Category: Biomarker

Circulating extracellular vesicles-microRNAs as potential biomarkers for the identification of ME/CFS: differentiating fatigue-related conditions

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating, multi-systemic condition that has gained renewed global attention due to its clinical overlap with the growing population of long COVID patients. Despite ongoing efforts to characterize the disease, definitive diagnostic molecular markers are yet to be fully established, posing challenges in clinically differentiating from idiopathic chronic fatigue (ICF) and depression (Dep). This study aimed to identify circulating extracellular vesicles (EVs)-associated microRNAs (miRNAs) that serve as both diagnostic signatures and windows into the disease’s underlying pathophysiology.

Methods: Circulating EVs from ME/CFS (n = 6), ICF (n = 6), and depression (n = 8) patients were analyzed using flow cytometry, nano-tracking analysis, and comprehensive miRNA analysis. Differentially expressed miRNAs were analyzed using KEGG pathway enrichment to identify ME/CFS-specific signatures. Key candidate biomarkers were further validated in an additional healthy control (HC) cohort (n = 4).

Results: ME/CFS-EVs exhibited a unique subpopulation with high calcein intensity and larger diameters. Initial global miRNA profiling (Volcano plot) identified miR-21-5p, let-7f-5p, miR-26b-5p, and miR-20a-5p as significantly dysregulated EV-miRNAs in ME/CFS compared to ICF and Dep. To explore systemic pathophysiology, we identified a 114 EV-miRNA signature that achieved 87.0 ± 4.8% sensitivity and 93.7 ± 2.4% specificity within repeated cross-validation of the discovery cohort. After adjusting for covariates, 91 miRNAs remained significant; pathway analysis of the 62 up-regulated EV-miRNAs revealed significant enrichment in neuro-systemic axes, encompassing cellular structural integrity (focal adhesion), core signaling hubs (PI3K-Akt), and systemic homeostasis (such as insulin signaling and endocrine functions). Preliminary evaluation confirmed that these target EV-miRNAs remained at minimal or undetectable levels in the HC group.

Conclusions: A 62 EV-miRNA signature provides insight into the interconnected neuro-systemic pathways disrupted in ME/CFS, particularly those governing neuronal connectivity and cellular scaffolding. Within this candidate EV-miRNA signature, the top-ranked miRNAs-miR-21-5p, let-7f-5p, miR-26b-5p, and miR-20a-5p-emerge as potential candidate biomarkers whose specific elevation was not shared by HC. These findings establish a valuable framework for targeted diagnosis and enhance our understanding of the molecular pathways involved in synaptic and structural alterations in ME/CFS.

Source: Eguchi A, Kuratsune H, Nakatomi Y, Yasui T, Nakagawa R, Watanabe Y, Fukuda S. Circulating extracellular vesicles-microRNAs as potential biomarkers for the identification of ME/CFS: differentiating fatigue-related conditions. J Transl Med. 2026 Jul 27;24(1):979. doi: 10.1186/s12967-026-08695-w. PMID: 42533331. https://link.springer.com/article/10.1186/s12967-026-08695-w (Full text)

Exploring differences in protein cargo of extracellular vesicles from ME/CFS patient plasma compared to healthy controls

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic and debilitating disease characterized by post-exertional malaise, fatigue and pain. Yet, its underlying biological mechanisms remain poorly understood. Extracellular vesicles (EVs) are nanoparticles carrying biological cargo and are involved in cell-cell communication. Plasma EVs reflect several disease states and may serve as minimally invasive biomarkers. In this exploratory study, we characterized the plasma EV profiles of ME/CFS patients (N = 49) and healthy controls (N = 50), by enriching for EVs by size-exclusion chromatography coupled to high-resolution quantitative proteomics.

The ME/CFS patients had significantly higher concentrations of EVs than healthy controls. Among the 424 detected proteins included for analyses, 11 had different levels in EVs from ME/CFS patients. The ME/CFS associated EV proteins appear to mainly originate from erythroid cells, hepatocytes and plasma B cells, based on their tissue expression. Albeit differences in EV protein levels did not withstand correction for multiple testing, our study is the largest to date, thereby encouraging future investigations on the role of EV and its cargo in ME/CFS.

