade": "early",
     "plain": "Gynaecological problems and early menopause are more common in women with ME/CFS. We do not know which comes first or why.",
     "detail": "Boneva 2015 (Georgia, 84 cases vs 73 controls) reported earlier menopause (37.6 vs 48.6 years; adjusted OR 1.22 per year), endometriosis 29.8% vs 12.3% (aOR 3.67), hysterectomy 54.8% vs 19.2% (aOR 3.23) and non-menstrual pelvic pain (aOR 12.0). The Wichita sample (36 vs 48) showed similar but mostly non-significant patterns. Surgical menopause after hysterectomy or oophorectomy confounds the 'early menopause' result, and recall bias is possible. 2026 survey work (Habermann-Horstmeier) describes different symptom structures by sex and a menopausal-status proxy, but it is cross-sectional and self-reported.",
     "studies": [
      {
       "cite": "Boneva 2015, Menopause",
       "n": "84 vs 73",
       "design": "population-based case-control",
       "result": "Earlier menopause, more endometriosis and hysterectomy",
       "url": "https://doi.org/10.1097/gme.0000000000000411",
       "verified": true
      },
      {
       "cite": "Boneva 2010, J Womens Health",
       "n": "36 vs 48",
       "design": "population-based case-control",
       "result": "More pelvic pain and endometriosis; menopause about 4 years earlier (ns)",
       "url": "https://doi.org/10.1089/jwh.2009.1900",
       "verified": true
      },
      {
       "cite": "Habermann-Horstmeier 2026, J Transl Med",
       "n": "survey cohort",
       "design": "cross-sectional factor analysis",
       "result": "Immune/GI symptom architecture differs by sex and menopausal proxy",
       "url": "https://doi.org/10.1186/s12967-026-08699-6",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does onset cluster at perimenopause after adjusting for age?",
      "Does HRT change ME/CFS course? No trial exists.",
      "Do symptoms vary with menstrual cycle in a measurable way?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Clinical gynaecological assessment; no ME/CFS-specific hormone test"
    },
    {
     "id": "endocrine-androgens-not-replicated",
     "subdomain": "Sex hormones",
     "finding": "Lower plasma androgenic steroids in ME/CFS (Cornell metabolomics) is mostly an age and sex artefact and does not replicate in BioMapAI.",
     "grade": "contested",
     "plain": "An earlier claim that patients have low 'male-type' steroid hormones mostly disappears once age and sex are accounted for.",
     "detail": "Across the two Cornell cohorts the androgenic steroid class was −0.42 SD, but in Germain 2022 the effect shrinks after age/sex/BMI adjustment. BioMapAI (n=236) shows −0.01 (p=0.90) and Che testosterone −0.18 (p=0.22) (Solving ME/CFS re-analysis). Cleare 2004 measured DHEA/DHEA-S and their response to CRH as a separate question.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets 03_steroids_crosscheck)",
       "n": "4 cohorts",
       "design": "re-analysis with covariate adjustment",
       "result": "Androgen deficit not replicated after adjustment",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Are sex steroids altered within defined subgroups (e.g. men, post-menopausal women)?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Yes: testosterone, SHBG, DHEA-S (not diagnostic for ME/CFS)"
    },
    {
     "id": "endocrine-melatonin-circadian",
     "subdomain": "Melatonin and circadian rhythm",
     "finding": "Melatonin may help the subgroup with delayed melatonin onset, and melatonin plus zinc modestly reduced physical fatigue in one small RCT.",
     "grade": "early",
     "plain": "Melatonin is the night-time hormone. A small trial and an open study suggest it can help some patients, especially those whose body clock runs late.",
     "detail": "van Heukelom 2006 (open-label, n=29 with DLMO after 21:30; 5 mg 5 h before DLMO for 3 months) improved fatigue, concentration and activity, more when DLMO was after 22:00. Fatigue normalised in 8/27. Castro-Marrero 2021 (RCT, n=50, melatonin 1 mg plus zinc 10 mg for 16 weeks) reduced physical fatigue at the last visit and improved SF-36 physical summary. Multiple outcomes were tested and the trial was not replicated. An older trial of melatonin and phototherapy reported no benefit (Williams 2002, from memory).",
     "studies": [
      {
       "cite": "Castro-Marrero 2021, Antioxidants",
       "n": "50",
       "design": "RCT",
       "result": "Reduced physical fatigue vs placebo (p<0.05)",
       "url": "https://doi.org/10.3390/antiox10071010",
       "verified": true
      },
      {
       "cite": "van Heukelom 2006, Eur J Neurol",
       "n": "29",
       "design": "open-label, DLMO-selected",
       "result": "Fatigue improved, mainly if DLMO >22:00",
       "url": "https://doi.org/10.1111/j.1468-1331.2006.01132.x",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Williams 2002, Eur J Clin Invest",
       "n": "about 30",
       "design": "randomised crossover (melatonin, phototherapy)",
       "result": "No benefit (from memory)",
       "url": "",
       "verified": false
      }
     ],
     "open_questions": [
      "What proportion of patients have delayed DLMO?",
      "Does timed low-dose melatonin help in a blinded DLMO-selected trial?"
     ],
     "hypotheses": [
      "hpa-endocrine"
     ],
     "test_available": "Research/private: salivary dim-light melatonin onset (DLMO); actigraphy"
    },
    {
     "id": "endocrine-genetic-pituitary",
     "subdomain": "Genetics of hormone systems",
     "finding": "ME/CFS genetic risk is enriched in brain tissues and the pituitary, but genetically predicted morning cortisol does not change risk.",
     "grade": "early",
     "plain": "Genes linked to ME/CFS are especially active in the brain and the pituitary, the master hormone gland. Genetically high or low cortisol does not seem to cause ME/CFS.",
     "detail": "In a DecodeME + Million Veteran Program meta-analysis (Maccallini 2026 preprint, 19,470 cases), tissue enrichment implicated multiple brain regions and the pituitary, with no peripheral tissue. MR using a single morning-cortisol instrument gave OR 1.04 (Solving ME/CFS re-analysis), which is underpowered. The pituitary result could reflect neural gene expression shared with brain rather than endocrine function.",
     "studies": [
      {
       "cite": "Maccallini 2026, Research Square preprint",
       "n": "19,470 vs 699,111",
       "design": "GWAS meta-analysis + enrichment",
       "result": "Brain regions and pituitary enriched",
       "url": "https://doi.org/10.21203/rs.3.rs-9702020/v1",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "DecodeME 15,579 cases",
       "design": "Mendelian randomisation",
       "result": "Morning cortisol (1 SNP) OR 1.04, null",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do pituitary-specific eQTLs colocalise with ME/CFS loci?",
      "Can a multi-SNP cortisol instrument test causality properly?"
     ],
     "hypotheses": [
      "hpa-endocrine",
      "pituitary-adh",
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "hpa-endocrine",
     "for": "A meta-analysis of 46 studies shows mildly low free cortisol and enhanced feedback. The genetic signal is enriched in the pituitary. Adrenal/pituitary diagnoses are enriched in Finnish registries.",
     "against": "The effects are small and overlap normal ranges. A similar pattern appears in other fatigue and pain conditions. Hydrocortisone and fludrocortisone trials were weak or null. Cortisol MR is null. In long COVID, low cortisol did not replicate in timed samples."
    },
    {
     "id": "pituitary-adh",
     "for": "One large uncontrolled series found undetectable vasopressin despite high osmolality. Miwa found low ADH and partial response to desmopressin.",
     "against": "Copeptin, the more reliable marker, was normal or low-normal. There is no controlled water-deprivation study and no pituitary lesions on MRI."
    },
    {
     "id": "low-blood-volume-preload",
     "for": "The renin-aldosterone paradox in POTS and ME, plus low ADH, would explain failure to restore low plasma volume.",
     "against": "Resting proteomic renin, ACE and BNP are normal. Fludrocortisone, which replaces aldosterone action, failed in RCTs."
    },
    {
     "id": "sickness-behaviour",
     "for": "Hypocortisolism with enhanced feedback fits a chronic post-infectious or inflammatory state.",
     "against": "This cannot be separated from deconditioning or sleep effects in cross-sectional data."
    }
   ],
   "glossary": [
    {
     "term": "HPA axis",
     "plain": "The hypothalamus-pituitary-adrenal chain that controls cortisol release."
    },
    {
     "term": "Cortisol awakening response",
     "plain": "The normal rise in cortisol in the 30–45 minutes after waking, measured in saliva."
    },
    {
     "term": "Synacthen (ACTH stimulation) test",
     "plain": "An injection of synthetic ACTH, with cortisol measured afterwards, to check whether the adrenal glands work."
    },
    {
     "term": "Dexamethasone suppression test",
     "plain": "A dose of a strong steroid to see how much the body's own cortisol is switched off (feedback sensitivity)."
    },
    {
     "term": "Vasopressin (ADH)",
     "plain": "The pituitary hormone that makes the kidneys keep water."
    },
    {
     "term": "Copeptin",
     "plain": "A stable fragment released with vasopressin. It is easier to measure accurately than vasopressin itself."
    },
    {
     "term": "Renin and aldosterone",
     "plain": "Kidney and adrenal hormones that make the body hold salt and water when blood volume is low."
    },
    {
     "term": "Low T3 syndrome",
     "plain": "Low active thyroid hormone with normal TSH, often seen in chronic illness rather than thyroid disease."
    },
    {
     "term": "IGF-1",
     "plain": "A blood marker of growth hormone activity."
    },
    {
     "term": "DLMO",
     "plain": "Dim-light melatonin onset, the evening time melatonin starts rising. It is a marker of body-clock timing."
    }
   ],
   "key_reviews": [
    {
     "cite": "Woo 2026, Molecular Psychiatry",
     "url": "https://doi.org/10.1038/s41380-026-03608-1",
     "why": "Most comprehensive meta-analysis of HPA findings (46 studies, all sample types and challenge tests)."
    },
    {
     "cite": "Zhang & Wang 2024, Front Endocrinol",
     "url": "https://doi.org/10.3389/fendo.2024.1373748",
     "why": "Review of HPA changes and HPA-targeted treatments in CFS."
    },
    {
     "cite": "Wirth 2026, Preprints.org",
     "url": "https://doi.org/10.20944/preprints202602.1993.v1",
     "why": "Hypothesis review of why vasopressin and RAAS fail to correct hypovolaemia (not peer reviewed)."
    },
    {
     "cite": "Miwa 2026, Front Med",
     "url": "https://doi.org/10.3389/fmed.2026.1744154",
     "why": "Clinical update on small heart, orthostatic intolerance and down-regulated volume hormones."
    }
   ],
   "gaps": [
    "No controlled copeptin study with a standard stimulus (water deprivation, hypertonic saline or arginine) in PEM-defined ME/CFS.",
    "No study pairs measured blood volume with supine/upright renin, aldosterone and copeptin in the same patients.",
    "No longitudinal study shows whether low cortisol precedes illness, as it could in post-infection cohorts.",
    "No modern RCT of any hormone therapy using PEM-based criteria or biomarker selection, for example hydrocortisone only in Synacthen low-responders, or desmopressin with copeptin stratification.",
    "There are no trials of HRT or menstrual-cycle-linked interventions, and no prospective data on onset around perimenopause.",
    "Low T3 has not been replicated and never tested as a treatment target.",
    "Prevalence of true adrenal insufficiency or hypopituitarism among clinic ME/CFS patients is unknown."
   ]
  },
  {
   "domain": "gut",
   "title": "Gut and microbiome",
   "overview": "Gut bacteria differ between people with ME/CFS and healthy controls more consistently than almost any blood measure. Fewer butyrate-producing species (Faecalibacterium, Roseburia, Coprococcus, Anaerostipes, Eubacterium rectale) and more Ruminococcus gnavus-type species appear in at least four independent cohorts across three labs, including a pre-specified test in the NIH cohort. The same pattern appears in long COVID. Blood levels of a gut-made metabolite, indole-3-propionate (IPA), are lower in four of four plasma cohorts, and re-analysis traces this to the R. gnavus-type overgrowth. What is not established is whether any of this causes symptoms. Dysbiosis is milder in long-standing illness, does not track severity reliably, and genetically predicted gut metabolite levels do not change ME/CFS risk. Markers of a 'leaky gut' (LPS, sCD14) come from a handful of studies, and the only randomised faecal transplant trial (n=11) was negative. Larger trials are under way.",
   "findings": [
    {
     "id": "gut-butyrate-producer-loss",
     "subdomain": "Dysbiosis",
     "finding": "Stool from ME/CFS patients has fewer butyrate-producing bacteria (notably Faecalibacterium prausnitzii, Eubacterium rectale, Roseburia, Coprococcus) and reduced microbial capacity to make butyrate.",
     "grade": "replicated",
     "plain": "Some 'good' gut bacteria that feed the gut lining by making butyrate are consistently scarcer in ME/CFS.",
     "detail": "Guo 2023 (106 vs 91, shotgun metagenomics, qPCR and stool SCFA) found lower F. prausnitzii and E. rectale and lower butyrate synthesis capacity. Its classifier was robust across US sites and to a validation cohort. Xiong 2023 (75 short-term, 79 long-term vs 79) found reduced butyrate biosynthesis genes and lower plasma butyrate, mostly in short-term patients. Giloteaux 2016 (16S, 48 vs 39) found lower diversity and fewer Firmicutes. Nagy-Szakal 2017 (50 vs 50) found lower Faecalibacterium in the IBS subgroup. In the NIH Walitt cohort (15 vs 17), a pre-specified 14-species signature built on other cohorts separated patients with AUC 0.87; all butyrate producers combined were −0.87 SD (Solving ME/CFS re-analysis). The NIH authors' own per-species tests did not pass FDR (min q 0.088).",
     "studies": [
      {
       "cite": "Guo 2023, Cell Host Microbe",
       "n": "106 vs 91",
       "design": "case-control, shotgun metagenomics + SCFA metabolomics",
       "result": "Reduced F. prausnitzii, E. rectale and butyrate synthesis capacity",
       "url": "https://doi.org/10.1016/j.chom.2023.01.004",
       "verified": true
      },
      {
       "cite": "Xiong 2023, Cell Host Microbe",
       "n": "154 vs 79",
       "design": "case-control, metagenomics + plasma metabolomics",
       "result": "Reduced butyrate biosynthesis; dysbiosis strongest in short-term illness",
       "url": "https://doi.org/10.1016/j.chom.2023.01.001",
       "verified": true
      },
      {
       "cite": "Giloteaux 2016, Microbiome",
       "n": "48 vs 39",
       "design": "case-control, 16S",
       "result": "Lower diversity, fewer Firmicutes",
       "url": "https://doi.org/10.1186/s40168-016-0171-4",
       "verified": true
      },
      {
       "cite": "Nagy-Szakal 2017, Microbiome",
       "n": "50 vs 50",
       "design": "case-control, metagenomics",
       "result": "Dysbiosis, with distinct taxa depending on IBS status",
       "url": "https://doi.org/10.1186/s40168-017-0261-y",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis of Walitt 2024 NIH stool",
       "n": "15 vs 17",
       "design": "pre-specified signature test",
       "result": "AUC 0.87; butyrate producers −0.87 SD (p=0.015)",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Walitt 2024, Nat Commun (authors' own analysis)",
       "n": "15 vs 17",
       "design": "case-control, per-species tests",
       "result": "No species significant after FDR",
       "url": "https://doi.org/10.1038/s41467-024-45107-3",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (3-cohort species meta)",
       "n": "NIH, Raijmakers, Columbia",
       "design": "random-effects meta-analysis",
       "result": "Effect sizes agree NIH vs Columbia (rho 0.57) but not NIH vs Netherlands (rho 0.07)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Is the change a cause, or a consequence of diet, inactivity, medication or IBS?",
      "Does it appear before or at onset in prospective post-infection cohorts?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Private shotgun stool metagenomics only; no validated clinical test or reference range"
    },
    {
     "id": "gut-rgnavus-bloom",
     "subdomain": "Dysbiosis",
     "finding": "Ruminococcus gnavus, Flavonifractor plautii, Ruthenibacterium lactatiformans and Enterocloster species are increased in ME/CFS, consistently across BioMapAI, Guo 2023 and NIH cohorts and in long COVID.",
     "grade": "several",
     "plain": "Alongside the loss of 'good' species, a group of bacteria often linked to gut inflammation is overgrown.",
     "detail": "In BioMapAI/JAX data (143 vs 88 people), R. lactatiformans (β +0.69), R. gnavus (+0.68) and F. plautii (+0.60) were raised (all q<1e-3). Guo 2023 agreed in direction for 14/17 shared species (binomial p=0.013; Solving ME/CFS re-analysis), but Guo listed only nominally significant species. In the NIH cohort, R. gnavus was +0.80 SD (p=0.014), E. bolteae +0.91 and C. symbiosum +0.77. Long COVID shows the same direction: Liu 2022 (106 patients followed 6 months) found higher R. gnavus and lower F. prausnitzii in post-acute COVID syndrome.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis of BioMapAI (Xiong 2025)",
       "n": "143 vs 88",
       "design": "species association, adjusted",
       "result": "R. gnavus, F. plautii, R. lactatiformans raised",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      },
      {
       "cite": "Liu 2022, Gut",
       "n": "106 COVID patients, 68 controls",
       "design": "prospective cohort, metagenomics",
       "result": "PACS linked to higher R. gnavus, lower F. prausnitzii",
       "url": "https://doi.org/10.1136/gutjnl-2021-325989",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does R. gnavus produce inflammatory polysaccharides or interfere with tryptophan metabolism in these patients?",
      "Is the bloom driven by antibiotics, diet or low gut transit?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "innate-hyperactivation"
     ],
     "test_available": "Private stool metagenomics only"
    },
    {
     "id": "gut-low-ipa",
     "subdomain": "Microbial metabolites",
     "finding": "Plasma indole-3-propionate (IPA), a gut-bacteria product of tryptophan, is lower in ME/CFS in 4 of 4 plasma cohorts (pooled −0.33 SD), and the deficit is fully explained by stool composition, mainly the R. gnavus-type overgrowth.",
     "grade": "replicated",
     "plain": "A protective molecule that only gut bacteria make is slightly low in patients' blood. The overgrown bacteria seem to crowd out the microbes that make it.",
     "detail": "Pooled across Germain 2020, Germain 2022, Che 2022 and BioMapAI: −0.33 SD (95% CI −0.49 to −0.16), p=0.0001. As a test it is weak (AUC 0.57–0.60). In 231 people with paired stool and plasma, a stool model predicted plasma IPA (CV Spearman 0.44), and adjusting for it removed the patient-control gap (−0.36 → −0.06). R. gnavus, F. plautii and R. lactatiformans were each inversely related to IPA. The IPA-producing C. sporogenes guild was not reduced. Fruit intake tracks IPA. No severity association survived FDR (all Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (datasets 02, deep2 microbiome_function)",
       "n": "4 plasma cohorts; 231 paired stool-plasma",
       "design": "meta-analysis + cross-validated prediction",
       "result": "IPA −0.33 SD; gap explained by stool composition",
       "url": "",
       "verified": true
      },
      {
       "cite": "Xiong 2025 (BioMapAI), Nat Med",
       "n": "249",
       "design": "longitudinal multi-omics",
       "result": "Altered microbiome-tryptophan-metabolite links",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (Mendelian randomisation)",
       "n": "DecodeME 15,579; MVP",
       "design": "two-sample MR",
       "result": "Genetically higher IPA does not lower ME/CFS risk (OR 1.19, 0.93–1.53)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Does raising IPA (diet, fibre, polyphenols) change any symptom?",
      "Does low IPA replicate with paired stool in a second cohort?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "metabolic-trap-ido"
     ],
     "test_available": "Research metabolomics only"
    },
    {
     "id": "gut-aromatic-lactates-host",
     "subdomain": "Microbial metabolites",
     "finding": "The replicated deficit of aromatic lactates (indolelactate, phenyllactate, 4-hydroxyphenyllactate) in ME/CFS blood is not predicted by stool bacteria or bacterial genes, so it is probably host-side or due to absorption, not dysbiosis.",
     "grade": "early",
     "plain": "Another set of low blood molecules looks like gut products, but the gut bacteria data do not explain them, so probiotics aimed at them are unlikely to help.",
     "detail": "Aromatic-lactate score −0.55 SD (p 6.5e-6) in BioMapAI, and −0.53 SD across 5 cohorts. Stool species and gene models predict it at zero (CV Spearman 0.01). 0/4,429 KEGG genes and 0/1,696 taxa associate with it, and Bifidobacterium plus Lactobacillus abundance is uncorrelated (rho −0.01). Adjusting for diet, medications, IBS and kidney function makes the deficit larger, not smaller. In Esteban 2026, stool aromatic metabolites were higher in patients, which hints at reduced absorption (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 microbiome_function)",
       "n": "231 paired; 5 plasma cohorts",
       "design": "cross-validated prediction, hypothesis-free scans",
       "result": "No gut predictor of the lactate deficit",
       "url": "",
       "verified": true
      },
      {
       "cite": "Esteban 2026, MicrobiologyOpen",
       "n": "19 vs 23",
       "design": "case-control, targeted stool metabolomics + AHR assay",
       "result": "Nine tryptophan metabolites elevated in stool; AHR activation not different",
       "url": "https://doi.org/10.1002/mbo3.70333",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is it host transamination/LDH handling, small-intestine microbes (not sampled by stool) or uptake?",
      "D- vs L-phenyllactate would distinguish bacterial from human origin."