Source: Rydland A, Yran ES, Nyman TA, Strand EB, Trøseid AS, Øvstebø R, Heinicke F, Lie BA, Viken MK. Exploring differences in protein cargo of extracellular vesicles from ME/CFS patient plasma compared to healthy controls. Biochem Biophys Rep. 2026 Jun 20;47:102679. doi: 10.1016/j.bbrep.2026.102679. PMID: 42375682; PMCID: PMC13312568. https://pmc.ncbi.nlm.nih.gov/articles/PMC13312568/ (Full text)

Raman Spectroscopy Combined with Machine Learning Reveals Myalgic Encephalomyelitis–Associated Biomolecular Signatures at Rest and After Standardized Stress

Abstract:

Myalgic encephalomyelitis (ME) is characterized by profound fatigue, post-exertional malaise (PEM), and cognitive dysfunction. Despite its clinical significance, the pathophysiology of PEM and disease heterogeneity remain unclear, and no validated biomarkers are available for rapid diagnosis or monitoring. We aimed to develop a screening approach combining label-free Raman spectroscopy (RS) and machine learning modeling (ML) to detect biomolecular changes in blood plasma and differentiate patients with ME from sedentary healthy controls.
Blood plasma was collected from 115 patients with ME and 45 controls at rest (T0) and 90 min after a standardized, non-invasive stress test designed to induce PEM. Plasma samples were analyzed by RS, and ML models were developed independently at each time point to differentiate patients with ME and controls.
The RS-ML models identified spectral features consistent with contributions from proteins, lipids, and low-molecular-weight metabolites. At T0 and T90, the area under the receiver operating characteristic curve, accuracy, specificity and sensitivity were 0.85 and 0.83, 79% and 84%, 82% and 90%, and 73% and 69%, respectively. RS-ML provides a rapid, low-cost approach to detect ME-associated biomolecular signatures in plasma and capture biochemical alterations associated with standardized stress.
Source: Heidarifard M, Moezzi A, Dallaire F, Ember K, Elremaly W, Caraus I, Franco A, Leblond F, Moreau A, Dehaes M. Raman Spectroscopy Combined with Machine Learning Reveals Myalgic Encephalomyelitis–Associated Biomolecular Signatures at Rest and After Standardized Stress. International Journal of Molecular Sciences. 2026; 27(11):4937. https://doi.org/10.3390/ijms27114937 https://www.mdpi.com/1422-0067/27/11/4937 (Full text)

Plasma Extracellular Vesicle Surface Marker Profiling Reveals Immune Cell-Associated Mitochondrial Membrane Potential Alterations in Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Background: Long COVID (LC) is characterized by symptoms persisting at least 3 months after SARS-CoV-2 infection and affecting multiple organ systems. Diagnosis relies on subjective criteria without established biomarkers. Immune dysregulation and mitochondrial dysfunction are implicated in LC pathophysiology. Given clinical overlap with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we investigated whether plasma extracellular vesicles (EVs) capture shared molecular signatures.

Methods: Plasma EVs from 125 individuals across pandemic-era and prepandemic cohorts were analyzed. The pandemic-era cohort included COVID-Recovered, LC with ME/CFS phenotype (LC-ME/CFS), and ME/CFS without infection (pan-ME/CFS). The prepandemic cohort included ME/CFS and matched controls. Extracellular vesicles were isolated using size-exclusion chromatography. Concentration and size were assessed by nanoparticle tracking analysis, and surface markers and mitochondrial membrane potential were evaluated by flow cytometry.