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "mitochondrial-energy"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-duration-dependence",
     "subdomain": "Dysbiosis",
     "finding": "Gut dysbiosis is strongest in the first years of illness and largely resolves in patients ill for more than 10 years, while their metabolic and clinical abnormalities persist.",
     "grade": "early",
     "plain": "Gut differences are clearest early in the illness and fade later, even though patients stay ill. This argues against gut bacteria alone keeping the illness going.",
     "detail": "Xiong 2023: short-term (<4 years, n=75) patients showed significant microbial dysbiosis, while long-term (>10 years, n=79) patients had largely resolved dysbiosis but more severe metabolic and clinical aberrations. This comes from one cross-sectional comparison of two cohorts, not follow-up of the same people. The NIH cohort, which is mostly under 5 years ill, still shows the signature (AUC 0.87).",
     "studies": [
      {
       "cite": "Xiong 2023, Cell Host Microbe",
       "n": "75 short, 79 long vs 79",
       "design": "cross-sectional two-cohort comparison",
       "result": "Dysbiosis mainly in short-term illness",
       "url": "https://doi.org/10.1016/j.chom.2023.01.001",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do the same patients' microbiomes normalise over time?",
      "Is early dysbiosis a consequence of the triggering infection or antibiotics?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-severity-link",
     "subdomain": "Dysbiosis and symptoms",
     "finding": "Links between specific gut bacteria and symptom severity are inconsistent: F. prausnitzii tracked fatigue in one cohort, but nothing survived correction for multiple testing in a 143-patient re-analysis.",
     "grade": "contested",
     "plain": "It is not yet clear that people with more abnormal gut bacteria are sicker.",
     "detail": "Guo 2023 found F. prausnitzii abundance inversely associated with fatigue severity. Nagy-Szakal 2017 reported correlations between distinct taxa and pain, fatigue and motivation within subgroups. In BioMapAI (143 patients), no taxon or metabolite module associated with severity, orthostatic intolerance or duration at FDR. The best nominal result was lower IPA with worse GI symptoms (p=0.005, q=0.41) (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Guo 2023, Cell Host Microbe",
       "n": "106 vs 91",
       "design": "case-control",
       "result": "F. prausnitzii inversely associated with fatigue",
       "url": "https://doi.org/10.1016/j.chom.2023.01.004",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis of BioMapAI",
       "n": "143 patients",
       "design": "within-patient association",
       "result": "No severity, OI or duration association at FDR",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Do within-person changes in microbiome track PEM episodes?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-ibs-overlap",
     "subdomain": "IBS and GI symptoms",
     "finding": "IBS is about seven times more common in ME/CFS (pooled OR 7.20; prevalence about 37%), and IBS status drives much of the measured microbiome difference.",
     "grade": "replicated",
     "plain": "Many people with ME/CFS also have irritable bowel syndrome. Studies need to separate the two, because IBS changes gut bacteria by itself.",
     "detail": "Rajabi 2026 meta-analysis: 22 studies, 18 pooled. OR 7.20 (2.77–18.76), I² 72%; pooled IBS prevalence 37% (23–54%), varying by ME/CFS criteria. Brown 2026 (116 vs 80): 53% vs 8% prior IBS diagnosis, with GI symptom burden tracking fatigue, cognitive, pain and flu-like symptoms. In Nagy-Szakal 2017, IBS co-morbidity was the strongest factor separating microbiome networks, and the top marker taxa differed between IBS and non-IBS subgroups. Genetically, ME/CFS correlates with IBS (rg 0.57) and its heritability is enriched in enteric neurons (Lee 2026 medRxiv; from Solving ME/CFS genetics summary).",
     "studies": [
      {
       "cite": "Rajabi 2026, Front Med",
       "n": "18 studies pooled",
       "design": "systematic review and meta-analysis",
       "result": "OR 7.20 for IBS; prevalence 37%",
       "url": "https://doi.org/10.3389/fmed.2026.1881784",
       "verified": true
      },
      {
       "cite": "Brown 2026, J Transl Med",
       "n": "116 vs 80",
       "design": "case-control",
       "result": "IBS 53% vs 8%; GI burden correlates with CRP and core symptoms",
       "url": "https://doi.org/10.1186/s12967-026-08442-1",
       "verified": true
      },
      {
       "cite": "Nagy-Szakal 2017, Microbiome",
       "n": "50 vs 50",
       "design": "case-control",
       "result": "IBS status strongest driver of microbiome separation",
       "url": "https://doi.org/10.1186/s40168-017-0261-y",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is ME/CFS dysbiosis independent of IBS in properly stratified analyses?",
      "Are enteric-neuron genetics a shared gut-brain mechanism?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "genetic-neuronal",
      "autonomic-small-fibre"
     ],
     "test_available": "Clinical: Rome IV IBS criteria; faecal calprotectin to exclude IBD"
    },
    {
     "id": "gut-translocation-lps",
     "subdomain": "Gut permeability and translocation",
     "finding": "Blood markers of bacterial translocation (LPS, LPS-binding protein, sCD14, IgA/IgM to enterobacterial LPS) are raised in several small ME/CFS studies, but standardised replication is lacking.",
     "grade": "contested",
     "plain": "Some studies suggest bits of gut bacteria leak into the blood ('leaky gut') and trigger inflammation. The evidence is small, uses different tests, and comes mostly from a few labs.",
     "detail": "Giloteaux 2016 (48 vs 39) found LPS, LBP and sCD14 elevated and correlated with each other. I-FABP (gut-cell damage) was also measured. Maes 2007 reported higher serum IgA and IgM against LPS of gram-negative enterobacteria, correlated with severity and IBS symptoms. De Meirleir 2018 (140 vs 140, retrospective) found sCD14 among the most discriminating serum markers. In the Solving ME/CFS tally, 5 groups report raised LPS/sCD14 and 2 do not. Brown 2026 found higher IgM to LPS only in the subgroup with more flu-like symptoms. Endotoxin assays are contamination-prone, sCD14 is a general monocyte activation marker, and no study has used a direct permeability test (lactulose/mannitol). Maes' 'leaky gut diet' studies were uncontrolled.",
     "studies": [
      {
       "cite": "Giloteaux 2016, Microbiome",
       "n": "48 vs 39",
       "design": "case-control",
       "result": "Raised LPS, LBP, sCD14",
       "url": "https://doi.org/10.1186/s40168-016-0171-4",
       "verified": true
      },
      {
       "cite": "Maes 2007, J Affect Disord",
       "n": "CFS, partial CFS and controls",
       "design": "case-control",
       "result": "Raised IgA/IgM to enterobacterial LPS",
       "url": "https://doi.org/10.1016/j.jad.2006.08.021",
       "verified": true
      },
      {
       "cite": "De Meirleir 2018, J Transl Med",
       "n": "140 vs 140",
       "design": "retrospective case-control",
       "result": "sCD14 among best discriminating markers",
       "url": "https://doi.org/10.1186/s12967-018-1696-z",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Maes & Leunis 2008, Neuro Endocrinol Lett",
       "n": "41",
       "design": "uncontrolled before-after",
       "result": "Claimed improvement with 'leaky gut' supplements; no control group",
       "url": "https://pubmed.ncbi.nlm.nih.gov/19112401/",
       "verified": true
      },
      {
       "cite": "Seton 2023, medRxiv (Quadram)",
       "n": "5 severe patients vs 5 household controls",
       "design": "feasibility case-control (IgG-Seq)",
       "result": "Reduced, not increased, serum IgG reactivity to stool microbes",
       "url": "https://doi.org/10.1101/2023.05.21.23290299",
       "verified": true
      }
     ],
     "open_questions": [
      "Does a direct permeability test (sugar absorption, zonulin is unreliable) show a difference?",
      "Do translocation markers rise after exertion (PEM)?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "innate-hyperactivation"
     ],
     "test_available": "Research only (LPS, LBP, sCD14); commercial 'leaky gut' panels are not validated"
    },
    {
     "id": "gut-quadram-virome-immune",
     "subdomain": "Virome and mucosal immunity (Quadram Institute)",
     "finding": "Quadram Institute studies of severe ME/CFS with household-matched controls report altered gut phage communities and reduced serum IgG reactivity to gut microbes, in very small samples.",
     "grade": "early",
     "plain": "A Norwich team is studying the viruses that infect gut bacteria, and the immune response to gut microbes, comparing patients with people in the same household. Their samples are tiny so far.",
     "detail": "Hsieh 2023 combined virus-like-particle and whole-metagenome sequencing in a small severe-ME/CFS cohort. It defined a core of tailed Caudoviricetes phages and predicted ME/CFS-specific virus-host links involving Anaerotruncus. Seton 2023 (5 pairs with same-household controls) found reduced serum IgG capacity and reactivity to stool microbes. In a twin pair discordant for ME/CFS, Giloteaux 2016 reported lower diversity and expanded Caudovirales phages in the ill twin. The virome of the Norwegian Comeback FMT trial (Quadram/UEA thesis 2024) and a RESTORE-ME trial virome analysis are in progress, and no results are published.",
     "studies": [
      {
       "cite": "Hsieh 2023, Int J Mol Sci",
       "n": "small severe cohort + healthy",
       "design": "virome + metagenome case-control",
       "result": "Core phage virome; predicted disease-associated virus-host interactions",
       "url": "https://doi.org/10.3390/ijms242417267",
       "verified": true
      },
      {
       "cite": "Seton 2023, medRxiv",
       "n": "5 vs 5 household",
       "design": "feasibility case-control",
       "result": "Reduced serum IgG reactivity to gut microbes",
       "url": "https://doi.org/10.1101/2023.05.21.23290299",
       "verified": true
      },
      {
       "cite": "Giloteaux 2016, Am J Case Rep",
       "n": "1 MZ twin pair",
       "design": "case report",
       "result": "Ill twin: lower diversity, Caudovirales expansion",
       "url": "https://doi.org/10.12659/ajcr.900314",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do phage shifts replicate in a powered cohort?",
      "Does reduced anti-microbial IgG reflect immune exhaustion or sampling noise?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "immune-exhaustion",
      "viral-persistence"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-fmt-trials",
     "subdomain": "Treatment: faecal transplant",
     "finding": "The only published randomised faecal microbiota transplant trial in CFS (n=11) found no benefit, and a larger Norwegian RCT (n=80) has not yet reported.",
     "grade": "early",
     "plain": "Transferring stool from a healthy donor has been tried once in a proper small trial and did not help. A bigger trial is under way.",
     "detail": "Salonen 2023 (Finland, donor FMT n=5 vs autologous n=6 by colonoscopy) found no change in VAS, MFIS, 15D or EQ-5D at 1 or 6 months, and no adverse events. It was underpowered. Kenyon 2019 reported improvement in a retrospective non-randomised series (from memory). The Comeback Study (NCT03691987, Harstad; 80 participants randomised 1:1:2 to two donors or autologous placebo, primary outcome FSS at 3 months) published its protocol in 2024. As of October 2026 I found no results.",
     "studies": [
      {
       "cite": "Salonen 2023, J Transl Med",
       "n": "11",
       "design": "RCT (donor vs autologous FMT)",
       "result": "No improvement in fatigue or HRQOL",
       "url": "https://doi.org/10.1186/s12967-023-04227-y",
       "verified": true
      },
      {
       "cite": "Skjevling 2024, BMJ Open (Comeback protocol)",
       "n": "80 planned",
       "design": "RCT protocol",
       "result": "Results pending",
       "url": "https://doi.org/10.1136/bmjopen-2023-073275",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Kenyon 2019, Human Microbiome Journal",
       "n": "42 (21 FMT)",
       "design": "retrospective, non-randomised",
       "result": "Reported improvement with FMT (from memory)",
       "url": "",
       "verified": false
      }
     ],
     "open_questions": [
      "Will the Comeback RCT show benefit?",
      "Should FMT target patients with the butyrate-loss / R. gnavus signature?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Not applicable (treatment; trials only)"
    },
    {
     "id": "gut-probiotics",
     "subdomain": "Treatment: probiotics and diet",
     "finding": "Probiotic trials in CFS are small; one 39-person RCT of Lactobacillus casei Shirota reduced anxiety but did not target fatigue.",
     "grade": "early",
     "plain": "No probiotic has been shown to improve ME/CFS itself.",
     "detail": "Rao 2009 (n=39, 24 billion CFU daily for 2 months) raised stool Lactobacillus and Bifidobacteria and reduced Beck anxiety scores (p=0.01). Fatigue was not the primary outcome. Venturini 2019 was an uncontrolled pilot. Re-analysis argues against Bifidobacterium/Lactobacillus products for the indolelactate deficit, because their abundance does not track it. Fibre and polyphenols have a better mechanistic case for IPA, but there are no trials (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Rao 2009, Gut Pathog",
       "n": "39",
       "design": "RCT",
       "result": "Reduced anxiety; stool lactobacilli up",
       "url": "https://doi.org/10.1186/1757-4749-1-6",
       "verified": true
      },
      {
       "cite": "Venturini 2019, Oxid Med Cell Longev",
       "n": "small",
       "design": "uncontrolled pilot",
       "result": "Changes in immune/oxidative markers",
       "url": "https://doi.org/10.1155/2019/1684198",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would a butyrate-producer consortium or fibre intervention shift symptoms in signature-positive patients?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Not applicable"
    },
    {
     "id": "gut-genetics-not-causal",
     "subdomain": "Genetics of the gut-brain axis",
     "finding": "ME/CFS genetic risk overlaps with IBS and is enriched in enteric neurons, but genetically predicted gut-derived metabolites (IPA, indolelactate, kynurenine, bile acids) do not change ME/CFS risk.",
     "grade": "early",
     "plain": "The genes involved overlap with those for irritable bowel syndrome and gut nerves. But inheriting higher or lower levels of gut metabolites does not change ME/CFS risk, so those metabolites are probably effects, not causes.",
     "detail": "Solving ME/CFS MR (DecodeME discovery, MVP replication): IPA OR 1.19 (0.93–1.53), indolelactate 0.99, kynurenine 1.01, glycocholate 1.02, all null. Lee 2026 (medRxiv, from Solving ME/CFS genetics summary): rg with IBS 0.57 and heritability enrichment in enteric neurons (FDR 0.004). Wielscher 2026 (preprint): 'barrier-microbiome' traits show among the strongest genetic overlap, with IBS contributing most pleiotropy. OLFM4, a DecodeME locus, is highly expressed in ileal stem cells and neutrophils.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "15,579 + MVP",
       "design": "two-sample MR",
       "result": "All gut-metabolite exposures null",
       "url": "",
       "verified": true
      },
      {
       "cite": "Wielscher 2026, Research Square preprint",
       "n": "DecodeME 15,579",
       "design": "genetic correlation + MR",
       "result": "Barrier-microbiome traits and IBS share pleiotropy with ME/CFS",
       "url": "https://doi.org/10.21203/rs.3.rs-9363637/v2",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is the shared IBS genetics neuronal (enteric nervous system) rather than microbial?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-longcovid-parallel",
     "subdomain": "Long COVID",
     "finding": "In long COVID, the gut microbiome at hospital admission predicted persistent symptoms, and at 6 months patients had more R. gnavus and fewer butyrate producers, mirroring ME/CFS.",
     "grade": "several",
     "plain": "Long COVID shows a gut pattern similar to ME/CFS, and in one study the gut profile early in COVID predicted who stayed ill.",
     "detail": "Liu 2022 (Hong Kong, 106 patients, 258 serial stool samples; 68 controls) reported that admission microbiota composition was associated with PACS. Patients who recovered had normalised microbiomes, while PACS showed higher R. gnavus and B. vulgatus and lower F. prausnitzii. Butyrate producers (B. pseudocatenulatum, F. prausnitzii) showed the largest inverse correlation with PACS. Antibiotic use and hospital care in severe COVID are confounders.",
     "studies": [
      {
       "cite": "Liu 2022, Gut",
       "n": "106 vs 68",
       "design": "prospective cohort",
       "result": "Admission microbiome associated with PACS; R. gnavus up, F. prausnitzii down",
       "url": "https://doi.org/10.1136/gutjnl-2021-325989",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does pre-infection microbiome predict long COVID in community (non-hospitalised) cohorts?"
     ],
     "hypotheses": [
      "gut-dysbiosis",
      "viral-persistence"
     ],
     "test_available": "Research only"
    },
    {
     "id": "gut-machine-learning-classifiers",
     "subdomain": "Multi-omics classifiers",
     "finding": "Microbiome-based machine-learning classifiers distinguish ME/CFS from controls in held-out and external cohorts (BioMapAI, Guo 2023), but they are research tools with modest real-world specificity.",
     "grade": "several",
     "plain": "Computer models can tell patient and control stool apart better than chance, but this is not a diagnostic test yet.",
     "detail": "BioMapAI (Xiong 2025, Nat Med; 249 participants over 4 years) integrated metagenomics, plasma metabolomics, immune profiling and labs to predict severity. It classified held-out and external cohorts and linked SCFA, BCAA, tryptophan and benzoate metabolism to MAIT/γδT inflammation. Guo 2023's classifier generalised across US sites. Classifiers have not been tested against disease controls (IBS, depression, MS, fibromyalgia), so specificity is unknown. Within-cohort cross-validation in the plasma metabolome transferred poorly (AUC 0.56; Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Xiong 2025, Nat Med",
       "n": "249",
       "design": "longitudinal multi-omics, deep learning",
       "result": "Classifies ME/CFS in held-out and external cohorts",
       "url": "https://doi.org/10.1038/s41591-025-03788-3",
       "verified": true
      },
      {
       "cite": "Guo 2023, Cell Host Microbe",
       "n": "106 vs 91 + validation",
       "design": "case-control classifier",
       "result": "Robust to geography; generalised to validation cohort",
       "url": "https://doi.org/10.1016/j.chom.2023.01.004",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "How do classifiers perform against IBS and other fatigue conditions?"
     ],
     "hypotheses": [
      "gut-dysbiosis"
     ],
     "test_available": "Research only"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "gut-dysbiosis",
     "for": "Loss of butyrate producers and an R. gnavus-type bloom are seen in at least 4 cohorts and 3 labs, and a pre-specified NIH test gave AUC 0.87. Low IPA in 4/4 cohorts is explained by stool composition. The same pattern occurs in long COVID, where the admission microbiome predicted persistence. There is a genetic overlap with IBS and enteric neurons.",
     "against": "Dysbiosis fades in long-standing illness while symptoms persist and does not track severity at FDR. MR shows gut metabolites are not causal. IBS confounds the comparison. The only FMT RCT was null (n=11). The translocation markers are small, lab-specific and use contamination-prone assays."
    },
    {
     "id": "innate-hyperactivation",
     "for": "Raised LPS/LBP/sCD14 in some studies. In BioMapAI, the microbiome links to MAIT/γδT inflammatory cells.",
     "against": "Re-analysis found 0/622 immune-module associations at q<0.1. The Quadram feasibility study found reduced anti-microbial IgG."
    },
    {
     "id": "genetic-neuronal",
     "for": "Heritability is enriched in enteric neurons and the genetic correlation with IBS is about 0.57, suggesting a gut-brain neural link.",
     "against": "These are preprints, and the NOS1 region shared with IBS does not colocalise."
    }
   ],
   "glossary": [
    {
     "term": "Microbiome",
     "plain": "The community of bacteria, viruses and fungi living in the gut."
    },
    {
     "term": "Dysbiosis",
     "plain": "An imbalance in the gut microbial community compared with healthy people."
    },
    {
     "term": "Butyrate",
     "plain": "A short-chain fatty acid made by some gut bacteria from fibre. It fuels the cells lining the colon and dampens inflammation."
    },
    {
     "term": "Shotgun metagenomics",
     "plain": "Sequencing all DNA in stool to identify species and their genes, more detailed than 16S sequencing."
    },
    {
     "term": "16S rRNA sequencing",
     "plain": "A cheaper method that identifies bacteria by one marker gene, usually only to genus level."
    },
    {
     "term": "Indole-3-propionate (IPA)",
     "plain": "A protective tryptophan breakdown product made only by certain gut bacteria."
    },
    {
     "term": "LPS / sCD14 / LBP",
     "plain": "LPS is a component of bacterial walls. sCD14 and LBP are proteins the body makes in response to it, used as markers of bacteria leaking into blood."
    },
    {
     "term": "Virome / phages",
     "plain": "The viruses in the gut, most of which infect bacteria (bacteriophages)."
    },
    {
     "term": "FMT",
     "plain": "Faecal microbiota transplantation: transferring stool from a healthy donor to a patient."
    },
    {
     "term": "IBS",
     "plain": "Irritable bowel syndrome, a disorder of gut-brain interaction causing pain and altered bowel habit."
    }
   ],
   "key_reviews": [
    {
     "cite": "König 2022, Front Immunol",
     "url": "https://doi.org/10.3389/fimmu.2021.628741",
     "why": "Broad review of microbiome hypotheses (dysbiosis, permeability, SCFA, D-lactate, tryptophan) and interventions."
    },
    {
     "cite": "Nikolova 2025, Int J Mol Sci",
     "url": "https://doi.org/10.3390/ijms27010425",
     "why": "Recent overview of gut microbiome mechanisms in ME/CFS."
    },
    {
     "cite": "Rajabi 2026, Front Med",
     "url": "https://doi.org/10.3389/fmed.2026.1881784",
     "why": "First meta-analysis quantifying IBS overlap."
    },
    {
     "cite": "Perera 2025, Int J Mol Sci (Quadram)",
     "url": "https://doi.org/10.3390/ijms262211161",
     "why": "Argues that mucosal viruses are an under-studied piece."
    }
   ],
   "gaps": [
    "No prospective study samples stool before or at the triggering infection and follows people into ME/CFS, outside the hospitalised COVID cohort of Liu 2022.",
    "No microbiome study of ME/CFS uses disease controls (IBS without ME/CFS, depression, MS, deconditioned controls) to test specificity.",
    "No direct intestinal permeability measurement (lactulose/mannitol or similar) in a powered ME/CFS cohort; no study of translocation markers before and after exertion.",
    "Results of the 80-person Comeback FMT RCT and the RESTORE-ME virome work are not yet published.",
    "No intervention trial selects patients by microbiome signature (e.g. butyrate-producer loss) or tests fibre/polyphenols to raise IPA.",
    "Small-intestinal microbiota (which stool does not sample) has not been studied, though it could explain the aromatic-lactate deficit.",
    "Within-person longitudinal sampling is needed to test whether microbiome shifts track PEM or relapses."
   ]
  },
  {
   "domain": "genetics",
   "title": "Genetics",
   "overview": "ME/CFS runs in families, and twin, genealogy and three-generation cohort studies put familial or heritable risk at roughly 20 to 50%. In 2025 DecodeME (15,579 cases) found the first eight robust genome-wide loci, with SNP heritability of about 8 to 10%. Each variant shifts risk only slightly (odds ratios 1.07 to 1.10), so no gene test can diagnose or predict ME/CFS. The overall genetic signal replicates in independent biobanks (MVP, FinnGen, UK Biobank), but the individual loci mostly do not yet, partly because those biobanks use loose case definitions. Across several analyses the signal points to neurons in the brain (and possibly the pituitary), not blood immune cells. It overlaps strongly with chronic pain, fibromyalgia, IBS and migraine, and partly with depression, though the specific ME/CFS loci do not share causal variants with depression. Earlier small candidate-gene and HLA findings mostly failed to replicate. Several 2025–2026 follow-ups (Mendelian randomisation, rare variants, a UK Biobank/All of Us GWAS) are preprints that still need confirmation.",
   "findings": [
    {
     "id": "genetics-familial-heritability",
     "subdomain": "Heritability and family studies",
     "finding": "ME/CFS aggregates in families: relatives of cases have about 2-3x the risk, MZ twins are more concordant than DZ twins, and familiality in a 153,803-person Dutch cohort was 42%.",
     "grade": "replicated",
     "plain": "Having a close relative with ME/CFS raises your own risk, which means genes (and perhaps shared environment) play a part.",
     "detail": "In the Utah Population Database (Albright 2011), relative risk was 2.70 (1.56–4.66) for first-degree relatives, 2.34 for second-degree and 1.93 for third-degree, with excess distant relatedness (p=0.010). Buchwald 2001 (146 female twin pairs) found 55% MZ vs 19% DZ concordance for idiopathic chronic fatigue and estimated heritability up to 51%. Its confidence intervals are wide, and it measured chronic fatigue, not strict ME/CFS. Bos 2025 (Lifelines, 153,803 people, 90,397 with a first-degree relative) found recurrence risk ratio 1.45–2.23 within disorders and familiality 42% (33–50) for ME/CFS. Familial correlations were 0.97 with fibromyalgia and 0.83 with depression. Familiality includes shared household environment.",
     "studies": [
      {
       "cite": "Albright 2011, BMC Neurology",
       "n": "Utah genealogy-linked health records",
       "design": "familial aggregation",
       "result": "First-degree RR 2.70; third-degree RR 1.93",
       "url": "https://doi.org/10.1186/1471-2377-11-62",
       "verified": true
      },
      {
       "cite": "Buchwald 2001, Psychosom Med",
       "n": "146 female twin pairs",
       "design": "classic twin study",
       "result": "MZ 55% vs DZ 19% concordance; h² up to 51%",
       "url": "https://doi.org/10.1097/00006842-200111000-00012",
       "verified": true
      },
      {
       "cite": "Bos 2025, Psychol Med",
       "n": "153,803",
       "design": "three-generation cohort familiality",
       "result": "Familiality 42% for ME/CFS; rf 0.97 with fibromyalgia, 0.83 with MDD",
       "url": "https://doi.org/10.1017/s003329172500100x",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "How much of familiality is genetic vs shared environment or infection exposure?",
      "Does the familial risk differ between infection-onset and gradual-onset cases?"
     ],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "Family history only"
    },
    {
     "id": "genetics-decodeme-loci",
     "subdomain": "Genome-wide association (GWAS)",
     "finding": "DecodeME (15,579 PEM-defined cases vs 259,909 controls) identified 8 genome-wide significant loci, each with a small effect (OR about 1.07-1.10), and SNP heritability of about 8-10%.",
     "grade": "several",
     "plain": "The largest DNA study of ME/CFS found eight regions of the genome linked to the illness. Each nudges risk only a tiny amount.",
     "detail": "Lead genes as assigned: RABGAP1L, BTN2A2/TRIM38, FBXL4, SUDS3, OLFM4, CCPG1, CA10, ARFGEF2/CSE1L/ZNFX1 (the chr20 signal p=1e-11). Four loci (RABGAP1L, FBXL4, OLFM4, CA10) were nominally associated (p<0.05) with PEM-plus-fatigue cases in UK Biobank and Lifelines, but none passed multiple-testing correction in external sets. There was no sex-biased locus, and ARFGEF2 and CA10 replicated in males. The CA10 signal colocalises with multisite chronic pain. As of October 2026 this is still a medRxiv preprint; I found no peer-reviewed version. Gene assignments are weak at some loci: the 6q16 lead is about 890 kb from FBXL4, and the chr1 lead sits inside DARS2 (Solving ME/CFS re-analysis). An absolute-risk calculation shows one risk copy moves baseline risk from about 0.9% to about 0.97–0.99%.",
     "studies": [
      {
       "cite": "DecodeME Genetics Delivery Team 2025, medRxiv",
       "n": "15,579 vs 259,909",
       "design": "GWAS (European ancestry)",
       "result": "8 loci; no colocalisation with depression/anxiety",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (MVP-only and FinnGen replication)",
       "n": "3,891 MVP cases; 283 FinnGen",
       "design": "replication of lead SNPs",
       "result": "Lead loci 5/8 same direction in MVP (p=0.13), 4/8 in FinnGen; BTN2A2, OLFM4, CA10 wrong way in MVP",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Which genes are causal at each locus (fine-mapping, functional work)?",
      "Will loci replicate in a cohort with PEM-based phenotyping?"
     ],
     "hypotheses": [
      "genetic-neuronal",
      "viral-persistence",
      "innate-hyperactivation"
     ],
     "test_available": "Not useful individually (no clinical genetic test)"
    },
    {
     "id": "genetics-polygenic-replication",
     "subdomain": "Replication in biobanks",
     "finding": "The genome-wide DecodeME signal replicates in independent cohorts: genetic correlation 0.65-0.73 with the Million Veteran Program CFS GWAS and 0.80-0.90 with UK Biobank CFS, plus concordant directions for sub-threshold SNPs in FinnGen.",
     "grade": "replicated",
     "plain": "Even though single hits do not yet repeat everywhere, the overall genetic fingerprint of ME/CFS shows up again in other large populations, including mostly male US veterans.",
     "detail": "Solving ME/CFS re-analysis derived MVP-only statistics (3,891 independent cases) from the public DecodeME+MVP meta. rg 0.73 ± 0.16. DecodeME SNPs at p<1e-5 were concordant 42/63 in MVP (p=0.006) and 41/61 in FinnGen postviral fatigue (p=0.005). Pan-UKB self-report CFS gave rg 0.90, but it shares controls. An independent GitHub re-analysis (trafalmadorian97) reported rg 0.65 with MVP and 0.80 with Neale UKB. The MVP cohort is mostly male, so polygenic risk applies across sexes.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "DecodeME 15,579; MVP 3,891; FinnGen 283",
       "design": "LDSC rg and sign concordance",
       "result": "rg 0.73; concordance p=0.006 (MVP), 0.005 (FinnGen)",
       "url": "",
       "verified": true
      },
      {
       "cite": "Maccallini 2026, Research Square preprint",
       "n": "19,470 vs 699,111",
       "design": "DecodeME + MVP meta-analysis",
       "result": "5 loci incl. new TSHZ3 region; brain and pituitary enrichment",
       "url": "https://doi.org/10.21203/rs.3.rs-9702020/v1",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "How much does loose biobank phenotyping (billing codes, self-report) dilute single-locus replication?"
     ],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-brain-neuron-enrichment",
     "subdomain": "Tissue and cell-type enrichment",
     "finding": "Genome-wide ME/CFS heritability is enriched in brain tissues and neurons (including glutamatergic synapse genes and subcortical/cerebellar neuron types), with no enrichment in blood immune cells.",
     "grade": "several",
     "plain": "The genes involved are mainly active in nerve cells in the brain, not in blood immune cells. This suggests the brain is central to inherited risk.",
     "detail": "DecodeME: MAGMA gene-property enrichment in all 13 GTEx brain tissues and no others. Independent S-LDSC re-analysis (trafalmadorian97 GitHub): CNS tissues and chromatin only. ImmGen and blood-cell ATAC-seq were null, and medium spiny neuron clusters were implicated. Maccallini 2026 (DecodeME+MVP): brain regions and pituitary, glutamatergic synapse gene sets, with 7/12 gene sets replicating in rare-variant genes from HEAL2. Lee 2026 (medRxiv): inhibitory neurons (p=1.2e-7) and enteric neurons. Immune abnormalities may still be downstream of illness. A locus-level permutation with only 8 loci could not place any tissue (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "DecodeME 2025, medRxiv",
       "n": "15,579 cases",
       "design": "GWAS + MAGMA",
       "result": "All 13 brain tissues enriched; no others",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      },
      {
       "cite": "Maccallini 2026, Research Square preprint",
       "n": "19,470 cases",
       "design": "meta-GWAS enrichment + rare-variant replication",
       "result": "Glutamatergic synapse; subcortical and cerebellar neurons; pituitary",
       "url": "https://doi.org/10.21203/rs.3.rs-9702020/v1",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (locus tissue permutation)",
       "n": "8 loci",
       "design": "permutation",
       "result": "No tissue survives FDR at locus level (expected with 8 loci)",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Which brain circuits (basal ganglia, cerebellum, claustrum) matter, and do imaging studies of genetic-risk extremes show differences?"
     ],
     "hypotheses": [
      "genetic-neuronal",
      "neuroinflammation"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-pain-overlap",
     "subdomain": "Shared genetics with other conditions",
     "finding": "ME/CFS genetics overlaps most with chronic pain, fibromyalgia, IBS and migraine, and substantially with depression, but none of the 8 DecodeME loci share a causal variant with depression.",
     "grade": "several",
     "plain": "Genes that raise ME/CFS risk also raise risk of chronic pain and gut-brain conditions. This is shared biology, not evidence that ME/CFS is depression.",
     "detail": "The CA10 locus colocalises with multisite chronic pain (PPH4 92.8%). Re-analyses report the highest rg with fibromyalgia, then multisite pain, as well as syncope, migraine and asthma. Lee 2026 medRxiv: rg with major depression 0.60 and with IBS 0.57. MR in Solving ME/CFS re-analysis: depression OR 1.82 (MVP 1.36) and multisite pain OR 3.3, but reverse MR is also positive (ME/CFS → depression OR 1.15) and heterogeneity is high, which indicates shared liability rather than one-way causation. A UK Biobank MR (Song 2025) found no causal effect of depression on ME/CFS.",
     "studies": [
      {
       "cite": "DecodeME 2025, medRxiv",
       "n": "15,579 cases",
       "design": "GWAS colocalisation",
       "result": "CA10 shares causal variant with chronic pain; no colocalisation with depression/anxiety",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "DecodeME + MVP",
       "design": "bidirectional MR",
       "result": "Depression, pain, insomnia associated both ways; shared liability",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Song 2025, Sci Rep",
       "n": "UK Biobank CFS GWAS",
       "design": "two-sample MR",
       "result": "No causal effect of MDD on ME/CFS",
       "url": "https://doi.org/10.1038/s41598-025-85217-6",
       "verified": true
      }
     ],
     "open_questions": [
      "Which part of ME/CFS genetics is specific after subtracting pain genetics? A re-analysis suggests the chr6 BTN region and one striatal neuron cluster."
     ],
     "hypotheses": [
      "genetic-neuronal",
      "sickness-behaviour"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-infection-loci",
     "subdomain": "Infection response genes",
     "finding": "Three DecodeME loci lie near infection-response genes (OLFM4, RABGAP1L, BTN2A2/TRIM38), and the OLFM4 effect is larger in infection-onset cases, but infection- and non-infection-onset cases are genetically highly correlated.",
     "grade": "early",
     "plain": "Some risk genes help the body respond to infections, which fits the common post-viral onset. But people whose illness began without an obvious infection carry similar overall genetic risk.",
     "detail": "Infection-onset GWAS (9,738 cases): 3 loci (6p22.2, 20q13.13, OLFM4). The OLFM4 beta was 0.095±0.017 with infection onset vs 0.034±0.022 without. The no-infection GWAS (5,841) found nothing, which is a power issue. Independent re-analysis finds high rg between onset subgroups. OLFM4 is a neutrophil and ileal stem-cell protein. ZNFX1 (a dsRNA sensor) and CSE1L (needed for STAT1 import) sit at the chr20 locus. Genetically higher EBV DNA load gave OR 1.13 (ns) in DecodeME and 1.59 (p=0.012) in MVP (Solving ME/CFS re-analysis). MHC-based negative controls (VZV, HHV-6B) also aligned, so the result is not EBV-specific. The long COVID FOXP4 variant is not associated with ME/CFS (p=0.59).",
     "studies": [
      {
       "cite": "DecodeME 2025, medRxiv",
       "n": "9,738 infection-onset cases",
       "design": "stratified GWAS",
       "result": "OLFM4 effect larger with infection onset",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (EBV load MR)",
       "n": "DecodeME; MVP",
       "design": "MR with Kamitaki 2026 instruments",
       "result": "OR 1.13 (ns) / 1.59 (p=0.012)",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Lammi 2025, Nat Genet (long COVID GWAS)",
       "n": "6,450 cases",
       "design": "GWAS",
       "result": "FOXP4 lead variant; no association with ME/CFS in DecodeME",
       "url": "https://doi.org/10.1038/s41588-025-02100-w",
       "verified": true
      }
     ],
     "open_questions": [
      "Do OLFM4 risk-genotype carriers show different neutrophil or gut responses (recall-by-genotype)?",
      "Is self-reported onset type too noisy to stratify on?"
     ],
     "hypotheses": [
      "viral-persistence",
      "herpes-reactivation",
      "innate-hyperactivation"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-hla",
     "subdomain": "HLA",
     "finding": "Reported HLA associations are inconsistent: Lande 2020's HLA-C*07:04 and DQB1*03:03 risk alleles are not among DecodeME's hits, and DecodeME's protective HLA-DQA1*05:01 signal is barely visible at SNP level.",
     "grade": "contested",
     "plain": "HLA genes shape immune responses and are linked to autoimmune diseases. Claims of specific HLA types in ME/CFS have not held up consistently.",
     "detail": "Lande 2020 (426 Norwegian CCC cases vs 4,511 controls) reported HLA-C*07:04 (OR 2.1) and DQB1*03:03 (OR 1.5); carriers of either were 19.2% vs 12.2%. DecodeME reported DQA1*05:01 as protective (p=1.4e-10; 21.7% vs 23.2%), with no DRB1*03:01 or DQB1*02:01 signal. In Solving ME/CFS re-analysis, the best DQ2.5 tag SNP was OR 0.96 (p=0.025). That gap suggests the imputed-allele p-value is inflated, possibly because HLA was imputed separately for cases and controls, a problem the authors flagged. MHC z-scores correlate only 0.21 between DecodeME and MVP. A purely in-silico 2025 study modelled Lande alleles binding herpes peptides, with no patient data.",
     "studies": [
      {
       "cite": "Lande 2020, Sci Rep",
       "n": "426 vs 4,511",
       "design": "HLA NGS case-control",
       "result": "C*07:04 OR 2.1; DQB1*03:03 OR 1.5",
       "url": "https://doi.org/10.1038/s41598-020-62157-x",
       "verified": true
      },
      {
       "cite": "DecodeME 2025, medRxiv",
       "n": "15,579 vs 259,909",
       "design": "HLA imputation",
       "result": "DQA1*05:01 protective p=1.4e-10",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 HLA)",
       "n": "DecodeME + MVP",
       "design": "SNP-level MHC re-analysis",
       "result": "Best DQ2.5 tag OR 0.96; DecodeME-MVP MHC z correlation 0.21",
       "url": "",
       "verified": true
      }
     ],
     "open_questions": [
      "Will joint HLA imputation of cases and controls confirm any allele?"
     ],
     "hypotheses": [
      "autoimmunity",
      "herpes-reactivation"
     ],
     "test_available": "HLA typing is available but not useful for ME/CFS"
    },
    {
     "id": "genetics-kir-alleles",
     "subdomain": "NK-cell receptor genes",
     "finding": "Specific inhibitory KIR alleles (e.g. KIR3DL1*020, KIR3DL2*009) were more or less common in 418 Norwegian patients than 473 controls, though KIR gene content did not differ.",
     "grade": "early",
     "plain": "Variants in genes for natural killer cell 'brake' receptors may differ slightly in ME/CFS. This has not been checked in another cohort.",
     "detail": "Ramadan 2025 used high-resolution KIR sequencing with HLA ligand annotation. Patient-enriched alleles were KIR3DL3*002 (OR 1.43), KIR3DL1*020 (OR 2.20) and KIR3DL2*009 (OR 1.56); reduced alleles were KIR3DL3*013 (OR 0.60) and KIR3DL2*010 (OR 0.46, p=0.0005). There was no association with gene content or copy number. Nominal p-values without a replication cohort; KIR is poorly captured by GWAS arrays, so DecodeME cannot test it.",
     "studies": [
      {
       "cite": "Ramadan 2025, Brain Behav Immun",
       "n": "418 vs 473",
       "design": "case-control, KIR allele sequencing",
       "result": "Several inhibitory KIR alleles associated (nominal p 0.0005–0.01)",
       "url": "https://doi.org/10.1016/j.bbi.2025.106098",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Replication in UK or US cohorts; link to NK cytotoxicity measures."
     ],
     "hypotheses": [
      "immune-exhaustion",
      "herpes-reactivation"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-early-gwas-candidates",
     "subdomain": "Earlier GWAS and candidate genes",
     "finding": "Before DecodeME, small GWAS and candidate-gene studies (GRIK2, NPAS2, TPPP, TRA locus and many others) produced no association that replicated in UK Biobank or later cohorts.",
     "grade": "refuted",
     "plain": "Older genetic studies were too small. Their 'hits' did not hold up when tested in larger groups.",
     "detail": "Smith 2011 (40 vs 40, 116k SNPs plus expression) flagged GRIK2 and NPAS2. Hajdarevic 2022 (Norwegian 427 discovery, Danish 460, UKB 2,105) found no genome-wide significant loci; the best signal was TPPP (p=8.5e-7) and the top SNP did not replicate. Dibble 2020 concluded that most candidate gene associations are not replicated by the UK Biobank CFS cohort. Ueland 2022's T-cell receptor alpha signal did not replicate (from Solving ME/CFS genetics summary).",
     "studies": [
      {
       "cite": "Hajdarevic 2022, Brain Behav Immun",
       "n": "2,532 cases total",
       "design": "GWAS with replication",
       "result": "No genome-wide significant loci; TPPP not replicated",
       "url": "https://doi.org/10.1016/j.bbi.2022.03.010",
       "verified": true
      },
      {
       "cite": "Dibble 2020, Hum Mol Genet",
       "n": "review incl. UKB",
       "design": "critical review",
       "result": "Most candidate gene associations not replicated",
       "url": "https://doi.org/10.1093/hmg/ddaa169",
       "verified": true
      },
      {
       "cite": "Smith 2011, Neuropsychobiology",
       "n": "40 vs 40",
       "design": "convergent SNP + expression",
       "result": "GRIK2, NPAS2 nominal",
       "url": "https://doi.org/10.1159/000326692",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "No: commercial 'ME/CFS gene panels' are not supported"
    },
    {
     "id": "genetics-ukb-allofus-2026",
     "subdomain": "Genome-wide association (GWAS)",
     "finding": "A 2026 UK Biobank GWAS with stricter, multi-source case definitions (1,268 cases) reports 7 loci that replicate in disjoint UK Biobank or All of Us cohorts (near BICD1, GRIN2A, CSMD1, RORA, CLYBL), though none replicate in all three.",
     "grade": "early",
     "plain": "A new study using carefully confirmed cases in two biobanks found seven genetic regions that repeated in a second group. It is a preprint and the groups are small.",
     "detail": "Slaughter 2026 (medRxiv) used TarGene with electronic health records plus survey data to confirm case and control status. 176 variants passed FDR<5% in discovery, and replication cohorts had 319 (UKB) and 371 (All of Us) cases. Fine-mapping at one locus colocalised with reduced CLYBL expression in putamen, but the CLYBL stop-gain variant was not associated. No gene-by-sex or gene-by-deprivation interactions survived correction. FDR-based discovery in a small sample and no overlap with DecodeME's 8 loci call for caution. GRIN2A (an NMDA receptor subunit) fits the glutamatergic signal.",
     "studies": [
      {
       "cite": "Slaughter 2026, medRxiv",
       "n": "1,268 discovery; 319 + 371 replication",
       "design": "GWAS with two replications",
       "result": "7 replicated loci, none in all three cohorts",
       "url": "https://doi.org/10.64898/2026.09.09.26362115",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do these loci show any signal in DecodeME summary statistics?"
     ],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-mr-proteins-ephb1",
     "subdomain": "Mendelian randomisation",
     "finding": "Proteome-wide cis-MR finds genetically higher plasma EPHB1 (a pain-sensitisation receptor) and CLEC3B (tetranectin) associated with ME/CFS risk in DecodeME, replicating in the independent MVP cohort.",
     "grade": "early",
     "plain": "Using genes as a natural experiment, two blood proteins look like they may influence ME/CFS risk. One of them is involved in how the nervous system amplifies pain.",
     "detail": "Solving ME/CFS re-analysis of about 1,950 UKB-PPP and 1,690 deCODE plasma proteins (Wald ratio). EPHB1: OR 1.18/SD (p=2e-4), MVP 1.29 (p=0.0016), combined FDR 0.002; its pQTL is in LD with the regional DecodeME peak (r² 0.84). CLEC3B: OR 1.21, MVP 1.44 (p=0.0008), but not in LD with the regional peak. PRDX6 (1q25) was protective (0.75; MVP 0.70, p=0.037), and dermatopontin was nominal on both platforms. Only 4/93 nominal hits replicated, versus about 2.3 expected by chance, so these are leads. There was no formal colocalisation because full pQTL statistics need a login, and there are no usable drugs.",
     "studies": [
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "DecodeME 15,579; MVP 3,891",
       "design": "cis-pQTL MR with replication",
       "result": "EPHB1 and CLEC3B replicate; PRDX6 nominal",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Full colocalisation with deCODE/UKB-PPP region data; CSF/brain pQTLs for EPHB1."
     ],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-mr-biomarkers",
     "subdomain": "Mendelian randomisation",
     "finding": "MR of DecodeME suggests higher mitochondrial DNA copy number is protective and higher GlycA and mean platelet volume raise risk, while genetically predicted metabolite leads (IPA, kynurenine, ketones, carnitine, bile acids) and vitamin D, BMI and blood pressure are null or unreplicated.",
     "grade": "early",
     "plain": "Genetic natural experiments hint that inflammation and blood platelets may contribute to risk. Many blood molecules that are low in patients look like effects of being ill, not causes.",
     "detail": "Wielscher 2026 (Research Square, not peer reviewed) reported mtDNA copy number as protective and GlycA and MPV as risk-raising. It also found the strongest genetic overlap in barrier-microbiome, neurovascular and energetics traits. Solving ME/CFS MR found indolelactate OR 0.99, kynurenine 1.01 and 3-hydroxybutyrate 0.99, all null. Palmitoylcarnitine 0.88 did not replicate. Vitamin D OR 0.88 (p=0.03) was not replicated and had a pleiotropic Egger intercept. Many published two-sample MR papers on CFS use small UK Biobank self-report GWAS; one was retracted in 2025.",
     "studies": [
      {
       "cite": "Wielscher 2026, Research Square preprint",
       "n": "DecodeME 15,579",
       "design": "rg, pleiotropy, MR",
       "result": "mtDNA copy number protective; GlycA, MPV risk",
       "url": "https://doi.org/10.21203/rs.3.rs-9363637/v2",
       "verified": true
      },
      {
       "cite": "Solving ME/CFS re-analysis (deep2 genetics_mr)",
       "n": "DecodeME + MVP",
       "design": "MR with replication",
       "result": "Metabolite and risk-factor exposures null or unreplicated",
       "url": "",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Retraction notice 2025, Food Sci Nutr",
       "n": "-",
       "design": "retracted two-mediation MR study",
       "result": "Example of low-quality MR in CFS literature",
       "url": "https://doi.org/10.1002/fsn3.71097",
       "verified": true
      }
     ],
     "open_questions": [
      "Replicate GlycA/MPV/mtDNA MR in MVP; test platelet activation in patients."
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "complement-thromboinflammation",
      "innate-hyperactivation"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-rare-variants-heal2",
     "subdomain": "Rare variants and sequencing",
     "finding": "Whole-genome sequencing of 464 cases and 611 controls with a deep-learning rare-variant model (HEAL2) proposes 115 loss-of-function-intolerant risk genes, partly overlapping the common-variant synaptic signal.",
     "grade": "early",
     "plain": "Looking at rare DNA changes, one study suggests a set of genes, many active in nerves and immune cells. The method is new and the sample is small.",
     "detail": "Zhang 2025 (medRxiv) reports that a genetic score from personal rare variants tracked PEM, unrefreshing sleep, brain fog and muscle pain. Patient multi-omics showed reduced expression of the risk genes, including in cytotoxic CD4 T cells. Maccallini 2026 found 7 of 12 common-variant gene sets replicate among HEAL2 genes. Deep-learning gene prioritisation in about 1,000 genomes has high overfitting risk, and there has been no independent sequencing replication. No single rare variant has been shown to cause ME/CFS. A verified, specific rare-variant 'Mendelian ME/CFS' report does not exist to my knowledge.",
     "studies": [
      {
       "cite": "Zhang 2025, medRxiv (HEAL2)",
       "n": "464 vs 611",
       "design": "WGS + deep learning",
       "result": "115 risk genes; symptom links",
       "url": "https://doi.org/10.1101/2025.04.15.25325899",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Independent WGS replication; burden tests in DecodeME exomes when available."
     ],
     "hypotheses": [
      "genetic-neuronal",
      "immune-exhaustion"
     ],
     "test_available": "Research only; clinical exome only for red-flag mimics (mitochondrial, HLH, immunodeficiency)"
    },
    {
     "id": "genetics-combinatorial-precisionlife",
     "subdomain": "Combinatorial analyses",
     "finding": "Proprietary combinatorial SNP analyses (PrecisionLife) report thousands of multi-SNP signatures in UK Biobank and DecodeME, with top vs bottom decile of signature count giving OR 1.64.",
     "grade": "contested",
     "plain": "A company's method finds combinations of DNA variants linked to ME/CFS, but the method is closed and hard for others to check.",
     "detail": "Das 2022 (2,382 UKB cases) mapped 84 SNP combinations to 14 genes, including CLOCK, SLC15A4, INSR and KCNB1. Sardell 2026 (J Transl Med, DecodeME vs UKB controls) reported 22,411 signatures from 7,555 SNPs in 2,311 genes, reproducible across three disjoint DecodeME case sets (p=4e-21 for count), 259 core genes, and 76/180 long-COVID genes overlapping. 'Reproducible' here means within related cohorts sharing UK Biobank controls; the method is proprietary and the gene list is very broad.",
     "studies": [
      {
       "cite": "Sardell 2026, J Transl Med",
       "n": "DecodeME cohorts vs UKB controls",
       "design": "combinatorial genetics",
       "result": "OR 1.64 top vs bottom decile of signature count",
       "url": "https://doi.org/10.1186/s12967-026-08167-1",
       "verified": true
      },
      {
       "cite": "Das 2022, J Transl Med",
       "n": "2,382 cases",
       "design": "combinatorial genetics (UKB)",
       "result": "14 genes; 5 SNPs replicated in a UKB CFS set",
       "url": "https://doi.org/10.1186/s12967-022-03815-8",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do signatures replicate in an external cohort with different controls (MVP, FinnGen, All of Us)?"
     ],
     "hypotheses": [
      "genetic-neuronal"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-trpm3",
     "subdomain": "TRPM3 ion channel",
     "finding": "TRPM3 ion channel dysfunction in NK cells and TRPM3 SNPs are reported repeatedly by one research group, but the SNP studies are tiny and no TRPM3 association appears in GWAS.",
     "grade": "contested",
     "plain": "A calcium channel called TRPM3 is reported to work poorly in immune cells of ME/CFS patients. Nearly all of this comes from one team, and large DNA studies do not point to the TRPM3 gene.",
     "detail": "Marshall-Gradisnik 2016 genotyped B cells from 11 patients vs 11 controls and reported SNPs/genotypes in TRPM3 and acetylcholine receptor genes. An earlier NK-cell SNP study had about 115 vs 90 (from memory). Functional work: Sasso 2026 used patch-clamp at two sites run by the same consortium (36 vs 42) and found reduced TRPM3-like currents in NK cells. Independent-lab replication and blinding details are lacking, and TRPM3 is not among DecodeME, MVP or Slaughter 2026 loci. Functional changes could be acquired rather than inherited.",
     "studies": [
      {
       "cite": "Sasso 2026, Front Med",
       "n": "36 vs 42",
       "design": "case-control patch-clamp, two sites",
       "result": "Reduced TRPM3 function in NK cells",
       "url": "https://doi.org/10.3389/fmed.2025.1703924",
       "verified": true
      },
      {
       "cite": "Marshall-Gradisnik 2016, J Int Med Res",
       "n": "11 vs 11",
       "design": "SNP/genotype case-control",
       "result": "TRPM3 and AChR gene SNPs reported",
       "url": "https://doi.org/10.1177/0300060516671622",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "DecodeME 2025, medRxiv",
       "n": "15,579 cases",
       "design": "GWAS",
       "result": "No TRPM3 locus among genome-wide hits",
       "url": "https://doi.org/10.1101/2025.08.06.25333109",
       "verified": true
      }
     ],
     "open_questions": [
      "Will an independent lab, blinded to case status, reproduce reduced TRPM3 currents?",
      "Is TRPM3 gene-level association present in DecodeME MAGMA results?"
     ],
     "hypotheses": [
      "sodium-calcium-overload",
      "immune-exhaustion"
     ],
     "test_available": "Research only"
    },
    {
     "id": "genetics-hypermobility-overlap",
     "subdomain": "Connective tissue",
     "finding": "Joint hypermobility and Ehlers-Danlos syndrome are over-represented in ME/CFS (Beighton ≥4 in 60% of children with CFS vs 24% of controls; 50% in a Swedish severe cohort; EDS about 100x expected in Finnish registries).",
     "grade": "several",
     "plain": "People with very flexible joints, or hypermobile Ehlers-Danlos syndrome, seem more likely to have ME/CFS or long COVID with PEM. No gene test exists for the hypermobile type.",
     "detail": "Barron 2002 (58 incident paediatric CFS vs 58 controls): median Beighton 4 vs 1, OR 3.5 (1.6–7.5). Bragée 2020 (229 severe CCC patients, no controls): 50% hypermobile, with high rates of possible intracranial hypertension and craniocervical findings on MRI. Finnish FinnGen/FinRegistry postviral fatigue shows EDS O/E 106–153, which is crude and inflated by healthcare use (Solving ME/CFS re-analysis). Lubell 2026 (online survey, 1,816 people after COVID) found extreme hypermobility OR 3.15 for long COVID with high post-exertional symptom exacerbation, rising to OR 9.46 with a pre-COVID orthostatic/neurocognitive profile. Hypermobile EDS has no known causal gene, and connective-tissue genes are not among GWAS hits. Collagen breakdown markers are raised in 4/4 Metabolon cohorts (Solving ME/CFS re-analysis).",
     "studies": [
      {
       "cite": "Barron 2002, J Pediatr",
       "n": "58 vs 58",
       "design": "matched case-control",
       "result": "Hypermobility 60% vs 24%; OR 3.5",
       "url": "https://doi.org/10.1067/mpd.2002.127496",
       "verified": true
      },
      {
       "cite": "Bragée 2020, Front Neurol",
       "n": "229",
       "design": "retrospective cross-sectional, no controls",
       "result": "50% hypermobile",
       "url": "https://doi.org/10.3389/fneur.2020.00828",
       "verified": true
      },
      {
       "cite": "Lubell 2026, medRxiv",
       "n": "1,816",
       "design": "retrospective online survey",
       "result": "Extreme hypermobility OR 3.15 for long COVID with high PESE",
       "url": "https://doi.org/10.64898/2026.06.24.26356475",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Prevalence in population-based ME/CFS cohorts with blinded Beighton scoring?",
      "Does hypermobility define a subgroup with distinct orthostatic or craniocervical features?"
     ],
     "hypotheses": [
      "connective-tissue",
      "brainstem-craniocervical",
      "low-blood-volume-preload"
     ],
     "test_available": "Yes: Beighton score and 2017 hEDS criteria (clinical); genetic testing only for other EDS types"
    },
    {
     "id": "genetics-mtdna-haplogroups",
     "subdomain": "Mitochondrial DNA",
     "finding": "Mitochondrial DNA haplogroups and common mtDNA SNPs are not associated with ME/CFS status, though some variants were associated with particular symptoms.",
     "grade": "early",
     "plain": "Inherited types of mitochondrial DNA do not differ between patients and controls.",
     "detail": "Billing-Ross 2016 (Cornell cohorts) found no haplogroup or SNP association with disease status but reported variant links to symptom severity. Hanson 2016 is the authors' reply clarifying this. MR suggesting higher mtDNA copy number is protective (Wielscher 2026) concerns copy number, not haplogroup. The chr1 DecodeME lead lies in DARS2, a mitochondrial tRNA synthetase, which is a weak hint.",
     "studies": [
      {
       "cite": "Hanson 2016, J Transl Med (reply on Billing-Ross 2016)",
       "n": "Cornell cohorts",
       "design": "mtDNA sequencing case-control",
       "result": "No association with disease status; symptom associations",
       "url": "https://doi.org/10.1186/s12967-016-1104-5",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Heteroplasmy and somatic mtDNA changes in muscle have not been studied at scale."
     ],
     "hypotheses": [
      "mitochondrial-energy"
     ],
     "test_available": "Research only"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "genetic-neuronal",
     "for": "Brain-only tissue enrichment (DecodeME and independent re-analyses), glutamatergic synapse and specific neuron types (Maccallini 2026), CA10 shared with chronic pain, EPHB1 MR, and GRIN2A in Slaughter 2026.",
     "against": "Most results are preprints. Individual loci do not yet replicate. Neuronal enrichment is common to many brain-related traits, and the overlap with pain and depression may reflect shared symptom liability."
    },
    {
     "id": "viral-persistence",
     "for": "Infection-response genes at 3 loci, a larger OLFM4 effect in infection onset, and suggestive EBV-load MR (MVP p=0.012).",
     "against": "Infection and non-infection onset are genetically similar, EBV MR control viruses align too, and immune cell types are not enriched."
    },
    {
     "id": "autoimmunity",
     "for": "HLA associations reported in Norway (Lande 2020) and DQA1*05:01 in DecodeME.",
     "against": "The HLA findings disagree with each other and the DecodeME signal is weak at SNP level. Common-variant genetics shows no immune-cell enrichment."
    },
    {
     "id": "connective-tissue",
     "for": "Hypermobility and EDS are enriched clinically and in registries, collagen breakdown markers are raised, and hypermobility is a risk factor for long COVID with PEM.",
     "against": "No connective-tissue genes appear among GWAS loci, the hEDS gene is unknown, and the studies are mostly clinic- or survey-based with selection bias."
    },
    {
     "id": "mitochondrial-energy",
     "for": "MR suggests mtDNA copy number is protective, and the DARS2 gene sits at the chr1 lead.",
     "against": "There is no muscle tissue enrichment, mtDNA haplogroups are not associated, and the FBXL4 assignment is distant."
    }
   ],
   "glossary": [
    {
     "term": "GWAS",
     "plain": "Genome-wide association study: compares millions of DNA letters between cases and controls to find regions linked to a disease."
    },
    {
     "term": "Locus (loci)",
     "plain": "A region of the genome. A GWAS 'hit' is a locus, not necessarily a known gene."
    },
    {
     "term": "Odds ratio per allele",
     "plain": "How much one copy of a variant changes the odds of disease. 1.08 means 8% higher odds, which is a very small effect."
    },
    {
     "term": "SNP heritability",
     "plain": "The share of variation in risk explained by common DNA variants measured on chips."
    },
    {
     "term": "Genetic correlation (rg)",
     "plain": "How much the genetic risk for two traits overlaps (from −1 to 1)."
    },
    {
     "term": "Mendelian randomisation (MR)",
     "plain": "Using inherited variants as a natural experiment to test whether a factor (such as a protein level) causes a disease."
    },
    {
     "term": "Colocalisation",
     "plain": "A test of whether two genetic signals in the same region share the same causal variant."
    },
    {
     "term": "HLA",
     "plain": "Immune genes that present fragments of microbes to T cells; often linked to autoimmune disease."
    },
    {
     "term": "Beighton score",
     "plain": "A 9-point bedside test of joint flexibility; 4 or more (adults often 5) suggests generalised hypermobility."
    },
    {
     "term": "Polygenic",
     "plain": "Risk spread across thousands of variants, each with a tiny effect."
    }
   ],
   "key_reviews": [
    {
     "cite": "Dibble 2020, Hum Mol Genet",
     "url": "https://doi.org/10.1093/hmg/ddaa169",
     "why": "Critical review of pre-DecodeME genetics showing candidate genes do not replicate."
    },
    {
     "cite": "DecodeME 2025, medRxiv preprint",
     "url": "https://doi.org/10.1101/2025.08.06.25333109",
     "why": "Primary report of the 8 loci, HLA, sex and onset analyses; summary statistics public on OSF."
    },
    {
     "cite": "Maccallini 2026, Research Square preprint",
     "url": "https://doi.org/10.21203/rs.3.rs-9702020/v1",
     "why": "DecodeME+MVP meta-analysis with tissue, cell-type and rare-variant cross-replication."
    }
   ],
   "gaps": [
    "DecodeME is still a preprint; peer review and independent locus-level replication in a PEM-phenotyped cohort are needed.",
    "No GWAS of PEM severity or of illness course; DecodeME questionnaires would allow one.",
    "No recall-by-genotype studies (e.g. OLFM4 or CA10 risk homozygotes) to link variants to measurable biology.",
    "Non-European ancestry is almost absent from ME/CFS genetics.",
    "HLA needs joint imputation or sequencing of cases and controls; KIR findings need replication.",
    "No independent, blinded replication of TRPM3 functional or SNP findings outside the originating consortium.",
    "Hypermobility prevalence in population-based ME/CFS with blinded assessment is unknown, and genetic studies of hypermobility-ME/CFS overlap have not been done.",
    "Rare-variant evidence rests on one deep-learning study of about 1,000 genomes; larger sequencing studies are needed."
   ]
  },
  {
   "domain": "trials",
   "title": "Treatment trials and natural history",
   "overview": "No treatment has replicated, placebo-controlled evidence of benefit in ME/CFS or post-COVID ME/CFS. Since 2019 the large, well-controlled trials have been negative: rituximab, Paxlovid (three trials), immunoadsorption against sham (two trials), ivabradine for long-COVID POTS, temelimab, and low-dose naltrexone for fatigue. The positive signals (daratumumab, cyclophosphamide, low-dose rapamycin, low-dose aripiprazole, single-dose pyridostigmine, 40-session HBOT) come from small, open-label or single-centre studies, and they need to be read against placebo response rates of 35-40% or more. Graded exercise is no longer recommended in England (NICE 2021), and patient surveys and a German rehab study report frequent worsening, while pacing and volume/compression measures are the most highly rated but untested by trials. Natural history data show low full recovery in adults (median ~5%), prevalence of ~0.2-1%, a female excess of 3-4:1, no clear rise in all-cause mortality, and a marked rise in suicide.",
   "findings": [
    {
     "id": "trials-rituximab",
     "subdomain": "Immune: B-cell depletion",
     "finding": "B-cell depletion with rituximab did not improve fatigue or function versus placebo in a 151-person phase III trial (RituxME).",
     "grade": "refuted",
     "plain": "A drug that removes antibody-making B cells looked promising in small Norwegian studies, but a large placebo-controlled trial found it worked no better than a dummy infusion.",
     "detail": "RituxME (Fluge 2019): 77 rituximab vs 74 placebo over 24 months; overall response 26.0% vs 35.1% (difference 9.2 points favouring placebo, 95% CI -5.5 to 23.3, p=0.22); no difference on any secondary endpoint; serious adverse events 26% vs 19%. Six-year follow-up (Rekeland 2024): SF-36 physical function 32.9 to 45.5 on rituximab vs 32.3 to 43.1 on placebo. The earlier positive phase II results (Fluge 2011, 2015) were small and partly open-label; the phase III result is the standard example of how large the placebo/natural-course improvement can be in ME/CFS trials.",
     "studies": [
      {
       "cite": "Fluge 2019, Ann Intern Med (RituxME)",
       "n": "77 vs 74",
       "design": "phase III double-blind RCT, 5 Norwegian centres",
       "result": "Response 26.0% rituximab vs 35.1% placebo, p=0.22; no secondary benefit",
       "url": "https://doi.org/10.7326/M18-1451",
       "verified": true
      },
      {
       "cite": "Rekeland 2024, PLoS One",
       "n": "RituxME 151 + CycloME 40 (75.7% / 94.4% followed)",
       "design": "6-year follow-up of two trials",
       "result": "SF-36 PF at 6 yr 45.5 rituximab vs 43.1 placebo; SF-36 PF >=70 in 27.6% vs 20.4%",
       "url": "https://doi.org/10.1371/journal.pone.0307484",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Fluge 2015, PLoS One (KTS-2-2010)",
       "n": "29",
       "design": "open-label phase II with maintenance",
       "result": "18/29 reported clinically significant responses (not controlled)",
       "url": "https://doi.org/10.1371/journal.pone.0129898",
       "verified": true
      }
     ],
     "open_questions": [
      "Is there a B-cell-dependent subgroup that a whole-population trial would dilute?",
      "Why did both arms improve by ~10 SF-36 points over 18 months?"
     ],
     "hypotheses": [
      "autoimmunity",
      "serum-factor"
     ],
     "test_available": "Not recommended outside trials"
    },
    {
     "id": "trials-cyclophosphamide",
     "subdomain": "Immune: broad immunosuppression",
     "finding": "In an open-label phase II study, 55% of 40 patients responded to six IV cyclophosphamide infusions, and many responders stayed improved at 6 years, but there was no control arm.",
     "grade": "early",
     "plain": "A chemotherapy drug that suppresses the immune system was followed by improvement in about half of a small Norwegian group, but without a comparison group the effect cannot be separated from natural improvement or placebo.",
     "detail": "CycloME (Rekeland 2020): 600-700 mg/m2 every 4 weeks x6; 22/40 (55%) responders; SF-36 PF 35.4 to 54.4 at 18 months and 56.7 at 6 years; 44.1% had SF-36 PF >=70 at 6 years versus 20.4% of RituxME placebo patients (different trial, not randomised). Nausea in ~90%; 11 serious adverse events in 8 patients. Response was higher in carriers of HLA-DQB1*03:03 and/or HLA-C*07:04 (83% vs 43%), an unreplicated post hoc finding. The authors themselves advise against use outside trials because of toxicity (infertility, bladder and cancer risk).",
     "studies": [
      {
       "cite": "Rekeland 2020, Front Med",
       "n": "40",
       "design": "open-label phase II",
       "result": "55% responders; 68% of responders still improved at 3-4 years",
       "url": "https://doi.org/10.3389/fmed.2020.00162",
       "verified": true
      },
      {
       "cite": "Rekeland 2024, PLoS One",
       "n": "36 of 40 followed",
       "design": "6-year follow-up",
       "result": "SF-36 PF 56.7 at 6 yr; 17.6% at >=90 (normal range)",
       "url": "https://doi.org/10.1371/journal.pone.0307484",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would a randomised trial against an active-placebo reproduce the effect?",
      "Does the HLA association replicate (HLA-C*07:04 also appears in other ME/CFS genetic reports)?"
     ],
     "hypotheses": [
      "autoimmunity",
      "immune-exhaustion"
     ],
     "test_available": "Not recommended outside trials"
    },
    {
     "id": "trials-daratumumab",
     "subdomain": "Immune: plasma-cell depletion",
     "finding": "In an open-label pilot, 6 of 10 women with moderate-to-severe ME/CFS improved markedly after the anti-CD38 antibody daratumumab, and low baseline NK-cell count predicted non-response.",
     "grade": "early",
     "plain": "A drug that removes long-lived antibody-producing plasma cells was followed by large, lasting improvements in six of ten patients. This is the most striking recent signal, but it is ten people with no placebo group.",
     "detail": "Fluge 2025: 4-7 subcutaneous injections after a 12-week run-in. Mean SF-36 PF 25.9 to 55.0 at 8-9 months (p=0.002); in the 6 responders 32.2 to 78.3; 5 sustained improvement (SF-36 PF ~88) with step counts above 10,000/day for weeks. Mean steps 3,359 to 5,862. IgG fell 54% in responders vs 40% in non-responders. Low baseline NK-cell counts associated with no response. No serious adverse events. A randomised trial is planned in Norway; Charité is pursuing a related approach with inebilizumab. Given RituxME's ~35% placebo response, n=10 open-label cannot establish efficacy.",
     "studies": [
      {
       "cite": "Fluge 2025, Front Med",
       "n": "10",
       "design": "open-label pilot (EudraCT 2022-000281-18)",
       "result": "6/10 marked improvement; SF-36 PF 25.9 to 55.0 (p=0.002)",
       "url": "https://doi.org/10.3389/fmed.2025.1607353",
       "verified": true
      },
      {
       "cite": "Rucker, The Sick Times 2026 (conference report)",
       "n": "10",
       "design": "follow-up talk, Berlin 2026",
       "result": "Fluge reported 5 of 6 responders still in remission at 2-3 years",
       "url": "https://thesicktimes.org/2026/05/26/international-me-cfs-conference-roundup-setbacks-and-new-hopes-for-therapeutic-research/",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does the response survive randomisation and blinding?",
      "Is NK-cell count a genuine predictor or a chance correlation in n=10?",
      "Which antibodies, if any, are being removed?"
     ],
     "hypotheses": [
      "autoimmunity",
      "serum-factor"
     ],
     "test_available": "Research only (trial planned in Norway)"
    },
    {
     "id": "trials-immunoadsorption",
     "subdomain": "Immune: antibody removal",
     "finding": "Removing antibodies by immunoadsorption was no better than a sham procedure in two controlled trials (IAMPOCO, n=40; IA-PACS-CFS, n=65), although open-label series had reported 50-70% responders.",
     "grade": "refuted",
     "plain": "Filtering antibodies out of the blood seemed to help in uncontrolled studies. When patients were compared with others who had a fake procedure, both groups improved similarly.",
     "detail": "Stein 2025 (open-label, post-COVID ME/CFS with raised beta2-adrenergic receptor antibodies, n=20): 70% 'responders', SF-36 PF +17.75 at 4 weeks; antibody fall did not distinguish responders. IAMPOCO (Mainz, tryptophan adsorbers, patient-blinded sham crossover, n=40): no difference on the Post-COVID Functional Scale (OR 1.17, p=0.771) or fatigue, cognition, grip strength; 24 vs 10 adverse events. IA-PACS-CFS (Charité, 44 IA vs 21 sham, 5 sessions): 'no statistically or clinically significant differences' on Chalder fatigue at day 60 (preliminary, conference). Critics note IA-PACS-CFS did not select for autoantibody-positive patients; IAMPOCO removed GPCR antibodies and still showed no benefit. A 2025 antigen-profiling study found no elevated GPCR autoantibodies in ME/CFS at all.",
     "studies": [
      {
       "cite": "Stein 2025, Lancet Reg Health Eur",
       "n": "20",
       "design": "prospective open-label cohort (repeat IA, beta2-AR antibody positive)",
       "result": "70% responders at 4 weeks; no control",
       "url": "https://doi.org/10.1016/j.lanepe.2024.101161",
       "verified": true
      },
      {
       "cite": "Stortz/Kraus et al. 2026, IAMPOCO (Mainz repository; Lancet Reg Health Eur correspondence)",
       "n": "40",
       "design": "randomised patient-blinded sham-controlled crossover",
       "result": "No difference: PCFS OR 1.17, p=0.771; no secondary benefit",
       "url": "https://openscience.ub.uni-mainz.de/handle/20.500.12030/16359",
       "verified": true
      },
      {
       "cite": "IA-PACS-CFS (Charité), presented 2026",
       "n": "44 vs 21",
       "design": "double-blind sham-controlled RCT",
       "result": "Failed primary endpoint (Chalder fatigue, day 60)",
       "url": "https://thesicktimes.org/2026/05/26/international-me-cfs-conference-roundup-setbacks-and-new-hopes-for-therapeutic-research/",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Stein, conference case series 2026",
       "n": "15",
       "design": "uncontrolled, autoantibody-positive",
       "result": "7/15 responded",
       "url": "https://thesicktimes.org/2026/05/26/international-me-cfs-conference-roundup-setbacks-and-new-hopes-for-therapeutic-research/",
       "verified": true
      },
      {
       "cite": "Wossidlo 2026, Infection",
       "n": "18",
       "design": "prospective exploratory, uncontrolled",
       "result": "Reports changes in autoantibodies and performance after 5 sessions",
       "url": "https://doi.org/10.1007/s15010-026-02914-8",
       "verified": true
      }
     ],
     "open_questions": [
      "Does an autoantibody-selected subgroup benefit in a sham-controlled design?",
      "Are the measured GPCR antibodies meaningful at all, given negative antigen-profiling data?"
     ],
     "hypotheses": [
      "autoimmunity",
      "serum-factor"
     ],
     "test_available": "Not recommended outside trials; GPCR antibody tests (CellTrend) are commercial but of unproven meaning"
    },
    {
     "id": "trials-bc007",
     "subdomain": "Immune: GPCR-antibody neutralisation",
     "finding": "The aptamer BC007 (rovunaptabin) failed to beat placebo in the company's 114-person long-COVID trial; a 30-person investigator-led trial reported secondary-endpoint gains.",
     "grade": "contested",
     "plain": "A drug designed to neutralise antibodies against blood-vessel and heart receptors was tested twice in long COVID. The larger trial found no benefit; a smaller exploratory one reported improvements on fatigue scales.",
     "detail": "reCOVer (Erlangen, Hohberger 2025): n=30 with GPCR autoantibodies, primary endpoint safety (9 vs 5 adverse events, NS); secondary endpoints improved (FACIT fatigue p=0.038, Bell score p=0.0004, FSS p=0.009) in an exploratory crossover analysis. BLOC (Berlin Cures, NCT05911009, ~114 participants, 19 sites): topline November 2024 showed no difference from placebo; data unpublished; the company stopped operating. The negative result is larger but unpublished; the positive one is small, exploratory and multiplicity-prone. Combined with the immunoadsorption sham trials and the negative antigen-profiling study, the GPCR-autoantibody route has weakened.",
     "studies": [
      {
       "cite": "Hohberger 2025, eClinicalMedicine (reCOVer)",
       "n": "30",
       "design": "phase IIa double-blind placebo-controlled crossover RCT",
       "result": "Safety primary met; secondary fatigue/QoL improvements",
       "url": "https://doi.org/10.1016/j.eclinm.2025.103358",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Berlin Cures BLOC trial topline, 2024 (NCT05911009)",
       "n": "~114",
       "design": "phase II double-blind RCT",
       "result": "No significant effect vs placebo (company announcement; unpublished)",
       "url": "https://clinicaltrials.gov/study/NCT05911009",
       "verified": false
      }
     ],
     "open_questions": [
      "Will the BLOC data be published?",
      "Is there any validated assay for functional GPCR antibodies?"
     ],
     "hypotheses": [
      "autoimmunity",
      "vascular-endothelial"
     ],
     "test_available": "Not available"
    },
    {
     "id": "trials-ivig",
     "subdomain": "Immune: immunoglobulin",
     "finding": "IVIG has no consistent controlled evidence: 1990s ME/CFS RCTs conflicted, and a 30-person trial in autoimmune POTS found no difference from albumin.",
     "grade": "contested",
     "plain": "Infusions of pooled antibodies from donors are sometimes used for severe patients. Old trials gave mixed answers and the most recent small trial in POTS found the same improvement with a volume-matched control infusion.",
     "detail": "iSTAND (Vernino 2024): 16 IVIG vs 14 albumin, 0.4 g/kg x8 over 12 weeks; COMPASS-31 change -5.5 vs -10.6 (p=0.63); responders 46.7% vs 38.5% (NS). Albumin also expands volume, so the comparator may have masked or matched a volume effect. 1990s ME/CFS RCTs: Lloyd 1990 (n=49) positive, Peterson 1990 (n=30) negative, Vollmer-Conna 1997 (n=99) negative. In the Eckey/Xiao 2025 patient survey IVIG was rated among the most helpful in the severe multisystemic cluster (self-report, selected users). The RECOVER-AUTONOMIC IVIG arm in long-COVID POTS has not yet reported (as of Oct 2026).",
     "studies": [
      {
       "cite": "Vernino 2024, Clin Auton Res (iSTAND)",
       "n": "16 vs 14",
       "design": "RCT vs albumin",
       "result": "No difference in COMPASS-31 (p=0.629)",
       "url": "https://doi.org/10.1007/s10286-024-01020-9",
       "verified": true
      },
      {
       "cite": "Lloyd 1990, Am J Med",
       "n": "49",
       "design": "RCT",
       "result": "Benefit reported",
       "url": "https://doi.org/10.1016/0002-9343(90)90173-b",
       "verified": false
      }
     ],
     "contradicting": [
      {
       "cite": "Vollmer-Conna 1997, Am J Med",
       "n": "99",
       "design": "RCT, adolescents/adults",
       "result": "No benefit over placebo",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Vollmer-Conna+1997+immunoglobulin+chronic+fatigue",
       "verified": false
      },
      {
       "cite": "Peterson 1990, Am J Med",
       "n": "30",
       "design": "RCT",
       "result": "No benefit",
       "url": "https://doi.org/10.1016/0002-9343(90)90172-a",
       "verified": false
      }
     ],
     "open_questions": [
      "What will RECOVER-AUTONOMIC's IVIG arm show?",
      "Is there a small-fibre-neuropathy subgroup that responds?"
     ],
     "hypotheses": [
      "autoimmunity",
      "autonomic-small-fibre"
     ],
     "test_available": "Prescribed in some specialist clinics; costly"
    },
    {
     "id": "trials-rintatolimod",
     "subdomain": "Immune: TLR3 agonist",
     "finding": "Rintatolimod (Ampligen) gave a modest placebo-adjusted gain in treadmill time in a 234-person phase III trial, but the result was borderline and the FDA declined approval.",
     "grade": "contested",
     "plain": "An immune-stimulating drug given by drip twice a week helped exercise time a little in one large company trial; regulators did not find this convincing.",
     "detail": "AMP-516 (Strayer 2012): 40 weeks IV 400 mg twice weekly; intention-to-treat placebo-adjusted exercise tolerance +21.3% (p=0.047), +28% after compliance correction; reduced use of symptomatic drugs (p=0.048). Lead authors employed by the manufacturer. FDA issued a complete response letter in 2009 and has not approved it. A 2020 post hoc analysis suggested better response in illness of 2-8 years' duration. Not replicated by an independent group.",
     "studies": [
      {
       "cite": "Strayer 2012, PLoS One",
       "n": "234",
       "design": "phase III double-blind RCT, 12 sites",
       "result": "Exercise tolerance +21.3% vs placebo (p=0.047)",
       "url": "https://doi.org/10.1371/journal.pone.0031334",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Strayer 2020, PLoS One (post hoc)",
       "n": "ITT population",
       "design": "post hoc subgroup",
       "result": "Benefit concentrated in 2-8 years' duration (hypothesis-generating)",
       "url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC7595369",
       "verified": false
      }
     ],
     "open_questions": [
      "Would an independent trial with a pre-specified duration subgroup replicate?"
     ],
     "hypotheses": [
      "innate-hyperactivation",
      "viral-persistence"
     ],
     "test_available": "Expanded access in the US only"
    },
    {
     "id": "trials-temelimab",
     "subdomain": "Immune/viral: HERV-W",
     "finding": "The anti-HERV-W envelope antibody temelimab did not improve fatigue versus placebo in 203 HERV-W-positive long-COVID patients.",
     "grade": "refuted",
     "plain": "A drug aimed at a dormant virus-like gene product that can be switched on after infection did not help long-COVID fatigue in a proper trial.",
     "detail": "GNC-501 (GeNeuro, 2024): IV temelimab vs placebo, primary endpoint 3-point fall in PROMIS Fatigue SF-7a at week 24; no clinically meaningful difference and most secondary endpoints negative; well tolerated. Company suggested shorter disease duration or changes in HERV-W status may matter (post hoc). Company press release; full peer-reviewed paper not seen.",
     "studies": [
      {
       "cite": "GeNeuro 2024, GNC-501 topline (company release)",
       "n": "203",
       "design": "phase II double-blind RCT",
       "result": "Primary fatigue endpoint not met",
       "url": "https://www.fiercebiotech.com/biotech/geneuros-long-covid-trial-fails-triggering-layoffs-and-search-strategic-alternatives",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Is HERV-W activation a marker without causal role?"
     ],
     "hypotheses": [
      "viral-persistence",
      "innate-hyperactivation"
     ],
     "test_available": "Not available"
    },
    {
     "id": "trials-rapamycin",
     "subdomain": "Immune/metabolic: mTOR inhibition",
     "finding": "Low-dose rapamycin (6 mg weekly) was followed by self-reported improvement in about three-quarters of completers in an uncontrolled decentralised study; randomised trials are pending.",
     "grade": "early",
     "plain": "A low weekly dose of an immune-modulating drug thought to restart cellular clean-up (autophagy) was followed by improvement in many patients, but no one in the study got a placebo.",
     "detail": "Ruan 2025: 86 enrolled, 70 reached day 30; 52 (74.3%) reported improvement in fatigue, PEM and orthostatic intolerance, with MFI and SF-36 gains; autophagy markers (pSer258-ATG13 down, BECLIN-1 up) moved and correlated with outcomes. No serious adverse events. Follow-on metabolomics (Gile 2026) in responders only. Uncontrolled response rates of this size are similar to placebo responses in RCTs (35-40%+) plus attrition bias. The Mount Sinai placebo-controlled trial in long COVID (NCT06960928, ~80-90) ends Nov 2026.",
     "studies": [
      {
       "cite": "Ruan 2025, J Transl Med",
       "n": "86 (70 assessed)",
       "design": "decentralised open-label",
       "result": "74.3% improved; autophagy markers changed",
       "url": "https://doi.org/10.1186/s12967-025-07213-8",
       "verified": true
      },
      {
       "cite": "Gile 2026, J Transl Med",
       "n": "responder subset",
       "design": "observational mechanistic follow-up",
       "result": "Purine metabolism and mitochondrial changes in responders",
       "url": "https://doi.org/10.1186/s12967-026-08575-3",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Will the Mount Sinai RCT confirm benefit?",
      "Can autophagy markers select responders prospectively?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "immune-exhaustion"
     ],
     "test_available": "Off-label prescription in some US clinics; research only in UK"
    },
    {
     "id": "trials-paxlovid",
     "subdomain": "Antiviral: SARS-CoV-2",
     "finding": "Three randomised trials of nirmatrelvir-ritonavir (Paxlovid) for 15-25 days in established long COVID (total ~1,200 people) found no benefit on any phenotype.",
     "grade": "refuted",
     "plain": "If leftover coronavirus were driving long COVID, an antiviral might help. Three good trials found it did not, at least at the doses and durations tested.",
     "detail": "RECOVER-VITAL (Baden 2026): 959 randomised to 15 or 25 days vs placebo-ritonavir, cognitive, autonomic and exercise phenotypes; no primary benefit (e.g. autonomic -6.4%, 95% CI -18.5 to 5.7). STOP-PASC (Stanford, n=155, 15 days) and PAX LC (Yale, n=100, 15 days, PROMIS-29) also null. STOP-PASC immune profiling found no EBV reactivation or microclot differences versus controls. The trials do not exclude a role for persistence in tissue reservoirs that this drug fails to reach, or effects in very early or highly selected patients.",
     "studies": [
      {
       "cite": "Baden 2026, Lancet Infect Dis (RECOVER-VITAL)",
       "n": "959",
       "design": "phase 2 double-blind RCT, 69 US sites",
       "result": "No benefit for any phenotype at day 90",
       "url": "https://doi.org/10.1016/S1473-3099(26)00406-8",
       "verified": true
      },
      {
       "cite": "Sawano 2025, Lancet Infect Dis (PAX LC)",
       "n": "100",
       "design": "phase 2 double-blind RCT",
       "result": "No change in PROMIS-29 physical score vs placebo",
       "url": "https://medicine.yale.edu/news-article/does-paxlovid-reduce-long-covid-symptoms-yale-led-trial-finds-out/",
       "verified": false
      },
      {
       "cite": "Geng 2024, JAMA Intern Med (STOP-PASC)",
       "n": "155",
       "design": "double-blind RCT",
       "result": "No benefit",
       "url": "https://doi.org/10.1001/jamainternmed.2024.2007",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would longer courses, combination antivirals or monoclonal antibodies (trials under way) reach tissue reservoirs?",
      "Is there a biomarker of persistent antigen that would select responders?"
     ],
     "hypotheses": [
      "viral-persistence"
     ],
     "test_available": "Not recommended for long COVID"
    },
    {
     "id": "trials-valganciclovir",
     "subdomain": "Antiviral: herpesviruses",
     "finding": "Valganciclovir in 30 patients selected for high HHV-6 and EBV antibody levels improved some secondary fatigue and cognition scores in a small RCT; it has not been replicated.",
     "grade": "early",
     "plain": "Antivirals against herpesviruses have been tried in patients with high antibody levels, with modest results in one small trial. High antibodies show past infection, not necessarily active virus.",
     "detail": "Montoya 2013: 2:1 randomised, 6 months; reported benefit on MFI mental fatigue (p=0.039), Fatigue Severity Scale (p=0.006) and cognition (p=0.025); primary endpoint detail not confirmed this session. Preceded by an open-label series (Kogelnik 2006, 9/12 near-resolution). Neutropenia risk requires monitoring. Lerner's valacyclovir/valganciclovir EBV-subset series are uncontrolled. Selection by IgG titre is a weak marker of active replication.",
     "studies": [
      {
       "cite": "Montoya 2013, J Med Virol",
       "n": "30",
       "design": "double-blind RCT 2:1",
       "result": "Secondary fatigue/cognition improvements",
       "url": "https://doi.org/10.1002/jmv.23713",
       "verified": false
      },
      {
       "cite": "Kogelnik 2006, J Clin Virol",
       "n": "12",
       "design": "open-label",
       "result": "9/12 near resolution",
       "url": "https://doi.org/10.1016/s1386-6532(06)70009-9",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would selection by viral DNA (PCR) rather than antibody titre identify responders?"
     ],
     "hypotheses": [
      "herpes-reactivation",
      "viral-persistence"
     ],
     "test_available": "EBV/CMV/HHV-6 PCR and serology are clinically available; antiviral use off-label"
    },
    {
     "id": "trials-ldn",
     "subdomain": "Neuro-immune: low-dose naltrexone",
     "finding": "The first sizeable placebo-controlled trial of low-dose naltrexone in post-COVID ME/CFS (66 vs 71, 16 weeks, up to 4.5 mg) found no fatigue benefit, with over 40% improving on placebo.",
     "grade": "refuted",
     "plain": "Low-dose naltrexone is widely used off-label and many patients report benefit. In a proper trial, the drug did not reduce fatigue more than placebo. Pain and subgroup results are still being analysed.",
     "detail": "The trial is Canadian (Nacul, University of British Columbia; NCT05430152), presented at the Berlin International ME/CFS Conference, May 2026; preliminary and unpublished. Earlier evidence was retrospective (Polo 2019, n=218, 74% self-reported benefit) or uncontrolled long-COVID series. I found no separate German LDN RCT; the 'German' attribution likely reflects the Berlin venue. The LIFT trial (pyridostigmine x LDN factorial, n=160, Harvard/OMF, NCT06366724) and a RECOVER paediatric trial are pending. Effects on pain, sleep or specific subgroups remain possible.",
     "studies": [
      {
       "cite": "Nacul et al., presented May 2026 (UBC; NCT05430152)",
       "n": "66 vs 71",
       "design": "double-blind placebo-controlled RCT, 16 weeks",
       "result": "Primary (fatigue) not met; >40% placebo responders",
       "url": "https://thesicktimes.org/2026/05/26/international-me-cfs-conference-roundup-setbacks-and-new-hopes-for-therapeutic-research/",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Polo 2019, Fatigue: Biomed Health Behav",
       "n": "218",
       "design": "retrospective chart review",
       "result": "74% reported benefit",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Polo+low+dose+naltrexone+chronic+fatigue+2019",
       "verified": false
      }
     ],
     "open_questions": [
      "Does LDN help pain or a pain-predominant subgroup?",
      "What will the LIFT factorial show?"
     ],
     "hypotheses": [
      "neuroinflammation",
      "innate-hyperactivation"
     ],
     "test_available": "Private/off-label prescription"
    },
    {
     "id": "trials-aripiprazole",
     "subdomain": "Neuro: low-dose aripiprazole",
     "finding": "In a retrospective single-clinic review, 74% of 101 patients on very low-dose aripiprazole (mostly 0.25-2 mg) reported improvement; there is no randomised trial.",
     "grade": "early",
     "plain": "A tiny dose of an antipsychotic drug, thought to act on dopamine and brain immune cells, was linked to improvement in a clinic's records, but this kind of study cannot rule out placebo or selective reporting.",
     "detail": "Crosby 2021 (Stanford): 101 patients; ~74% reported improvement in brain fog, fatigue, unrefreshing sleep or energy; side effects included insomnia, akathisia, vivid dreams. Retrospective, no control, outcomes from clinic notes. Rationale (microglial modulation, dopamine partial agonism) is speculative. No RCT reported as of Oct 2026.",
     "studies": [
      {
       "cite": "Crosby 2021, J Transl Med",
       "n": "101",
       "design": "retrospective single-clinic",
       "result": "74% improved",
       "url": "https://doi.org/10.1186/s12967-021-02721-9",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Would a placebo-controlled trial confirm benefit?"
     ],
     "hypotheses": [
      "neuroinflammation"
     ],
     "test_available": "Off-label prescription"
    },
    {
     "id": "trials-pyridostigmine",
     "subdomain": "Circulation: cholinergic / preload",
     "finding": "A single 60 mg pyridostigmine dose raised peak oxygen uptake relative to placebo on a repeat invasive exercise test in 45 ME/CFS patients with preload failure, largely because the placebo group got worse; longer trials are negative or pending.",
     "grade": "early",
     "plain": "Pyridostigmine boosts nerve signalling that tightens veins and returns blood to the heart. In a one-day lab study it stopped exercise capacity dropping on a second test. Whether it helps daily life is untested; the factorial LIFT trial should answer that.",
     "detail": "Joseph/Systrom 2022: 23 vs 22; peak VO2 +13.3 vs -40.2 mL/min (difference ~54 mL/min), with cardiac output and right atrial pressure maintained instead of falling; oxygen extraction unchanged. Same-day laboratory endpoint. São Paulo 2025 (ATS abstract, long COVID with RV preload dysfunction, n=32, up to 360 mg/day for 6-10 weeks): negative overall (p=0.35), borderline +103 mL in a post hoc subgroup. Kanjwal 2011 POTS retrospective (n=203): 43% improved, 19% stopped for GI effects. LIFT (n=160, 13 weeks, FUNCAP-55 and CPET endpoints) has not reported.",
     "studies": [
      {
       "cite": "Joseph 2022, Chest",
       "n": "45 (23 vs 22)",
       "design": "single-dose double-blind RCT between two iCPETs",
       "result": "Peak VO2 +13 vs -40 mL/min",
       "url": "https://doi.org/10.1016/j.chest.2022.04.146",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "São Paulo group 2025, ATS abstract",
       "n": "32",
       "design": "RCT 6-10 weeks, long COVID",
       "result": "Negative overall (p=0.35)",
       "url": "https://www.atsjournals.org/",
       "verified": false
      },
      {
       "cite": "Kanjwal 2011, Pacing Clin Electrophysiol",
       "n": "203",
       "design": "retrospective POTS",
       "result": "43% improved; 19% stopped for GI effects",
       "url": "https://doi.org/10.1111/j.1540-8159.2011.03047.x",
       "verified": false
      }
     ],
     "open_questions": [
      "Does LIFT show benefit on daily function?",
      "Does preload failure on iCPET predict response?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Licensed drug (myasthenia); off-label use; iCPET research only"
    },
    {
     "id": "trials-ivabradine",
     "subdomain": "Circulation: heart-rate control",
     "finding": "Ivabradine lowered standing heart rate but did not improve symptoms in 181 people with long-COVID POTS (RECOVER-AUTONOMIC), despite an earlier positive 22-person crossover in hyperadrenergic POTS.",
     "grade": "refuted",
     "plain": "Slowing a racing heart is not the same as making people feel better. A large trial in long-COVID POTS showed exactly that.",
     "detail": "Taub 2021: 22 hyperadrenergic POTS (noradrenaline >600 pg/mL), 1-month crossover; lower HR and better RAND-36 QoL. RECOVER-AUTONOMIC (2026, ACC late-breaker): 181 adults, 12 weeks; no meaningful change in orthostatic intolerance or long-COVID symptoms vs placebo, though supine-to-standing HR change fell significantly. The factorial coordinated non-pharmacological care arm (diet-mediated volume expansion, rehab, education) is reported separately. Full paper pending. Whether a hyperadrenergic subgroup benefits remains open.",
     "studies": [
      {
       "cite": "Taub 2021, J Am Coll Cardiol",
       "n": "22",
       "design": "double-blind crossover RCT",
       "result": "Lower HR and better QoL",
       "url": "https://doi.org/10.1016/j.jacc.2020.12.029",
       "verified": true
      },
      {
       "cite": "RECOVER-AUTONOMIC ivabradine, ACC 2026 (NIH release)",
       "n": "181",
       "design": "double-blind placebo-controlled RCT",
       "result": "No symptom benefit; HR change reduced",
       "url": "https://recovercovid.org/news/recover-autonomic-clinical-trial-results-shared-2026-acc-conference",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Does a noradrenaline-defined hyperadrenergic subgroup respond?"
     ],
     "hypotheses": [
      "autonomic-small-fibre",
      "low-blood-volume-preload"
     ],
     "test_available": "Licensed drug; off-label for POTS"
    },
    {
     "id": "trials-midodrine-fludrocortisone",
     "subdomain": "Circulation: vasoconstrictors and volume",
     "finding": "Fludrocortisone did not beat placebo in a 100-person RCT in CFS with neurally mediated hypotension; midodrine reduces orthostatic heart rate acutely in small POTS studies but has no ME/CFS outcome trial.",
     "grade": "refuted",
     "plain": "Two standard drugs for low blood pressure on standing have been tried. Fludrocortisone, which makes the body hold salt, did not help in a proper trial in CFS. Midodrine tightens blood vessels and helps some POTS patients in the short term, but it has not been properly tested in ME/CFS.",
     "detail": "Rowe 2001: fludrocortisone 0.1 mg vs placebo for 9 weeks in CFS + NMH on tilt; >=15-point wellness improvement in 14% vs 10% (p=0.76). Peterson 1998 (n=25) also negative. Midodrine: small acute crossover studies in POTS/orthostatic intolerance show lower standing HR (memory, not re-checked); risks include supine hypertension (relevant for bedbound patients). No ME/CFS RCT with function as endpoint. Grade refers to fludrocortisone; midodrine evidence is early.",
     "studies": [
      {
       "cite": "Rowe 2001, JAMA",
       "n": "100",
       "design": "double-blind RCT",
       "result": "14% vs 10% improved (p=0.76)",
       "url": "https://doi.org/10.1001/jama.285.1.52",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Ross 2014, Am J Physiol (midodrine in POTS)",
       "n": "small",
       "design": "acute crossover",
       "result": "Improved orthostatic tolerance acutely",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=midodrine+postural+tachycardia+randomized",
       "verified": false
      }
     ],
     "open_questions": [
      "Would a midodrine trial in ME/CFS with low-flow physiology show functional benefit?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "Licensed drugs; tilt test available on NHS"
    },
    {
     "id": "trials-coq10-nadh",
     "subdomain": "Metabolic: supplements",
     "finding": "Two Spanish RCTs of CoQ10 200 mg + NADH 20 mg (n=73, n=207) reported modest reductions in fatigue scores; CoQ10 alone failed in a 121-person long-COVID crossover trial.",
     "grade": "contested",
     "plain": "Supplements meant to support mitochondria produced small improvements in two trials from one group, with supplier involvement. CoQ10 on its own did nothing in long COVID.",
     "detail": "Castro-Marrero 2015: 8 weeks, n=73; FIS-40 improved (p<0.05) with higher NAD+/NADH, CoQ10, ATP in PBMCs. Castro-Marrero 2021: 12 weeks, 104 vs 103; cognitive fatigue and FIS-40 total improved (p~0.02), SF-36 improved. Same research group, industry links; effect sizes small and not replicated independently. Hansen 2023 (Denmark, CoQ10 500 mg/day, n=121 crossover) in long COVID: no benefit.",
     "studies": [
      {
       "cite": "Castro-Marrero 2015, Antioxid Redox Signal",
       "n": "73",
       "design": "double-blind RCT",
       "result": "FIS-40 improved (p<0.05)",
       "url": "https://doi.org/10.1089/ars.2014.6181",
       "verified": true
      },
      {
       "cite": "Castro-Marrero 2021, Nutrients",
       "n": "207",
       "design": "double-blind RCT",
       "result": "Cognitive fatigue and FIS-40 improved",
       "url": "https://doi.org/10.3390/nu13082658",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Hansen 2023, eClinicalMedicine",
       "n": "121",
       "design": "crossover RCT, long COVID",
       "result": "CoQ10 alone: no benefit",
       "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Hansen+coenzyme+Q10+post-COVID+condition+randomized",
       "verified": false
      }
     ],
     "open_questions": [
      "Would an independent group replicate the CoQ10+NADH result?"
     ],
     "hypotheses": [
      "mitochondrial-energy"
     ],
     "test_available": "Over-the-counter"
    },
    {
     "id": "trials-oxaloacetate",
     "subdomain": "Metabolic: supplements",
     "finding": "Oxaloacetate 2 g/day reduced Chalder fatigue more than control (−25% vs −10%) in an 82-person company-run RCT in ME/CFS; a 69-person long-COVID trial missed its primary endpoint.",
     "grade": "early",
     "plain": "A supplement that feeds a step in energy metabolism reduced fatigue in one trial run with the manufacturer. A similar trial in long COVID did not meet its main goal.",
     "detail": "RESTORE ME (Cash 2024): 90 days, 2,000 mg vs rice-flour control; fatigue −25% vs −10% (between-group p=0.0039); secondary analysis (Vernon 2025) showed cognitive gains at day 60. Sponsor (Terra Biological) involvement. REGAIN (long COVID, n=69, 42 days): primary Chalder negative, secondary cognition gains. No independent replication.",
     "studies": [
      {
       "cite": "Cash 2024, Front Neurol (RESTORE ME)",
       "n": "82",
       "design": "double-blind controlled RCT",
       "result": "Fatigue −25% vs −10%, p=0.0039",
       "url": "https://doi.org/10.3389/fneur.2024.1483876",
       "verified": true
      },
      {
       "cite": "Vernon 2025, Front Neurol",
       "n": "82",
       "design": "secondary analysis",
       "result": "Cognitive improvement at day 60 (p=0.034)",
       "url": "https://doi.org/10.3389/fneur.2025.1691147",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "REGAIN 2025 (long COVID)",
       "n": "69",
       "design": "RCT",
       "result": "Primary endpoint not met",
       "url": "https://clinicaltrials.gov/",
       "verified": false
      }
     ],
     "open_questions": [
      "Independent replication with objective endpoints (actigraphy)?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "metabolic-trap-ido"
     ],
     "test_available": "Over-the-counter"
    },
    {
     "id": "trials-hbot",
     "subdomain": "Physical: hyperbaric oxygen",
     "finding": "Forty daily sessions of hyperbaric oxygen at 2.0 ATA improved cognition and symptoms versus sham in a 73-person long-COVID trial, but ten sessions at 2.4 bar showed no benefit in an 80-person sham-controlled trial.",
     "grade": "contested",
     "plain": "Breathing pure oxygen under pressure helped in an Israeli trial with 40 sessions, but not in a Swedish trial with 10 sessions. Dose may matter, or the positive trial may not hold up. It needs daily travel, which many patients cannot do.",
     "detail": "Zilberman-Itskovich 2022 (Tel Aviv): 37 vs 36, 40 sessions, 5/week, 100% O2 at 2.0 ATA for 90 min with air breaks, sham at 1.03 ATA air; global cognition improved (d≈0.5, p=0.038), plus energy, sleep, psychiatric symptoms and pain; benefits persisted at ~1-year follow-up in 31 patients (Hadanny 2024); a cardiac substudy showed improved GLS. HOT-LoCO (Kjellberg 2025, Sweden): 40 vs 40, 10 sessions over 6 weeks, 100% O2 at 2.4 bar 90 min vs air at 1.2-1.34 bar; RAND-36 PF difference 0.63 (95% CI −7.0 to 8.3, p=0.87); both groups improved. Charité (Kedor, 2026 conference): 20 vs 40 sessions; improvements only in the 40-session group (no sham arm reported). The 'sham' pressure differs between trials and blinding of pressure is imperfect. No ME/CFS (non-COVID) RCT.",
     "studies": [
      {
       "cite": "Zilberman-Itskovich 2022, Sci Rep",
       "n": "73",
       "design": "double-blind sham RCT, 40 sessions at 2.0 ATA",
       "result": "Cognition, energy, sleep, pain improved",
       "url": "https://doi.org/10.1038/s41598-022-15565-0",
       "verified": true
      },
      {
       "cite": "Hadanny 2024, Sci Rep",
       "n": "31",
       "design": "1-year follow-up of RCT HBOT arm",
       "result": "Improvements maintained",
       "url": "https://doi.org/10.1038/s41598-024-53091-3",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Kjellberg 2025, BMJ Open (HOT-LoCO)",
       "n": "80",
       "design": "double-blind sham RCT, 10 sessions at 2.4 bar",
       "result": "No difference (RAND-36 PF diff 0.63, p=0.87)",
       "url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC11997836/",
       "verified": true
      }
     ],
     "open_questions": [
      "Is there a dose-response (sessions x pressure) in a single trial with proper sham?",
      "Does it apply to non-COVID ME/CFS?"
     ],
     "hypotheses": [
      "small-vessel-capillary",
      "mitochondrial-energy",
      "neuroinflammation"
     ],
     "test_available": "Private clinics; not NHS-funded for this use"
    },
    {
     "id": "trials-pace-get-cbt",
     "subdomain": "Rehabilitation: GET and CBT",
     "finding": "PACE (n=641) reported modest gains in self-rated fatigue and function with CBT and graded exercise, but these were not matched by objective measures, used Oxford criteria, and vanished between groups by 2.5 years.",
     "grade": "contested",
     "plain": "The largest trial of talking therapy and gradually increasing exercise said both helped moderately. Re-analysis using the trial's own original plan found the benefits much smaller and limited to questionnaires that unblinded patients filled in.",
     "detail": "White 2011: CBT and GET vs specialist medical care: Chalder fatigue −3.4 and −3.2 points, SF-36 PF +7.1 and +9.4 at 52 weeks; adaptive pacing no better than SMC. Long-term follow-up (Sharpe 2015): no between-group differences at median 31 months. Wilshire 2018 re-analysis using protocol-specified outcomes (data via FOI): CBT/GET did not significantly beat control after correction; recovery rates low (single digits to ~10%) and not different; objective measures (6-minute walk modest, step test, employment, benefits) largely null. Criticisms: Oxford criteria (fatigue only; PEM not required), outcome switching, unblinded self-report, mid-trial newsletter. Cochrane exercise review (Larun, re-dated 2024) rates evidence low-moderate and notes limited applicability to ME/CFS defined with PEM; a full update is pending.",
     "studies": [
      {
       "cite": "White 2011, Lancet (PACE)",
       "n": "641",
       "design": "4-arm RCT",
       "result": "Modest self-reported gains for CBT/GET; APT no effect",
       "url": "https://doi.org/10.1016/S0140-6736(11)60096-2",
       "verified": true
      },
      {
       "cite": "Sharpe 2015, Lancet Psychiatry",
       "n": "481 followed",
       "design": "long-term follow-up",
       "result": "No between-group differences at ~2.5 years",
       "url": "https://doi.org/10.1016/S2215-0366(15)00317-X",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Wilshire 2018, BMC Psychol",
       "n": "641 (re-analysis)",
       "design": "protocol-based re-analysis",
       "result": "CBT/GET not significantly better after correction; effects confined to self-report",
       "url": "https://doi.org/10.1186/s40359-018-0218-3",
       "verified": true
      },
      {
       "cite": "Larun 2019/2024, Cochrane (CD003200)",
       "n": "8 RCTs",
       "design": "systematic review",
       "result": "Exercise may slightly reduce fatigue; low-certainty; applicability to PEM-defined ME/CFS uncertain",
       "url": "https://doi.org/10.1002/14651858.CD003200.pub9",
       "verified": false
      }
     ],
     "open_questions": [
      "Would a blinded-assessor trial with objective endpoints in PEM-defined patients show any benefit?"
     ],
     "hypotheses": [
      "sickness-behaviour"
     ],
     "test_available": "n/a"
    },
    {
     "id": "trials-nice-2021-harms",
     "subdomain": "Rehabilitation: guidance and harms",
     "finding": "NICE's 2021 guideline (NG206) told clinicians not to offer graded exercise therapy, and patient surveys plus a 2026 German rehabilitation study report frequent worsening after exercise-based or even pacing-focused rehab.",
     "grade": "several",
     "plain": "England's health guidance body reviewed the evidence and concluded that the old exercise trials were low quality and that GET should not be offered. Large patient surveys and a German study suggest rehab programmes often make people worse.",
     "detail": "NICE NG206 (Oct 2021): rated the GET/CBT evidence 'low' or 'very low' quality; recommends against GET or any programme based on fixed incremental increases; CBT may be offered only as supportive therapy, not as cure. Eckey/Xiao 2025 survey (n≈3,925): GET had a net score of about −72% (most harmful item). CFS_CARE (Charité, 2026 conference): a specialised rehab programme including pacing left 42 of 97 participants worse on the Bell scale, few better. Survey data are self-selected; CFS_CARE is uncontrolled and preliminary. Nevertheless the direction is consistent across sources.",
     "studies": [
      {
       "cite": "NICE 2021, NG206",
       "n": "guideline",
       "design": "evidence review",
       "result": "Do not offer GET; CBT only as supportive",
       "url": "https://www.nice.org.uk/guidance/ng206",
       "verified": false
      },
      {
       "cite": "Eckey 2025, PNAS",
       "n": "~3,925",
       "design": "patient survey",
       "result": "GET net −72%",
       "url": "https://doi.org/10.1073/pnas.2426874122",
       "verified": true
      },
      {
       "cite": "Wittke/Scheibenbogen, CFS_CARE, presented 2026",
       "n": "97",
       "design": "uncontrolled rehab study",
       "result": "42/97 worsened",
       "url": "https://thesicktimes.org/2026/05/26/international-me-cfs-conference-roundup-setbacks-and-new-hopes-for-therapeutic-research/",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "White 2011, Lancet (PACE)",
       "n": "641",
       "design": "RCT",
       "result": "No excess serious adverse reactions reported with GET",
       "url": "https://doi.org/10.1016/S0140-6736(11)60096-2",
       "verified": true
      }
     ],
     "open_questions": [
      "What exactly distinguishes harmful from tolerable activity programmes?",
      "How common is lasting deterioration after rehab in a controlled study?"
     ],
     "hypotheses": [
      "sickness-behaviour",
      "mitochondrial-energy"
     ],
     "test_available": "n/a"
    },
    {
     "id": "trials-pacing",
     "subdomain": "Self-management: pacing",
     "finding": "Pacing (staying within one's energy limits) is the top-rated approach in patient surveys and NICE's recommended foundation, but it has never shown benefit in a controlled trial (adaptive pacing was no better than usual care in PACE).",
     "grade": "early",
     "plain": "Patients consistently rate pacing as the most helpful thing they do, and guidelines recommend it. It has not been properly tested in a trial designed with patients, so its benefit rests on consensus and self-report.",
     "detail": "Eckey/Xiao 2025: pacing net score ~75% (highest of >150 treatments). PACE APT arm (n=160): no difference vs SMC. Critics argue APT in PACE was not the envelope-style pacing patients use. Pacing is plausible as harm avoidance (preventing PEM) rather than a treatment. Heart-rate-guided pacing studies are small and uncontrolled.",
     "studies": [
      {
       "cite": "Eckey 2025, PNAS",
       "n": "~3,925",
       "design": "patient survey",
       "result": "Pacing net assessment ~75%",
       "url": "https://doi.org/10.1073/pnas.2426874122",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "White 2011, Lancet (PACE)",
       "n": "160 APT vs 160 SMC",
       "design": "RCT",
       "result": "APT no better than specialist care",
       "url": "https://doi.org/10.1016/S0140-6736(11)60096-2",
       "verified": true
      }
     ],
     "open_questions": [
      "Does HR- or wearable-guided pacing reduce PEM frequency in a randomised trial?"
     ],
     "hypotheses": [
      "mitochondrial-energy",
      "low-blood-volume-preload"
     ],
     "test_available": "Self-management; NHS ME/CFS services"
    },
    {
     "id": "trials-patient-surveys",
     "subdomain": "Patient-reported outcomes",
     "finding": "A 3,925-person survey rated >150 treatments; illness severity predicted perceived response more strongly than diagnosis, and symptom clusters (severe multisystemic, POTS-dominant, cognitive/pain, mild) preferred different treatments.",
     "grade": "early",
     "plain": "Thousands of patients rated what helped. The cheap supportive measures came top (pacing, fluids, compression), graded exercise came bottom, and different kinds of patients reported benefit from different things.",
     "detail": "Eckey, Li, Morrison, Davis, Xiao, PNAS 2025: ME/CFS and long COVID ratings correlated (R²=0.68). Top: pacing 75%, fluids/electrolytes ~69%, compression ~62%; GET −72%. Clusters: severe multisystemic (IVIG, lymphatic drainage), POTS-dominant (compression, autonomic modulators), cognitive (CNS stimulants), mild. Self-selected social-media sample, no placebo, recall bias; useful for prioritising trials, not for efficacy claims.",
     "studies": [
      {
       "cite": "Eckey 2025, PNAS",
       "n": "~3,925",
       "design": "online patient survey",
       "result": "Pacing, fluids, compression highest; GET lowest; severity shaped response",
       "url": "https://doi.org/10.1073/pnas.2426874122",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Do survey-defined clusters map onto biological subgroups?"
     ],
     "hypotheses": [
      "low-blood-volume-preload",
      "autonomic-small-fibre"
     ],
     "test_available": "n/a"
    },
    {
     "id": "trials-placebo-response",
     "subdomain": "Trial methodology",
     "finding": "Placebo and natural-course improvement in ME/CFS and long-COVID trials is large (35% responders in RituxME, >40% in the LDN trial, both arms improving in IVIG, HBOT and immunoadsorption trials), so uncontrolled response rates of 50-75% are uninterpretable.",
     "grade": "replicated",
     "plain": "In almost every controlled trial, many people on the dummy treatment also improve. This is why open-label studies that report most patients getting better cannot show a treatment works.",
     "detail": "RituxME placebo SF-36 PF 32.3 to 45.5 at 18 months; LDN >40% placebo responders; iSTAND albumin COMPASS −10.6; HOT-LoCO both arms improved; IAMPOCO both arms improved; BLOC failure attributed partly to placebo response. Contributing factors: regression to the mean (patients enrol when worse), natural fluctuation, expectation, and post-COVID spontaneous recovery in early cohorts.",
     "studies": [
      {
       "cite": "Fluge 2019, Ann Intern Med",
       "n": "151",
       "design": "RCT",
       "result": "35.1% placebo response",
       "url": "https://doi.org/10.7326/M18-1451",
       "verified": true
      },
      {
       "cite": "Vernino 2024, Clin Auton Res",
       "n": "30",
       "design": "RCT",
       "result": "Albumin arm improved as much as IVIG",
       "url": "https://doi.org/10.1007/s10286-024-01020-9",
       "verified": true
      },
      {
       "cite": "Kjellberg 2025, BMJ Open",
       "n": "80",
       "design": "RCT",
       "result": "Both arms improved",
       "url": "https://pmc.ncbi.nlm.nih.gov/articles/PMC11997836/",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What run-in periods and objective endpoints best reduce placebo noise?"
     ],
     "hypotheses": [],
     "test_available": "n/a"
    },
    {
     "id": "trials-recovery-rates",
     "subdomain": "Natural history: recovery",
     "finding": "Full recovery from established adult ME/CFS is uncommon: a systematic review of 14 criteria-based cohorts found a median full-recovery rate of 5% (range 0-31%) and median improvement of 39.5%.",
     "grade": "several",
     "plain": "Most adults with long-standing ME/CFS do not fully recover, although many improve somewhat over years. Children and people early in the illness do better.",
     "detail": "Cairns & Hotopf 2005: 28 studies, 14 with operational CFS criteria; full recovery median 5%, improvement 39.5% (8-63%); return to work 8-30%. Better outcome with lower baseline fatigue. Adolescents have better prognosis (studies report a majority improving or recovering over 5-10 years; memory, not re-checked). RituxME placebo arm at 6 years: 7.4% reached SF-36 PF >=90. Old cohorts used heterogeneous criteria; newer prospective data in post-COVID ME/CFS (Legler 2023) show little improvement to 20 months.",
     "studies": [
      {
       "cite": "Cairns & Hotopf 2005, Occup Med",
       "n": "14 CFS cohorts",
       "design": "systematic review",
       "result": "Median full recovery 5%; improvement 39.5%",
       "url": "https://doi.org/10.1093/occmed/kqi013",
       "verified": true
      },
      {
       "cite": "Rekeland 2024, PLoS One",
       "n": "placebo arm ~54",
       "design": "6-year follow-up",
       "result": "7.4% placebo at SF-36 PF >=90; 14.8% worsened >=20 points",
       "url": "https://doi.org/10.1371/journal.pone.0307484",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What are recovery rates in PEM-defined adult cohorts followed prospectively from onset?"
     ],
     "hypotheses": [],
     "test_available": "n/a"
    },
    {
     "id": "trials-post-covid-course",
     "subdomain": "Natural history: post-COVID ME/CFS",
     "finding": "About 4.5% of SARS-CoV-2-infected adults in RECOVER met IOM ME/CFS criteria (vs 0.6% uninfected; HR 4.93), and those meeting Canadian criteria at Charité stayed severely ill to 20 months while other long-COVID patients improved.",
     "grade": "several",
     "plain": "COVID roughly quintupled the rate of new ME/CFS. People whose long COVID meets ME/CFS criteria tend not to recover over the first two years, unlike milder long COVID.",
     "detail": "Vernon 2025 (RECOVER-Adult, n=11,785 infected, 1,439 uninfected): incidence 2.66 vs 0.93 per 100 person-years; 88.7% of post-COVID ME/CFS also met long-COVID criteria; criteria based on self-report. Legler 2023 (Charité, n=106, 3-8, 9-16, 17-20 months): PCS-ME/CFS persistently severe; lower initial grip strength predicted persistence. Kedor 2022 (n=42): about half met CCC at 6 months.",
     "studies": [
      {
       "cite": "Vernon 2025, J Gen Intern Med",
       "n": "11,785 infected vs 1,439",
       "design": "prospective cohort",
       "result": "4.5% vs 0.6% met ME/CFS criteria; HR 4.93",
       "url": "https://doi.org/10.1007/s11606-024-09290-9",
       "verified": true
      },
      {
       "cite": "Legler 2023, eClinicalMedicine",
       "n": "106",
       "design": "prospective cohort",
       "result": "PCS-ME/CFS severity persistent at 20 months",
       "url": "https://doi.org/10.1016/j.eclinm.2023.102146",
       "verified": true
      },
      {
       "cite": "Kedor 2022, Nat Commun",
       "n": "42",
       "design": "prospective observational",
       "result": "~half met CCC ME/CFS at 6 months",
       "url": "https://doi.org/10.1038/s41467-022-32507-6",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "What fraction of post-COVID ME/CFS recovers by 5 years?"
     ],
     "hypotheses": [
      "viral-persistence",
      "autoimmunity"
     ],
     "test_available": "n/a"
    },
    {
     "id": "trials-prevalence-sex",
     "subdomain": "Natural history: prevalence and sex ratio",
     "finding": "ME/CFS affects roughly 0.2-1% of adults depending on criteria (US 2021-22: 1.3% self-reported diagnosis; England: 0.16% with a hospital-recorded diagnosis), with women affected 3-4 times as often as men.",
     "grade": "replicated",
     "plain": "Somewhere between 1 in 100 and 1 in 500 adults have ME/CFS, depending on how it is counted. About three to four in every four patients are women.",
     "detail": "Lim 2020 meta-analysis (46 studies): pooled prevalence 0.39%, meta-analytic 0.68% (0.48-0.97); women ~1.5-2x more often in pooled data. Vahratian 2023 (NHIS 2021-22): 1.3% of US adults (women ~1.7% vs men ~0.9%) — self-report of a clinician diagnosis plus current illness. UK: Nacul 2011 primary-care minimum 0.2%; Samms & Ponting 2025 (Hospital Episode Statistics): 100,055 diagnosed in England (0.16%), F:M 3.88:1, peaking ~6:1 in the 40s; white people ~5x more likely diagnosed, suggesting under-diagnosis. DecodeME's cohort is ~83% female. Commonly cited UK total: ~250,000-400,000 (estimate). Long COVID: ONS estimated ~2 million self-reported (~3%) in England/Scotland in 2024 (memory).",
     "studies": [
      {
       "cite": "Lim 2020, J Transl Med",
       "n": "46 studies, >1 million participants",
       "design": "meta-analysis",
       "result": "Pooled 0.39%; meta-analytic 0.68%",
       "url": "https://doi.org/10.1186/s12967-020-02269-0",
       "verified": true
      },
      {
       "cite": "Vahratian 2023, NCHS Data Brief 488",
       "n": "NHIS 2021-22",
       "design": "national survey",
       "result": "1.3% of US adults",
       "url": "https://doi.org/10.15620/cdc:134504",
       "verified": false
      },
      {
       "cite": "Samms & Ponting 2025, BMC Public Health",
       "n": "100,055 diagnosed",
       "design": "national hospital records",
       "result": "0.16%; F:M 3.88:1",
       "url": "https://www.meresearch.org.uk/research/new-estimates-of-me-cfs-prevalence-in-the-uk/",
       "verified": false
      },
      {
       "cite": "Nacul 2011, BMC Med",
       "n": "3 English regions",
       "design": "primary care cross-sectional",
       "result": "Minimum prevalence 0.2%",
       "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170215/",
       "verified": false
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Why the female predominance (DecodeME found few sex-specific loci)?",
      "How much is under-diagnosis in men and ethnic minorities?"
     ],
     "hypotheses": [
      "genetic-neuronal",
      "autoimmunity"
     ],
     "test_available": "n/a"
    },
    {
     "id": "trials-severity",
     "subdomain": "Natural history: severity",
     "finding": "Roughly a quarter of people with ME/CFS are estimated to be housebound or bedbound, and severity, not diagnosis, was the strongest predictor of reported treatment response.",
     "grade": "early",
     "plain": "A large minority are too ill to leave home. Severely ill patients are under-represented in almost every trial, so trial results may not apply to them.",
     "detail": "The 25% severe figure (often cited from IOM 2015 and UK charity surveys) rests on clinic and survey estimates, not population sampling (memory). Eckey/Xiao 2025: severity shaped treatment ratings more than ME/CFS vs long COVID. RituxME and CycloME excluded the most severe; Fluge's daratumumab study included moderate-to-severe. The original Fluge 2016 serum effect came from severe patients and the 2026 replication used mostly moderate patients.",
     "studies": [
      {
       "cite": "IOM 2015, Beyond ME/CFS",
       "n": "report",
       "design": "evidence review",
       "result": "At least a quarter housebound/bedbound at some point (estimate)",
       "url": "https://nap.nationalacademies.org/catalog/19012",
       "verified": false
      },
      {
       "cite": "Eckey 2025, PNAS",
       "n": "~3,925",
       "design": "patient survey",
       "result": "Severity strongest correlate of response",
       "url": "https://doi.org/10.1073/pnas.2426874122",
       "verified": true
      }
     ],
     "contradicting": [],
     "open_questions": [
      "Population-based severity distribution?",
      "Trials designed for severe, bedbound patients (home-based endpoints)?"
     ],
     "hypotheses": [],
     "test_available": "n/a"
    },
    {
     "id": "trials-mortality",
     "subdomain": "Natural history: mortality",
     "finding": "In 2,147 secondary-care CFS patients in London, all-cause and cancer mortality were not raised, but suicide mortality was about seven times expected (SMR 6.85).",
     "grade": "several",
     "plain": "ME/CFS is not, on average, shown to shorten life in registry data, but suicide risk is markedly higher, and rare deaths from severe illness (for example malnutrition) are documented.",
     "detail": "Roberts 2016 (SLaM CRIS, 2007-2013): 17 deaths; all-cause SMR 1.14 (0.65-1.85); cancer SMR 1.39 (0.60-2.73); suicide SMR 6.85 (2.22-15.98). Jason 2006 memorial-list analysis reported younger age at death from heart failure, cancer and suicide, but the sample is self-selected and biased. Deaths from severe ME with nutritional failure (e.g. UK coroner cases) are reported but not systematically counted. Small numbers of deaths limit precision.",
     "studies": [
      {
       "cite": "Roberts 2016, Lancet",
       "n": "2,147",
       "design": "retrospective cohort",
       "result": "All-cause SMR 1.14 (NS); suicide SMR 6.85",
       "url": "https://doi.org/10.1016/S0140-6736(15)01223-4",
       "verified": true
      }
     ],
     "contradicting": [
      {
       "cite": "Jason 2006, Health Care Women Int",
       "n": "166 deaths (memorial list)",
       "design": "case series",
       "result": "Younger mean age at death for several causes; biased sample",
       "url": "https://doi.org/10.1080/07399330600803766",
       "verified": true
      }
     ],
     "open_questions": [
      "Mortality in severe/very severe cohorts?",
      "Suicide risk factors (disbelief, lack of care, pain)?"
     ],
     "hypotheses": [],
     "test_available": "n/a"
    }
   ],
   "hypotheses_notes": [
    {
     "id": "autoimmunity",
     "for": "Daratumumab 6/10 sustained responders with IgG fall (Fluge 2025); cyclophosphamide 55% open-label response; long-COVID IgG transfers pain-like symptoms to mice (Chen 2026).",
     "against": "Rituximab phase III negative; immunoadsorption failed against sham twice; BC007 company trial negative; GPCR autoantibodies not elevated on REAP/Luminex (Germain 2025)."
    },
    {
     "id": "viral-persistence",
     "for": "Post-COVID ME/CFS incidence HR 4.93 (Vernon 2025); rintatolimod modest effect.",
     "against": "Three nirmatrelvir RCTs null; temelimab null."
    },
    {
     "id": "herpes-reactivation",
     "for": "Valganciclovir small RCT positive on secondary endpoints (Montoya 2013).",
     "against": "Unreplicated; STOP-PASC found no EBV reactivation difference."
    },
    {
     "id": "low-blood-volume-preload",
     "for": "Pyridostigmine single-dose iCPET effect; patients rate fluids and compression highly.",
     "against": "Fludrocortisone RCT negative; ivabradine lowered HR without symptom benefit; no sham-controlled saline trial."
    },
    {
     "id": "mitochondrial-energy",
     "for": "Small CoQ10+NADH and oxaloacetate RCTs; rapamycin open-label with autophagy marker changes.",
     "against": "CoQ10 alone negative in long COVID; positive supplement trials sponsor-linked and unreplicated."
    },
    {
     "id": "sickness-behaviour",
     "for": "Large placebo responses show symptoms are modifiable by expectation and context (not specific to this hypothesis).",
     "against": "PACE effects confined to self-report and not durable; GET rated harmful by patients; rehab worsened 42/97 in CFS_CARE."
    },
    {
     "id": "small-vessel-capillary",
     "for": "HBOT 40 sessions positive in one long-COVID RCT.",
     "against": "HBOT 10 sessions negative in HOT-LoCO."
    },
    {
     "id": "neuroinflammation",
     "for": "Low-dose aripiprazole, LDN rationales; retrospective reports.",
     "against": "LDN RCT negative for fatigue; no controlled aripiprazole data."
    }
   ],
   "glossary": [
    {
     "term": "Open-label",
     "plain": "Everyone, patients and doctors, knows who got the drug. Prone to placebo and expectation effects."
    },
    {
     "term": "Sham-controlled",
     "plain": "The comparison group gets a fake version of a procedure (for example blood passed through a filter that removes nothing) so no one knows who got the real thing."
    },
    {
     "term": "Placebo response",
     "plain": "Improvement in people given a dummy treatment, from expectation, natural fluctuation and enrolling when at a low point."
    },
    {
     "term": "SF-36 physical function",
     "plain": "A 0-100 questionnaire score of physical ability; healthy adults score around 90-100, many ME/CFS patients 20-50."
    },
    {
     "term": "ATA / bar",
     "plain": "Units of pressure. 1 ATA is sea-level air pressure; HBOT trials use 2.0-2.4."
    },
    {
     "term": "iCPET",
     "plain": "Invasive cardiopulmonary exercise test: a bike test with catheters in the heart's arteries to measure filling pressures and oxygen use."
    },
    {
     "term": "Oxford criteria",
     "plain": "A 1991 definition of CFS needing only six months of disabling fatigue; it does not require post-exertional malaise and captures broader fatigue states."
    },
    {
     "term": "SMR",
     "plain": "Standardised mortality ratio: observed deaths divided by the number expected for people of the same age and sex."
    }
   ],
   "key_reviews": [
    {
     "cite": "NICE 2021, NG206 ME/CFS guideline",
     "url": "https://www.nice.org.uk/guidance/ng206",
     "why": "Most thorough independent appraisal of GET/CBT evidence; current UK standard of care."
    },
    {
     "cite": "Eckey 2025, PNAS: Patient-reported treatment outcomes in ME/CFS and long COVID",
     "url": "https://doi.org/10.1073/pnas.2426874122",
     "why": "Largest dataset on what patients use and rate; maps treatments to symptom clusters."
    },
    {
     "cite": "Wilshire 2018, BMC Psychology: PACE re-analysis",
     "url": "https://doi.org/10.1186/s40359-018-0218-3",
     "why": "Protocol-based re-analysis of the most influential behavioural trial."
    },
    {
     "cite": "Blitshteyn 2025, Front Cell Infect Microbiol: Immunotherapies for POTS and long COVID",
     "url": "https://doi.org/10.3389/fcimb.2025.1647203",
     "why": "Overview of immune treatments and the trials still needed."
    },
    {
     "cite": "Proal 2025, Lancet Infect Dis: Targeting the SARS-CoV-2 reservoir",
     "url": "https://doi.org/10.1016/S1473-3099(24)00769-2",
     "why": "Rationale for antiviral/monoclonal trials after the Paxlovid failures."
    },
    {
     "cite": "Cairns & Hotopf 2005, Occup Med: prognosis of CFS",
     "url": "https://doi.org/10.1093/occmed/kqi013",
     "why": "Still the standard systematic review of natural history."
    }
   ],
   "gaps": [
    "No completed randomised trial of daratumumab, low-dose rapamycin (ME/CFS), aripiprazole or IV saline.",
    "No trial designed for severe or very severe (bedbound) patients with home-based objective endpoints.",
    "Almost no trial pre-specifies a biological selection marker; subgroup claims (NK cells, HLA, preload failure, illness duration) are all post hoc.",
    "Objective outcomes (actigraphy, 2-day CPET, step counts) are rarely primary endpoints, leaving self-report vulnerable to placebo response.",
    "Several key trials remain unpublished (BLOC/BC007, IA-PACS-CFS, Nacul LDN, RECOVER-AUTONOMIC full results, LIFT).",
    "Long-term natural history of PEM-defined, adult-onset ME/CFS from onset (including post-COVID) beyond two years.",
    "Systematic counting of deaths and harms (including rehab-induced deterioration) in ME/CFS."
   ]
  }
 ],
 "hypotheses": [
  {
   "id": "vascular-endothelial",
   "name": "Endothelial dysfunction",
   "one_line": "The lining of blood vessels fails to dilate and regulate flow, so tissues are under-supplied during effort.",
   "proponents": [
    "Scheibenbogen and Wirth (Charité)",
    "Fluge and Mella (Bergen)",
    "Sandvik/Sørland (Bergen)"
   ],
   "claim": "Endothelial cells normally release nitric oxide and other signals so arteries widen when tissue needs more blood. In ME/CFS this response is reported to be blunted, possibly through autoantibodies, inflammation or oxidative stress. Poor flow regulation would leave muscle and brain short of oxygen during exertion and could set off PEM. It is a mechanism layer rather than a root cause, and it could result from several upstream triggers.",
   "predictions": [
    "Lower flow-mediated dilation and reactive hyperaemia in patients",
    "Endothelial markers or patient IgG effects on endothelial cells in vitro",
    "Vasoactive treatments improving exercise tolerance"
   ],
   "evidence_for": [
    "Flow-mediated dilation 5.1% vs 8.2% (Sandvik 2023, n=39); reduced endothelial function in CycloME substudy (Sørland 2021)",
    "Patient IgG fragments endothelial mitochondria vs MS and healthy IgG (Prusty 2026, single lab)",
    "Lower vessel-wall/connective-tissue proteins (ITGAV, NOTCH3, TGF-beta set) in 4 cohorts and long COVID (Solving ME/CFS re-analysis)"
   ],
   "evidence_against": [
    "Effects are average differences with large overlap; deconditioning not always excluded",
    "BC007 and immunoadsorption (aimed at vasoactive GPCR antibodies) failed controlled trials",
    "Vericiguat (VERI-LONG) results not reported"
   ],
   "status": "active",
   "decisive_tests": [
    "Within-person FMD before and during PEM versus sedentary controls",
    "Placebo-controlled trial of a vasoactive drug with 2-day CPET endpoint"
   ],
   "related": [
    "small-vessel-capillary",
    "sodium-calcium-overload",
    "autoimmunity",
    "complement-thromboinflammation"
   ],
   "key_refs": [
    {
     "cite": "Sørland 2021, Front Med",
     "url": "https://doi.org/10.3389/fmed.2021.642710",
     "verified": true
    },
    {
     "cite": "Wirth & Scheibenbogen 2021, J Transl Med",
     "url": "https://doi.org/10.1186/s12967-021-02833-2",
     "verified": true
    }
   ]
  },
  {
   "id": "small-vessel-capillary",
   "name": "Capillary and microvascular damage",
   "one_line": "Capillaries in muscle and elsewhere are structurally damaged or rarefied, limiting oxygen delivery.",
   "proponents": [
    "Wüst group (VU Amsterdam)",
    "Appelman/van Vugt (Amsterdam UMC)",
    "Systrom (high-flow preload failure)"
   ],
   "claim": "Muscle biopsies show thickened capillary basement membranes and fewer capillaries, and long-COVID studies show capillary loss under the tongue and in the retina. If blood reaches the tissue but bypasses capillary beds or cannot exchange oxygen, extraction falls, as seen in the 'high-flow' iCPET phenotype. This would explain exertional intolerance unrelated to fitness. Cause could be immune, complement or viral damage to pericytes.",
   "predictions": [
    "Low oxygen extraction on iCPET",
    "Basement membrane thickening and capillary rarefaction on biopsy",
    "Improvement with treatments that repair microvasculature"
   ],
   "evidence_for": [
    "Capillary basement membrane thickening and lower capillarisation in ME/CFS and post-COVID biopsies (Wüst group 2025 preprint)",
    "Muscle damage, amyloid deposits and mitochondrial drop after exercise in long COVID (Appelman 2024)",
    "Reduced oxygen extraction in preload failure (Joseph 2021)",
    "40-session HBOT improved symptoms in one RCT (Zilberman-Itskovich 2022)"
   ],
   "evidence_against": [
    "Biopsy studies are small and largely from one network",
    "10-session HBOT trial negative (HOT-LoCO 2025)"
   ],
   "status": "active",
   "decisive_tests": [
    "Blinded biopsy morphometry in large ME/CFS vs deconditioned controls",
    "Correlation of capillary measures with 2-day CPET in the same patients"
   ],
   "related": [
    "vascular-endothelial",
    "complement-thromboinflammation",
    "microclots",
    "mitochondrial-energy"
   ],
   "key_refs": [
    {
     "cite": "Appelman 2024, Nat Commun",
     "url": "https://doi.org/10.1038/s41467-023-44432-3",
     "verified": true
    },
    {
     "cite": "Zilberman-Itskovich 2022, Sci Rep",
     "url": "https://doi.org/10.1038/s41598-022-15565-0",
     "verified": true
    }
   ]
  },
  {
   "id": "low-blood-volume-preload",
   "name": "Low blood volume and preload failure",
   "one_line": "Too little blood returns to the heart, especially upright and during exercise, reducing cardiac output and brain flow.",
   "proponents": [
    "Systrom/Joseph (Harvard)",
    "van Campen/Visser (Netherlands)",
    "Levine/Fu (POTS)",
    "Rowe (Johns Hopkins)"
   ],
   "claim": "Isotope and CO-rebreathing studies show reduced plasma and red-cell volume, and invasive CPET shows low right-heart filling pressure during exercise in many patients. Upright, this lowers stroke volume and brain blood flow (seen on tilt even without POTS). It could stem from small-fibre neuropathy, low ADH/renin response, or reduced red-cell production. It explains orthostatic intolerance well; whether it explains PEM is less clear.",
   "predictions": [
    "Low measured blood volume",
    "Low RAP on iCPET",
    "CBF drop on tilt",
    "Symptom relief with fluids, compression and pyridostigmine"
   ],
   "evidence_for": [
    "Preload failure common in iCPET series (Joseph 2021, n=160)",
    "Cerebral blood flow falls on tilt in ~90% of patients (van Campen 2020)",
    "Single-dose pyridostigmine maintained cardiac output on repeat iCPET (Joseph 2022)",
    "Patients rate fluids and compression among the most helpful (Eckey 2025)",
    "Low reticulocytes with adequate iron in postviral fatigue (Solving ME/CFS re-analysis, FinnGen; unreplicated)"
   ],
   "evidence_against": [
    "Fludrocortisone negative in RCT (Rowe 2001)",
    "Ivabradine reduced HR without symptom benefit (RECOVER-AUTONOMIC 2026)",
    "No sham-controlled trial of IV saline",
    "Blood-volume hormones normal in proteomics (Solving ME/CFS re-analysis)"
   ],
   "status": "active",
   "decisive_tests": [
    "Sham-controlled IV saline crossover with function endpoints",
    "Results of LIFT (pyridostigmine x LDN)",
    "Blood volume measurement before/after illness onset in prospective post-COVID cohorts"
   ],
   "related": [
    "autonomic-small-fibre",
    "pituitary-adh",
    "vascular-endothelial"
   ],
   "key_refs": [
    {
     "cite": "Joseph 2022, Chest",
     "url": "https://doi.org/10.1016/j.chest.2022.04.146",
     "verified": true
    },
    {
     "cite": "Rowe 2001, JAMA",
     "url": "https://doi.org/10.1001/jama.285.1.52",
     "verified": true
    }
   ]
  },
  {
   "id": "autonomic-small-fibre",
   "name": "Autonomic dysfunction and small-fibre neuropathy",
   "one_line": "Damage to small autonomic and sensory nerves impairs vascular tone, heart rate control and pain processing.",
   "proponents": [
    "Oaklander (MGH)",
    "Systrom",
    "Novak",
    "Blitshteyn"
   ],
   "claim": "A sizeable minority of ME/CFS and POTS patients have reduced intraepidermal nerve fibre density on skin biopsy. Loss of sympathetic fibres to veins would cause venous pooling and preload failure; sensory fibre loss would add pain. The neuropathy may be immune-mediated after infection. It is a likely contributor in a subgroup rather than a universal mechanism.",
   "predictions": [
    "Low IENFD on skin biopsy",
    "Abnormal QSART/sudomotor tests",
    "Response to IVIG in immune-mediated subgroup"
   ],
   "evidence_for": [
    "Small-fibre neuropathy frequent in biopsied iCPET patients (Joseph 2021)",
    "POTS and low HRV common in ME/CFS"
   ],
   "evidence_against": [
    "IVIG negative vs albumin in autoimmune POTS (Vernino 2024)",
    "Prevalence estimates vary widely between centres",
    "Ivabradine trial negative for symptoms"
   ],
   "status": "active",
   "decisive_tests": [
    "Population-representative skin biopsy study with blinded counting",
    "IVIG trial restricted to biopsy-proven SFN with function endpoints"
   ],
   "related": [
    "low-blood-volume-preload",
    "autoimmunity"
   ],
   "key_refs": [
    {
     "cite": "Vernino 2024, Clin Auton Res",
     "url": "https://doi.org/10.1007/s10286-024-01020-9",
     "verified": true
    },
    {
     "cite": "Blitshteyn 2025, Front Cell Infect Microbiol",
     "url": "https://doi.org/10.3389/fcimb.2025.1647203",
     "verified": true
    }
   ]
  },
  {
   "id": "viral-persistence",
   "name": "Viral persistence",
   "one_line": "Virus or viral antigen persists in tissue reservoirs and drives ongoing immune activation.",
   "proponents": [
    "Proal and VanElzakker (PolyBio)",
    "Peluso and Deeks (UCSF)",
    "Iwasaki (Yale)"
   ],
   "claim": "After SARS-CoV-2 and possibly other infections, viral RNA or protein may persist in gut, lymphoid or nervous tissue. Persistent antigen would keep the immune system activated and could explain chronic symptoms. Evidence is strongest in long COVID, where viral proteins are detected months later. Whether it applies to classic ME/CFS is unknown.",
   "predictions": [
    "Viral RNA/protein in tissue biopsies",
    "Antivirals or monoclonals improving symptoms",
    "Antigen levels tracking symptoms"
   ],
   "evidence_for": [
    "SARS-CoV-2 antigen detected in tissue and plasma months after infection (reviewed Proal 2023)",
    "ME/CFS incidence ~5x higher after SARS-CoV-2 (Vernon 2025)"
   ],
   "evidence_against": [
    "Three nirmatrelvir-ritonavir RCTs null (RECOVER-VITAL 2026, STOP-PASC 2024, PAX LC 2025)",
    "Temelimab (HERV-W) null",
    "Antigen detected also in recovered people in some studies"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Trials of longer or combination antivirals and monoclonals with tissue antigen as a selection marker",
    "Tissue antigen load vs symptom severity in matched recovered controls"
   ],
   "related": [
    "herpes-reactivation",
    "innate-hyperactivation",
    "immune-exhaustion"
   ],
   "key_refs": [
    {
     "cite": "Proal 2023, Nat Immunol",
     "url": "https://doi.org/10.1038/s41590-023-01601-2",
     "verified": true
    },
    {
     "cite": "Baden 2026, Lancet Infect Dis (RECOVER-VITAL)",
     "url": "https://doi.org/10.1016/S1473-3099(26)00406-8",
     "verified": true
    }
   ]
  },
  {
   "id": "herpes-reactivation",
   "name": "Herpesvirus reactivation (EBV, HHV-6)",
   "one_line": "Latent herpesviruses reactivate after a trigger and drive immune and neurological dysfunction.",
   "proponents": [
    "Montoya",
    "Lerner",
    "Ariza",
    "Prusty",
    "Peluso (EBV in long COVID)"
   ],
   "claim": "EBV and HHV-6 are carried latently by most adults. In ME/CFS, abortive reactivation or high antibody responses are reported, and EBV is a common trigger of onset. Reactivated virus or its proteins (e.g. dUTPase) could activate innate immunity. Antivirals have shown modest benefit in small selected groups.",
   "predictions": [
    "Viral DNA or early-gene antibodies elevated",
    "Antivirals help selected patients",
    "Genetic predisposition to EBV control affects risk"
   ],
   "evidence_for": [
    "Glandular fever is a strong trigger of ME/CFS in prospective cohorts",
    "Valganciclovir RCT positive on secondary scales (Montoya 2013, n=30)",
    "Genetically higher EBV DNA load suggestive risk factor (OR 1.59, MVP; Solving ME/CFS re-analysis)",
    "Elevated EBV gp42 reactivity in antigen profiling (Germain 2025)"
   ],
   "evidence_against": [
    "STOP-PASC found no EBV reactivation differences (Maestri 2025 preprint)",
    "Antibody titres poor marker of active replication",
    "No replicated antiviral RCT"
   ],
   "status": "active",
   "decisive_tests": [
    "Antiviral RCT selected by viral PCR/early antigen, not IgG",
    "Prospective serial viral load from acute infection through ME/CFS onset"
   ],
   "related": [
    "viral-persistence",
    "innate-hyperactivation",
    "immune-exhaustion"
   ],
   "key_refs": [
    {
     "cite": "Montoya 2013, J Med Virol",
     "url": "https://doi.org/10.1002/jmv.23713",
     "verified": true
    },
    {
     "cite": "Germain 2025, Int J Mol Sci",
     "url": "https://doi.org/10.3390/ijms26062799",
     "verified": true
    }
   ]
  },
  {
   "id": "autoimmunity",
   "name": "Autoimmunity (functional autoantibodies)",
   "one_line": "Post-infectious autoantibodies, for example against adrenergic or muscarinic receptors, cause vascular and autonomic dysfunction.",
   "proponents": [
    "Scheibenbogen (Charité)",
    "Fluge and Mella (Bergen)",
    "Wallukat (GPCR antibodies)",
    "Iwasaki (IgG transfer)"
   ],
   "claim": "A subset of patients may develop antibodies that disturb receptor signalling or nerve function after infection, similar to other post-infectious autoimmune conditions. Female predominance, infection onset and immune genetics fit. Antibody removal or B-/plasma-cell depletion should then help. Recent controlled trials have narrowed the GPCR version, while plasma-cell depletion and IgG transfer keep a broader version alive.",
   "predictions": [
    "Elevated specific autoantibodies",
    "Benefit from antibody removal or plasma-cell depletion",
    "Symptom transfer with patient IgG"
   ],
   "evidence_for": [
    "Daratumumab 6/10 sustained responders with IgG fall (Fluge 2025, open-label)",
    "Long-COVID IgG transfers pain hypersensitivity to mice (Chen 2026; Mignolet 2026)",
    "Cyclophosphamide 55% open-label response with HLA association (Rekeland 2020)"
   ],
   "evidence_against": [
    "Rituximab phase III negative (Fluge 2019)",
    "Immunoadsorption no better than sham (IAMPOCO, IA-PACS-CFS 2026)",
    "No GPCR autoantibody elevation on REAP/Luminex, q>0.68 (Germain 2025)",
    "BC007 company trial negative (2024)"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Randomised placebo-controlled daratumumab trial",
    "IgG transfer from ME/CFS (non-COVID) patients with blinded readouts",
    "Pre-specified antibody-selected sham-controlled trial"
   ],
   "related": [
    "serum-factor",
    "autonomic-small-fibre",
    "vascular-endothelial"
   ],
   "key_refs": [
    {
     "cite": "Fluge 2019, Ann Intern Med",
     "url": "https://doi.org/10.7326/M18-1451",
     "verified": true
    },
    {
     "cite": "Fluge 2025, Front Med",
     "url": "https://doi.org/10.3389/fmed.2025.1607353",
     "verified": true
    },
    {
     "cite": "Chen 2026, Cell Rep Med",
     "url": "https://doi.org/10.1016/j.xcrm.2026.102693",
     "verified": true
    },
    {
     "cite": "Germain 2025, Int J Mol Sci",
     "url": "https://doi.org/10.3390/ijms26062799",
     "verified": true
    }
   ]
  },
  {
   "id": "immune-exhaustion",
   "name": "Immune (T-cell) exhaustion",
   "one_line": "Chronic antigen stimulation leaves CD8 T cells and NK cells exhausted and less able to clear infection.",
   "proponents": [
    "Hanson and Grimson (Cornell)",
    "Walitt/NIH intramural",
    "Unutmaz (JAX)"
   ],
   "claim": "Exhausted T cells express inhibitory receptors and lose function after prolonged stimulation. In ME/CFS, single-cell and epigenetic data suggest CD8 T cells are primed toward exhaustion, and NK cytotoxicity is reduced. This fits with a persistent antigen or reactivating virus. It may be consequence rather than cause.",
   "predictions": [
    "Exhaustion markers (PD-1, TOX) on T cells",
    "Reduced NK cytotoxicity",
    "Changes after exercise provocation"
   ],
   "evidence_for": [
    "CD8 T cells transcriptionally primed toward exhaustion (Iu 2024, PNAS)",
    "T-cell exhaustion signals in NIH deep phenotyping (Walitt 2024)",
    "Low NK count predicted non-response to daratumumab (Fluge 2025)"
   ],
   "evidence_against": [
    "Immune cell, methylation and repertoire data give few reproducible signals across cohorts (Solving ME/CFS re-analysis)",
    "NK cytotoxicity findings vary by assay"
   ],
   "status": "active",
   "decisive_tests": [
    "Replication of exhaustion signatures in independent cohorts",
    "Longitudinal tracking from infection to ME/CFS onset"
   ],
   "related": [
    "viral-persistence",
    "herpes-reactivation",
    "innate-hyperactivation"
   ],
   "key_refs": [
    {
     "cite": "Iu 2024, PNAS",
     "url": "https://doi.org/10.1073/pnas.2415119121",
     "verified": true
    },
    {
     "cite": "Walitt 2024, Nat Commun",
     "url": "https://doi.org/10.1038/s41467-024-45107-3",
     "verified": true
    }
   ]
  },
  {
   "id": "innate-hyperactivation",
   "name": "Innate immune hyperactivation",
   "one_line": "Ongoing activation of monocytes, interferon and cytokine pathways sustains symptoms.",
   "proponents": [
    "Hornig and Lipkin (Columbia)",
    "Klimas",
    "Iwasaki"
   ],
   "claim": "Cytokine profiles differ early in ME/CFS, with an activated pattern in the first three years that later fades. Persistent interferon or inflammasome activity could cause sickness symptoms and metabolic shifts. Effects in blood are inconsistent across studies.",
   "predictions": [
    "Raised cytokines early in illness",
    "Interferon signature",
    "Benefit from anti-inflammatory treatment"
   ],
   "evidence_for": [
    "Distinct cytokine signatures early in illness (Hornig 2015, n=298)",
    "Complement and myeloid activation in long COVID (Cervia-Hasler 2024)"
   ],
   "evidence_against": [
    "Cytokine findings poorly replicated across ME/CFS cohorts",
    "Methylprednisolone trial in long COVID stopped early without benefit (PoCoVIT 2026)",
    "Prednisolone/losartan negative (Myoflame-19 2026)"
   ],
   "status": "active",
   "decisive_tests": [
    "Standardised longitudinal cytokine/interferon panels from onset",
    "Targeted anti-cytokine RCT in a biomarker-defined subgroup"
   ],
   "related": [
    "sickness-behaviour",
    "neuroinflammation",
    "complement-thromboinflammation"
   ],
   "key_refs": [
    {
     "cite": "Hornig 2015, Sci Adv",
     "url": "https://doi.org/10.1126/sciadv.1400121",
     "verified": true
    }
   ]
  },
  {
   "id": "complement-thromboinflammation",
   "name": "Complement activation and thromboinflammation",
   "one_line": "Chronic complement activation damages vessel lining and platelets, so clotting and inflammation feed each other.",
   "proponents": [
    "Boyman and Cervia-Hasler (Zurich)",
    "Baillie (Oxford)",
    "Klein/Iwasaki"
   ],
   "claim": "The terminal complement complex is persistently activated in active long COVID, with signs of haemolysis, platelet activation and monocyte-platelet aggregates. This could injure endothelium and capillaries. It is the best-replicated blood abnormality in long COVID but is less studied in classic ME/CFS.",
   "predictions": [
    "Raised sC5b-9 and complement split products",
    "Endothelial injury markers",
    "Benefit from complement inhibitors"
   ],
   "evidence_for": [
    "Cervia-Hasler 2024 (Science), replicated in outline by Baillie 2024, Klein 2023, Barratt-Due 2024",
    "Higher C3 and complement variants reported in ME/CFS (CDC; Solving ME/CFS re-analysis)"
   ],
   "evidence_against": [
    "One adjusted null replication (Farztdinov preprint)",
    "No complement inhibitor trial yet",
    "Limited ME/CFS data"
   ],
   "status": "active",
   "decisive_tests": [
    "Complement inhibitor RCT in biomarker-positive long COVID",
    "Complement measures in non-COVID ME/CFS cohorts"
   ],
   "related": [
    "vascular-endothelial",
    "microclots",
    "small-vessel-capillary"
   ],
   "key_refs": [
    {
     "cite": "Cervia-Hasler 2024, Science",
     "url": "https://doi.org/10.1126/science.adg7942",
     "verified": true
    }
   ]
  },
  {
   "id": "microclots",
   "name": "Fibrin amyloid microclots",
   "one_line": "Fibrinolysis-resistant amyloid fibrin(ogen) microclots block capillaries and trap inflammatory molecules.",
   "proponents": [
    "Pretorius (Stellenbosch)",
    "Kell (Liverpool)"
   ],
   "claim": "Fluorescence microscopy of platelet-poor plasma shows amyloid microclots, reported in long COVID and ME/CFS. They could obstruct capillaries and reduce oxygen delivery. Anticoagulant 'triple therapy' was promoted on this basis. The method is difficult to standardise and independent labs disagree.",
   "predictions": [
    "Microclot counts higher in patients",
    "Clearance associated with improvement"
   ],
   "evidence_for": [
    "Pretorius 2021 and later papers from the same network"
   ],
   "evidence_against": [
    "STOP-PASC found no microclot differences vs controls (Maestri 2025 preprint)",
    "Assay not standardised; few independent replications",
    "No RCT of anticoagulation for microclots; bleeding risk"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Blinded multi-lab microclot quantification on shared samples",
    "Placebo-controlled trial with microclot endpoints"
   ],
   "related": [
    "complement-thromboinflammation",
    "small-vessel-capillary"
   ],
   "key_refs": [
    {
     "cite": "Pretorius 2021, Cardiovasc Diabetol",
     "url": "https://doi.org/10.1186/s12933-021-01359-7",
     "verified": true
    }
   ]
  },
  {
   "id": "cell-danger-response",
   "name": "Cell danger response (Naviaux)",
   "one_line": "Cells stuck in a protective low-energy 'danger' state after a threat fail to complete the healing cycle.",
   "proponents": [
    "Robert Naviaux (UCSD)"
   ],
   "claim": "The cell danger response (CDR) is a conserved metabolic reaction to threat in which mitochondria shift from energy production to defence, with extracellular ATP (purinergic) signalling. Naviaux proposes that ME/CFS is a persistent CDR, a hypometabolic state like dauer. His 2016 metabolomics found most altered metabolites decreased, consistent with hypometabolism. Antipurinergic drugs such as suramin are the proposed test.",
   "predictions": [
    "Hypometabolic metabolomic profile",
    "Raised extracellular ATP/purinergic signalling",
    "Response to antipurinergic therapy"
   ],
   "evidence_for": [
    "Naviaux 2016 PNAS: 80% of altered metabolites decreased (n=45 vs 39)",
    "Rapamycin responders show purine metabolism changes (Gile 2026)"
   ],
   "evidence_against": [
    "Metabolomic findings vary across cohorts; Hoel/Germain datasets do not show uniform hypometabolism",
    "No ME/CFS trial of suramin"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Low-dose suramin RCT in ME/CFS",
    "Replication of hypometabolic signature with standardised fasting samples"
   ],
   "related": [
    "mitochondrial-energy",
    "metabolic-trap-ido",
    "innate-hyperactivation"
   ],
   "key_refs": [
    {
     "cite": "Naviaux 2016, PNAS",
     "url": "https://doi.org/10.1073/pnas.1607571113",
     "verified": true
    },
    {
     "cite": "Naviaux 2019, Mitochondrion",
     "url": "https://doi.org/10.1016/j.mito.2018.08.001",
     "verified": true
    }
   ]
  },
  {
   "id": "mitochondrial-energy",
   "name": "Mitochondrial / energy metabolism impairment",
   "one_line": "Cells cannot produce energy normally, especially during exertion, causing early lactate and PEM.",
   "proponents": [
    "Fluge and Mella (PDH)",
    "Missailidis and Fisher (La Trobe)",
    "Myhill/Booth",
    "Hanson (Cornell)"
   ],
   "claim": "Several lines suggest impaired pyruvate dehydrogenase, Complex V inefficiency or reduced oxidative capacity. This would force early reliance on glycolysis and slow recovery. It may be intrinsic or secondary to poor perfusion or circulating factors.",
   "predictions": [
    "Low PDH flux markers",
    "Abnormal mitochondrial respiration in patient cells",
    "Benefit from metabolic supplements"
   ],
   "evidence_for": [
    "Amino-acid pattern implying impaired PDH (Fluge 2016, n=200 vs 102)",
    "Complex V inefficiency in lymphoblasts (Missailidis 2020)",
    "Post-exercise mitochondrial enzyme drop in long COVID muscle (Appelman 2024)",
    "Aromatic lactate deficit across 5 cohorts (Solving ME/CFS re-analysis)"
   ],
   "evidence_against": [
    "Serum effect on myoblast respiration failed replication at n=120 (Ryback/Ponting 2026)",
    "CoQ10 alone negative in long COVID",
    "Supplement trials small and sponsor-linked"
   ],
   "status": "active",
   "decisive_tests": [
    "Muscle 31P-MRS and biopsy respiration before/after PEM vs deconditioned controls",
    "Independent replication of lymphoblast Complex V defect"
   ],
   "related": [
    "cell-danger-response",
    "small-vessel-capillary",
    "serum-factor"
   ],
   "key_refs": [
    {
     "cite": "Fluge 2016, JCI Insight",
     "url": "https://doi.org/10.1172/jci.insight.89376",
     "verified": true
    }
   ]
  },
  {
   "id": "metabolic-trap-ido",
   "name": "IDO metabolic trap (Phair, Davis)",
   "one_line": "A bistable tryptophan-kynurenine circuit, set by IDO1/IDO2 variants and substrate inhibition, flips into a persistent low-tryptophan state.",
   "proponents": [
    "Robert Phair",
    "Ron Davis (OMF / Stanford)",
    "Alex Kashi"
   ],
   "claim": "IDO1 converts tryptophan to kynurenine and is inhibited by high tryptophan. Combined with a deleterious IDO2 variant, a mathematical model predicts two stable states, one of which traps cells with low tryptophan and serotonin and high kynurenine after an inflammatory trigger. Exiting the trap would need a perturbation. It is a mathematical model with limited direct data.",
   "predictions": [
    "Enrichment of IDO2 deleterious variants",
    "Altered intracellular tryptophan/kynurenine",
    "Bistable behaviour in patient cells"
   ],
   "evidence_for": [
    "Preliminary OMF data on IDO2 variants (unpublished/limited)",
    "Altered tryptophan metabolites in some metabolomic studies"
   ],
   "evidence_against": [
    "DecodeME GWAS did not highlight IDO loci",
    "Mendelian randomisation finds no causal effect of kynurenine on ME/CFS risk (Solving ME/CFS re-analysis)",
    "Plasma kynurenine findings inconsistent"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Intracellular tryptophan/kynurenine measurement in patient cells with IDO genotypes",
    "Test of predicted bistability in culture"
   ],
   "related": [
    "cell-danger-response",
    "mitochondrial-energy",
    "omf-models"
   ],
   "key_refs": [
    {
     "cite": "Kashi, Davis, Phair 2019, Diagnostics",
     "url": "https://doi.org/10.3390/diagnostics9030082",
     "verified": true
    }
   ]
  },
  {
   "id": "hpa-endocrine",
   "name": "HPA axis blunting",
   "one_line": "The stress-hormone axis is under-active, with low cortisol and enhanced feedback.",
   "proponents": [
    "Cleare (King's)",
    "Demitrack (early work)"
   ],
   "claim": "Mild hypocortisolism, a flattened diurnal rhythm and enhanced negative feedback are reported in ME/CFS. This is probably a consequence of illness, inactivity or sleep disturbance rather than a cause, but it could amplify symptoms. Low-dose hydrocortisone had small effects and suppressed the adrenal axis.",
   "predictions": [
    "Low salivary awakening cortisol",
    "Enhanced dexamethasone suppression",
    "Response to low-dose hydrocortisone"
   ],
   "evidence_for": [
    "Repeated reports of mild hypocortisolism (2026 meta-analysis per atlas)",
    "Cleare 1999 hydrocortisone 5-10 mg: 28% vs 9% improved"
   ],
   "evidence_against": [
    "McKenzie 1998 hydrocortisone RCT: adrenal suppression, little benefit",
    "Changes small and nonspecific"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Longitudinal cortisol from pre-illness in prospective cohorts"
   ],
   "related": [
    "pituitary-adh",
    "sickness-behaviour"
   ],
   "key_refs": [
    {
     "cite": "Cleare 1999, Lancet",
     "url": "https://doi.org/10.1016/S0140-6736(98)04074-4",
     "verified": true
    }
   ]
  },
  {
   "id": "pituitary-adh",
   "name": "Pituitary / vasopressin deficiency",
   "one_line": "Low ADH (vasopressin) secretion despite concentrated blood leads to fluid loss and low blood volume.",
   "proponents": [
    "Hanson-adjacent clinical groups",
    "Bragée/Huhmar/Polo (2025 report)"
   ],
   "claim": "A 2025 study reported undetectable vasopressin in most patients despite high plasma osmolality. If true, this would explain polyuria, thirst and low blood volume, and suggest desmopressin for a subgroup. DecodeME tissue enrichment pointing to pituitary gives indirect support.",
   "predictions": [
    "Low copeptin relative to osmolality",
    "Response to desmopressin"
   ],
   "evidence_for": [
    "Vasopressin undetectable in 82% in an uncontrolled 2025 series",
    "DecodeME genetic signal enriched in brain and pituitary tissues"
   ],
   "evidence_against": [
    "Uncontrolled, assay concerns",
    "Desmopressin evidence only acute in POTS (Coffin 2012)"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Controlled copeptin study with water deprivation/hypertonic saline testing"
   ],
   "related": [
    "low-blood-volume-preload",
    "hpa-endocrine",
    "genetic-neuronal"
   ],
   "key_refs": [
    {
     "cite": "Huhmar/Bragée/Polo 2025, Endocr Pract",
     "url": "https://www.endocrinepractice.org/article/S1530-891X(25)01349-7/fulltext",
     "verified": false
    }
   ]
  },
  {
   "id": "neuroinflammation",
   "name": "Neuroinflammation (microglial activation)",
   "one_line": "Activated microglia and astrocytes in the brain produce fatigue, cognitive problems and pain.",
   "proponents": [
    "Nakatomi/Watanabe (Japan)",
    "VanElzakker",
    "Younger"
   ],
   "claim": "PET and MR spectroscopy studies suggest brain inflammation in ME/CFS and long COVID. Inflammation in brainstem or limbic regions could generate sickness behaviour and PEM. Results conflict and tracer studies are small.",
   "predictions": [
    "Raised TSPO PET signal",
    "Raised brain lactate/choline",
    "Benefit from glial modulators (LDN, aripiprazole)"
   ],
   "evidence_for": [
    "Nakatomi 2014 TSPO PET positive (n=9)",
    "Long-COVID TSPO increase (VanElzakker 2024)",
    "Raised ventricular lactate (Shungu/Mathew)"
   ],
   "evidence_against": [
    "Raijmakers 2022 TSPO PET negative",
    "James (Stanford) 2026: no brain TSPO signal, strong muscle signal",
    "LDN fatigue RCT negative (2026)"
   ],
   "status": "active",
   "decisive_tests": [
    "Large multi-tracer PET studies with standardised analysis",
    "CSF inflammatory markers vs matched controls"
   ],
   "related": [
    "sickness-behaviour",
    "brainstem-craniocervical",
    "innate-hyperactivation"
   ],
   "key_refs": [
    {
     "cite": "Walitt 2024, Nat Commun",
     "url": "https://doi.org/10.1038/s41467-024-45107-3",
     "verified": true
    }
   ]
  },
  {
   "id": "brainstem-craniocervical",
   "name": "Brainstem and craniocervical instability",
   "one_line": "Brainstem dysfunction, sometimes from craniocervical instability or Chiari, disrupts autonomic and arousal control.",
   "proponents": [
    "Henderson/Bolognese (surgeons)",
    "Bragée (Stockholm)",
    "Shan (brainstem MRI)"
   ],
   "claim": "Some patients, often with hypermobility, are reported to have craniocervical instability or intracranial hypertension compressing the brainstem. Surgery has been followed by improvement in case series. Imaging studies of brainstem volume and connectivity are small. This may explain a minority subgroup at most.",
   "predictions": [
    "Upright MRI abnormalities more common",
    "Surgical benefit in controlled studies"
   ],
   "evidence_for": [
    "Case series of fusion surgery improvement",
    "Bragée 2020: CCI and intracranial hypertension common in a referral sample"
   ],
   "evidence_against": [
    "No controlled trial; surgery high risk",
    "Imaging criteria not standardised; selection bias"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Blinded imaging comparing ME/CFS with matched hypermobile controls",
    "Prospective registry of surgical outcomes with controls"
   ],
   "related": [
    "connective-tissue",
    "neuroinflammation",
    "autonomic-small-fibre"
   ],
   "key_refs": [
    {
     "cite": "Bragée 2020, Front Neurol",
     "url": "https://doi.org/10.3389/fneur.2020.00828",
     "verified": true
    }
   ]
  },
  {
   "id": "sickness-behaviour",
   "name": "Persistent sickness behaviour / vagal signalling",
   "one_line": "The brain's sickness response, normally triggered by infection signals (e.g. via the vagus), stays switched on.",
   "proponents": [
    "VanElzakker (vagus nerve infection hypothesis)",
    "Dantzer (sickness behaviour biology)"
   ],
   "claim": "During infection, cytokines signal to the brain and cause fatigue, malaise and withdrawal. If a local infection or inflammation near the vagus nerve persists, or the brain's set point changes, sickness behaviour could persist. This frames many symptoms but is hard to test directly.",
   "predictions": [
    "Vagal or brainstem markers of immune signalling",
    "Symptoms reduced by blocking cytokine signalling"
   ],
   "evidence_for": [
    "Symptoms closely resemble sickness behaviour",
    "Post-infectious onset common"
   ],
   "evidence_against": [
    "Anti-inflammatory trials negative in long COVID",
    "Direct evidence of vagal infection absent"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Vagal nerve imaging/biopsy studies; vagal stimulation RCTs with sham"
   ],
   "related": [
    "neuroinflammation",
    "innate-hyperactivation"
   ],
   "key_refs": [
    {
     "cite": "VanElzakker 2013, Med Hypotheses",
     "url": "https://doi.org/10.1016/j.mehy.2013.05.034",
     "verified": true
    }
   ]
  },
  {
   "id": "gut-dysbiosis",
   "name": "Gut dysbiosis",
   "one_line": "Altered gut microbiota and barrier function drive immune activation and metabolic changes.",
   "proponents": [
    "Lipkin and Hornig (Columbia)",
    "Unutmaz (JAX)",
    "Hong Kong SIM01 group"
   ],
   "claim": "Butyrate producers such as Faecalibacterium prausnitzii are reduced in ME/CFS, and short-duration patients show stronger dysbiosis. Microbial products may affect immunity and metabolism. A synbiotic improved several long-COVID symptoms in one trial. Dysbiosis could also be a consequence of illness, diet and inactivity.",
   "predictions": [
    "Reduced butyrate producers",
    "Microbial metabolites tracking symptoms",
    "Benefit from microbiome interventions"
   ],
   "evidence_for": [
    "Reduced butyrate producers (Guo 2023, n=106 vs 91)",
    "Dysbiosis stronger early (Xiong 2023)",
    "R. gnavus/F. plautii overgrowth replicates and explains low IPA (Solving ME/CFS re-analysis)",
    "SIM01 synbiotic positive in long COVID (Lau 2024, n=463)"
   ],
   "evidence_against": [
    "MR finds no causal effect of IPA and related metabolites (Solving ME/CFS re-analysis)",
    "Aromatic lactate deficit not explained by gut bacteria (Solving ME/CFS re-analysis)",
    "Faecal transplant data anecdotal"
   ],
   "status": "active",
   "decisive_tests": [
    "Replication of SIM01-type trial in ME/CFS",
    "Prospective microbiome sampling before onset"
   ],
   "related": [
    "innate-hyperactivation",
    "mitochondrial-energy"
   ],
   "key_refs": [
    {
     "cite": "Guo 2023, Cell Host Microbe",
     "url": "https://doi.org/10.1016/j.chom.2023.01.004",
     "verified": true
    },
    {
     "cite": "Xiong 2023, Cell Host Microbe",
     "url": "https://doi.org/10.1016/j.chom.2023.01.001",
     "verified": true
    }
   ]
  },
  {
   "id": "genetic-neuronal",
   "name": "Genetic susceptibility acting through neurons",
   "one_line": "Inherited variants affecting nervous-system genes raise risk, pointing to brain-based mechanisms.",
   "proponents": [
    "Ponting and DecodeME consortium (Edinburgh)"
   ],
   "claim": "DecodeME (15,579 cases) found genome-wide significant loci, with tissue enrichment in brain and pituitary rather than immune tissues. The overall genetic signal replicates in the Million Veteran Program and FinnGen. Candidate genes include pain-sensitisation and immune-response genes. Genetics marks susceptibility, not necessarily the ongoing mechanism.",
   "predictions": [
    "Loci near neuronal genes",
    "Replication in independent cohorts",
    "Mendelian randomisation links to specific proteins"
   ],
   "evidence_for": [
    "DecodeME 2025 loci; tissue enrichment brain/pituitary",
    "Genetic correlation rg 0.73 with MVP; EPHB1 and CLEC3B replicate by MR (Solving ME/CFS re-analysis)"
   ],
   "evidence_against": [
    "Most individual loci not yet replicated",
    "Small effect sizes; heritability modest"
   ],
   "status": "active",
   "decisive_tests": [
    "Independent GWAS replication of individual loci",
    "Functional studies of implicated genes"
   ],
   "related": [
    "neuroinflammation",
    "pituitary-adh",
    "innate-hyperactivation"
   ],
   "key_refs": [
    {
     "cite": "DecodeME 2025, medRxiv",
     "url": "https://doi.org/10.1101/2025.08.06.25333109",
     "verified": true
    }
   ]
  },
  {
   "id": "serum-factor",
   "name": "Circulating (serum) factor",
   "one_line": "Something in patient blood, such as IgG or exosomes, impairs healthy cells.",
   "proponents": [
    "Davis/Esfandyarpour (nanoneedle)",
    "Fluge and Mella",
    "Prusty (Würzburg)",
    "Ramon-Azcon (IBEC)"
   ],
   "claim": "Several experiments put patient plasma or serum onto healthy cells and observed altered impedance, respiration, mitochondrial morphology or muscle force. If replicated, this would point to a transferable factor and allow bioassays. The best-known muscle mitochondrial version failed a well-powered replication in 2026.",
   "predictions": [
    "Patient plasma alters healthy cells reproducibly",
    "Effect removed by IgG depletion",
    "Effect disappears with recovery"
   ],
   "evidence_for": [
    "Plasma-swap nanoneedle impedance (Esfandyarpour 2019, n=20 vs 20)",
    "IgG fragments endothelial mitochondria vs MS and healthy IgG (Prusty 2026)",
    "Long-COVID IgG transfers symptoms to mice (Chen 2026)"
   ],
   "evidence_against": [
    "Ryback/Ponting 2026: no serum effect on myoblast respiration (n=67 vs 53, d≥0.52 excluded)",
    "IBEC 3D muscle study n=4/5/4 with pseudoreplication risk",
    "Nanoneedle unreplicated in peer review"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Multi-lab blinded serum bioassay with disease controls and PEM-timed samples",
    "IgG depletion and rescue experiments"
   ],
   "related": [
    "autoimmunity",
    "mitochondrial-energy",
    "vascular-endothelial"
   ],
   "key_refs": [
    {
     "cite": "Fluge 2016, JCI Insight",
     "url": "https://doi.org/10.1172/jci.insight.89376",
     "verified": true
    },
    {
     "cite": "Ryback/Ponting 2026, PLoS One (preprint bioRxiv 2025.06.03.657595)",
     "url": "https://doi.org/10.1101/2025.06.03.657595",
     "verified": true
    }
   ]
  },
  {
   "id": "sodium-calcium-overload",
   "name": "Sodium-calcium overload in muscle (Wirth & Scheibenbogen)",
   "one_line": "Under-perfused muscle accumulates sodium and then calcium via the sodium-calcium exchanger, damaging mitochondria and causing PEM.",
   "proponents": [
    "Klaus Wirth",
    "Carmen Scheibenbogen (Charité)"
   ],
   "claim": "The model proposes that beta2-adrenergic dysfunction (perhaps via autoantibodies) and vasoconstriction reduce muscle blood flow. Hypoxic muscle relies on glycolysis; protons are exported by NHE1, loading the cell with sodium, which reverses the sodium-calcium exchanger and raises intracellular calcium, injuring mitochondria. This cycle explains delayed, prolonged PEM and suggests drugs that block NHE1 or reduce sodium loading. Elements are testable with 23Na-MRI.",
   "predictions": [
    "Raised muscle sodium on 23Na-MRI",
    "Calcium overload and mitochondrial damage after exertion",
    "Benefit from NHE1 inhibitors or vasodilators"
   ],
   "evidence_for": [
    "Higher muscle sodium on 23Na-MRI (Petter 2022, 6 vs 6)",
    "Post-exertional muscle damage and mitochondrial drop (Appelman 2024)",
    "Endothelial dysfunction findings"
   ],
   "evidence_against": [
    "Upstream GPCR-antibody step weakened by negative antigen profiling and failed IA/BC007 trials",
    "Sodium MRI sample tiny",
    "No interventional test yet"
   ],
   "status": "active",
   "decisive_tests": [
    "Larger 23Na-MRI studies before and after exertion vs deconditioned controls",
    "Pilot trial of NHE1 inhibition"
   ],
   "related": [
    "vascular-endothelial",
    "small-vessel-capillary",
    "mitochondrial-energy",
    "autoimmunity"
   ],
   "key_refs": [
    {
     "cite": "Wirth & Scheibenbogen 2021, J Transl Med",
     "url": "https://doi.org/10.1186/s12967-021-02833-2",
     "verified": true
    },
    {
     "cite": "Wirth & Scheibenbogen 2020, Autoimmun Rev",
     "url": "https://doi.org/10.1016/j.autrev.2020.102527",
     "verified": true
    }
   ]
  },
  {
   "id": "mast-cell",
   "name": "Mast cell activation",
   "one_line": "Inappropriate mast-cell mediator release causes flushing, tachycardia, GI and cognitive symptoms.",
   "proponents": [
    "Afrin",
    "Weinstock",
    "Glynne (long COVID)"
   ],
   "claim": "Mast cell activation syndrome overlaps clinically with POTS, hypermobility and ME/CFS. Histamine and other mediators could cause vasodilation and orthostatic symptoms. Treatment with H1/H2 antihistamines is common. Diagnostic criteria are contested and data are observational.",
   "predictions": [
    "Raised tryptase/histamine metabolites during flares",
    "Response to mast-cell stabilisers"
   ],
   "evidence_for": [
    "Observational improvement with H1+H2 blockers in long COVID (Glynne 2022)",
    "Clinical overlap with POTS/hEDS"
   ],
   "evidence_against": [
    "No RCTs; mediator tests rarely positive",
    "Diagnostic criteria inconsistent"
   ],
   "status": "speculative",
   "decisive_tests": [
    "RCT of antihistamines with mediator-defined selection"
   ],
   "related": [
    "connective-tissue",
    "autonomic-small-fibre"
   ],
   "key_refs": [
    {
     "cite": "Glynne 2022, J Investig Med",
     "url": "https://doi.org/10.1136/jim-2021-002051",
     "verified": true
    }
   ]
  },
  {
   "id": "connective-tissue",
   "name": "Connective tissue / hypermobility",
   "one_line": "Hypermobility and connective-tissue weakness predispose to venous pooling, instability and dysautonomia.",
   "proponents": [
    "Rowe",
    "Eccles"
   ],
   "claim": "Joint hypermobility is more common in ME/CFS and POTS. Lax connective tissue in veins could worsen pooling, and in the neck could contribute to craniocervical problems. Lower blood levels of vessel-wall/extracellular-matrix proteins have been reported.",
   "predictions": [
    "Higher hypermobility prevalence",
    "Matrix protein changes",
    "Venous compliance increased"
   ],
   "evidence_for": [
    "Hypermobility overrepresented in clinic samples",
    "Lower ITGAV/NOTCH3/TGF-beta set proteins (Solving ME/CFS re-analysis)"
   ],
   "evidence_against": [
    "Clinic-based selection; population data lacking",
    "No mechanism-specific treatment"
   ],
   "status": "speculative",
   "decisive_tests": [
    "Population-based hypermobility prevalence in ME/CFS",
    "Venous compliance measurement"
   ],
   "related": [
    "brainstem-craniocervical",
    "mast-cell",
    "low-blood-volume-preload"
   ],
   "key_refs": [
    {
     "cite": "Eccles 2021, Clin Med (hypermobility and ME/CFS/POTS)",
     "url": "https://pubmed.ncbi.nlm.nih.gov/?term=Eccles+hypermobility+chronic+fatigue",
     "verified": false
    }
   ]
  },
  {
   "id": "integrated-hanson",
   "name": "Cornell integrated multi-omics model (Hanson)",
   "one_line": "PEM reflects a maladaptive multi-system response to exertion visible across immune, metabolic and proteomic layers.",
   "proponents": [
    "Maureen Hanson",
    "Arnaud Germain",
    "Cornell ME/CFS Collaborative Research Center"
   ],
   "claim": "Using 2-day CPET with blood sampling, the Cornell group reports that patients fail to mount and resolve normal exercise responses in plasma proteins, metabolites and immune cells, with CD8 T-cell exhaustion features. They frame ME/CFS as an integrated failure of recovery across systems rather than a single lesion. The model is descriptive and generates leads rather than one causal claim.",
   "predictions": [
    "Divergent post-exertion trajectories vs sedentary controls",
    "Changes larger on day 2"
   ],
   "evidence_for": [
    "Plasma proteome maladaptation after exertion in 79 vs 53 (Germain 2025, Mol Cell Proteomics)",
    "CD8 exhaustion priming (Iu 2024)"
   ],
   "evidence_against": [
    "Re-analysis of the 2022 metabolomics: within-person exercise responses barely differ; 'growing divergence' does not hold (Solving ME/CFS re-analysis, PEM)",
    "Clean negative autoantibody profiling from the same group (Germain 2025) narrows immune explanations"
   ],
   "status": "active",
   "decisive_tests": [
    "Pre-registered within-person analyses in a new 2-day CPET cohort",
    "Matching deconditioned and disease controls"
   ],
   "related": [
    "immune-exhaustion",
    "mitochondrial-energy",
    "omf-models"
   ],
   "key_refs": [
    {
     "cite": "Germain 2025, Mol Cell Proteomics",
     "url": "https://doi.org/10.1016/j.mcpro.2025.101467",
     "verified": true
    },
    {
     "cite": "Germain 2022, JCI Insight",
     "url": "https://doi.org/10.1172/jci.insight.157621",
     "verified": true
    }
   ]
  },
  {
   "id": "omf-models",
   "name": "Open Medicine Foundation models (Davis, Xiao, Systrom)",
   "one_line": "A cluster of OMF-funded models: a circulating plasma factor, the IDO metabolic trap, and neurovascular preload failure.",
   "proponents": [
    "Ron Davis",
    "Wenzhong Xiao",
    "David Systrom",
    "Robert Phair"
   ],
   "claim": "OMF has backed several ideas rather than one: plasma-swap nanoneedle evidence for a blood factor; the IDO metabolic trap; and Systrom's neurovascular dysregulation with preload failure, tested by pyridostigmine. Its data centre and the 2025 treatment survey aim to link subgroups to treatments. The common thread is a measurable, possibly reversible physiological state.",
   "predictions": [
    "Plasma factor in bioassays",
    "IDO2 variants/trap state",
    "Pyridostigmine benefit in preload failure"
   ],
   "evidence_for": [
    "Nanoneedle plasma-swap (Esfandyarpour 2019)",
    "Pyridostigmine single-dose iCPET RCT (Joseph 2022)",
    "Patient survey subgroups (Eckey 2025)"
   ],
   "evidence_against": [
    "No peer-reviewed nanoneedle replication",
    "IDO trap not supported by GWAS/MR",
    "Longer pyridostigmine trial in long COVID negative (2025 abstract)"
   ],
   "status": "active",
   "decisive_tests": [
    "LIFT trial results",
    "Independent replication of nanoneedle assay"
   ],
   "related": [
    "metabolic-trap-ido",
    "serum-factor",
    "low-blood-volume-preload"
   ],
   "key_refs": [
    {
     "cite": "Esfandyarpour 2019, PNAS",
     "url": "https://doi.org/10.1073/pnas.1901274116",
     "verified": true
    },
    {
     "cite": "Eckey 2025, PNAS",
     "url": "https://doi.org/10.1073/pnas.2426874122",
     "verified": true
    }
   ]
  },
  {
   "id": "komaroff-lipkin-synthesis",
   "name": "Multisystem post-infectious model (Komaroff & Lipkin reviews)",
   "one_line": "ME/CFS and long COVID share a set of biological abnormalities across brain, immune, energy and vascular systems, triggered by infection.",
   "proponents": [
    "Anthony Komaroff (Harvard)",
    "W. Ian Lipkin (Columbia)"
   ],
   "claim": "Two influential reviews catalogue overlapping findings: neuroinflammation, immune activation and exhaustion, autoantibodies, impaired energy metabolism, endothelial and autonomic dysfunction, and gut dysbiosis. They argue the two illnesses are variants of the same post-infectious process and that research on each informs the other. It is a synthesis, not a single falsifiable mechanism.",
   "predictions": [
    "Shared abnormalities between ME/CFS and long COVID",
    "Similar treatment responses"
   ],
   "evidence_for": [
    "Treatment ratings correlate between ME/CFS and long COVID (R²=0.68; Eckey 2025)",
    "Post-COVID ME/CFS incidence data (Vernon 2025)"
   ],
   "evidence_against": [
    "Many catalogued abnormalities are unreplicated or contested individually",
    "Breadth makes the model hard to falsify"
   ],
   "status": "active",
   "decisive_tests": [
    "Head-to-head cohorts with identical assays in ME/CFS, long COVID and recovered controls"
   ],
   "related": [
    "viral-persistence",
    "autoimmunity",
    "neuroinflammation",
    "mitochondrial-energy"
   ],
   "key_refs": [
    {
     "cite": "Komaroff & Lipkin 2021, Trends Mol Med",
     "url": "https://doi.org/10.1016/j.molmed.2021.06.002",
     "verified": true
    },
    {
     "cite": "Komaroff & Lipkin 2023, Front Med",
     "url": "https://doi.org/10.3389/fmed.2023.1187163",
     "verified": true
    }
   ]
  },
  {
   "id": "effort-preference",
   "name": "Altered 'effort preference' (NIH)",
   "one_line": "Patients choose fewer hard-effort options in a decision task, which the NIH team interpreted as a defining feature rather than fatigue or weakness.",
   "proponents": [
    "Walitt, Nath and the NIH intramural team"
   ],
   "claim": "In deep phenotyping of 17 post-infectious ME/CFS patients, participants chose the hard task less often in the Effort-Expenditure for Rewards Task, and the authors proposed altered effort preference linked to low CSF catecholamines and motor-network changes. They framed fatigue as arising from brain integrative circuits. Critics argue the task was not validated in ME/CFS and that choosing less effort is a rational response to PEM, not a preference.",
   "predictions": [
    "Reduced hard-task choices independent of capacity",
    "Brain activity differences during effort tasks"
   ],
   "evidence_for": [
    "Fewer hard-task choices (Walitt 2024, n=17 vs 21)",
    "Low CSF catecholamines and altered motor-cortex activity in the same study"
   ],
   "evidence_against": [
    "Tiny sample; task not validated; PEM-avoidance alternative explanation",
    "Patients and many researchers objected to the framing; findings not replicated"
   ],
   "status": "weakened",
   "decisive_tests": [
    "Replication with tasks that separate anticipated PEM cost from motivation",
    "Compare with other disabling illnesses with exertional limits"
   ],
   "related": [
    "neuroinflammation",
    "cognitive-behavioural-model"
   ],
   "key_refs": [
    {
     "cite": "Walitt 2024, Nat Commun",
     "url": "https://doi.org/10.1038/s41467-024-45107-3",
     "verified": true
    }
   ]
  },
  {
   "id": "cognitive-behavioural-model",
   "name": "Psychosomatic / cognitive-behavioural (fear-avoidance) model",
   "one_line": "An initial illness is perpetuated by unhelpful beliefs, activity avoidance and deconditioning, so CBT and graded exercise should reverse it.",
   "proponents": [
    "Wessely, Sharpe, Chalder, White (UK)",
    "Knoop and Bleijenberg (Netherlands)"
   ],
   "claim": "The model holds that after a trigger, beliefs that activity is harmful lead to avoidance, deconditioning and symptom focus, maintaining disability. It predicted that CBT and graded exercise would produce recovery. PACE and Dutch trials reported modest self-reported benefits. The model is now widely disputed: objective outcomes were null and PEM is not explained by beliefs.",
   "predictions": [
    "CBT/GET produce recovery including on objective measures",
    "Beliefs predict outcome",
    "No biological abnormality beyond deconditioning"
   ],
   "evidence_for": [
    "PACE: modest self-reported fatigue and function gains with CBT/GET at 52 weeks (White 2011)",
    "Belief/attribution associated with prognosis in older cohorts (Cairns & Hotopf 2005)",
    "Large placebo responses show context and expectation affect symptom reports"
   ],
   "evidence_against": [
    "Effects confined to self-report and not durable at 2.5 years (Sharpe 2015; Wilshire 2018)",
    "NICE 2021 rated evidence low/very low and advised against GET",
    "Patients rate GET most harmful (Eckey 2025); rehab worsened 42/97 (CFS_CARE 2026)",
    "Reproducible biological differences (2-day CPET, genetics, muscle pathology) not explained by beliefs",
    "Critique of the model's evidence base (Geraghty 2019)"
   ],
   "status": "largely refuted",
   "decisive_tests": [
    "Blinded-assessor trial with objective endpoints in PEM-defined patients",
    "Prospective test of whether beliefs precede and predict onset"
   ],
   "related": [
    "deconditioning",
    "effort-preference",
    "sickness-behaviour"
   ],
   "key_refs": [
    {
     "cite": "White 2011, Lancet (PACE)",
     "url": "https://doi.org/10.1016/S0140-6736(11)60096-2",
     "verified": true
    },
    {
     "cite": "Wilshire 2018, BMC Psychol",
     "url": "https://doi.org/10.1186/s40359-018-0218-3",
     "verified": true
    },
    {
     "cite": "Geraghty 2019, Health Psychol Open",
     "url": "https://doi.org/10.1177/2055102919838907",
     "verified": true
    }
   ]
  },
  {
   "id": "deconditioning",
   "name": "Deconditioning",
   "one_line": "Exercise intolerance is simply loss of fitness from inactivity.",
   "proponents": [
    "Implicit in rehabilitation models; early long-COVID exercise studies"
   ],
   "claim": "If symptoms reflect detraining, patients should look like sedentary controls on exercise testing, recover normally between tests, and improve with graded training. Deconditioning certainly coexists in severe illness. The question is whether it explains the disease.",
   "predictions": [
    "Normal reproducibility on repeat CPET",
    "Muscle findings match sedentary controls",
    "Training improves capacity"
   ],
   "evidence_for": [
    "Lower peak VO2 in patients is partly explained by fitness",
    "Some long-COVID CPET studies attributed limitation to deconditioning"
   ],
   "evidence_against": [
    "Day-2 CPET decline in many patients, not seen in sedentary controls (Keller 2024, n=84; memory)",
    "Post-exertional muscle damage and mitochondrial drop not explained by fitness (Appelman 2024)",
    "iCPET shows preload failure and poor extraction atypical of detraining (Joseph 2021, 2023 review)",
    "Lower muscle proteins in blood not explained by steps or lying time (Solving ME/CFS re-analysis)",
    "GET harmful in surveys"
   ],
   "status": "largely refuted",
   "decisive_tests": [
    "Matched-activity controls (bedrest or immobilised volunteers) on 2-day CPET and biopsy"
   ],
   "related": [
    "cognitive-behavioural-model",
    "small-vessel-capillary",
    "low-blood-volume-preload"
   ],
   "key_refs": [
    {
     "cite": "Joseph 2023, Chest",
     "url": "https://doi.org/10.1016/j.chest.2023.03.049",
     "verified": true
    },
    {
     "cite": "Appelman 2024, Nat Commun",
     "url": "https://doi.org/10.1038/s41467-023-44432-3",
     "verified": true
    }
   ]
  }
 ]
}