Results: Both pan-ME/CFS and LC-ME/CFS exhibited elevated EV concentrations compared with COVID-recovered controls after false discovery rate (FDR) correction (q = 0.0042 and 0.0024). Leukocyte-, monocyte/macrophage-, and platelet-derived EVs were increased, whereas B cell-derived EVs were reduced in both groups. Compared with controls, pan-ME/CFS demonstrated increased mitochondrial membrane potential in B cell-, monocyte/macrophage-, and NK cell-derived subsets after FDR correction, whereas no significant differences were observed in LC-ME/CFS. Prepandemic ME/CFS showed a nominal increase in leukocyte-derived EVs that did not persist after correction, whereas elevated mitochondrial membrane potential in B cell-derived EV subsets remained significant.

Conclusions: ME/CFS and LC-ME/CFS demonstrate partially overlapping immune cell-associated EV alterations. Mitochondrial membrane potential alterations within selected immune-derived EV subsets, particularly B cell-associated EVs, suggest immune-metabolic involvement. Plasma EV profiling may inform future biomarker development.

Source: Ikeda G, Koike-Ieki M, Inoue H, Dadhania AV, El Kamari V, Jagannathan P, Geng LN, Miglis MG, Shafer RW, Yang PC, Bonilla HF. Plasma Extracellular Vesicle Surface Marker Profiling Reveals Immune Cell-Associated Mitochondrial Membrane Potential Alterations in Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Open Forum Infect Dis. 2026 May 12;13(5):ofag209. doi: 10.1093/ofid/ofag209. PMID: 42131622; PMCID: PMC13166156. https://pmc.ncbi.nlm.nih.gov/articles/PMC13166156/ (Full text)

Involvement of autoantibodies against G protein-coupled receptors in post-COVID condition and Chronic Fatigue Syndrome

Abstract:

Post-COVID condition (PCC) and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) are chronic disorders marked by fatigue, autonomic dysfunction, and cognitive impairment. Autoantibodies (AAbs) targeting adrenergic and muscarinic receptors have been implicated in their pathophysiology. This study aimed to investigate the association between these AAbs, autonomic nervous system (ANS) function, and cognitive performance in PCC and ME/CFS.

We included 96 PCC patients, 59 ME/CFS patients, and 36 healthy controls (HCs). Plasma AAbs against α1, β1, β2 adrenergic and M1-M4 muscarinic receptors were measured via ELISA. ANS function was evaluated using COMPASS-31, Sudoscan, hemodynamic tests (deep breathing, Valsalva, tilt test), and heart rate variability. Cognitive domains assessed included attention, fluency, processing speed, memory, visuoconstruction, perception, and executive functions.

ME/CFS patients had significantly higher β2 adrenergic AAb titers than PCC and HCs (F₂,₁₈₆ = 3.15, p = 0.046). PCC patients showed more borderline/pathological M3 muscarinic AAb results compared to HCs. β2 AAb levels correlated with increased autonomic symptoms in PCC (r = 0.27, p = 0.048) and sympathovagal imbalance in ME/CFS (r = 0.45, p = 0.001). In ME/CFS, M1, M3, and M4 AAb titers positively correlated with verbal and working memory performance.

Distinct AAb profiles in PCC and ME/CFS suggest potential differences in immunological mechanisms. β2 adrenergic receptor AAbs were associated with measures of autonomic dysfunction in PCC patients, and with sympathovagal parameters in ME/CFS patients. Muscarinic AAbs were correlated with cognitive performance in ME/CFS, supporting a potential role of these autoantibodies in autonomic and cognitive dysfunction. These findings support further investigation of AAbs as biomarkers and therapeutic targets.

Source: Azcue N, Prada A, Del Pino R, Acera M, Fernández-Valle T, Ayo-Mentxakatorre N, Pérez-Concha T, Murueta-Goyena A, Lafuente JV, López de Munain A, Ruiz Irastorza G, Ribacoba L, Gabilondo I, Tijero-Merino B, Gómez-Esteban JC. Involvement of autoantibodies against G protein-coupled receptors in post-COVID condition and Chronic Fatigue Syndrome. Sci Rep. 2026 May 5. doi: 10.1038/s41598-026-49131-9. Epub ahead of print. PMID: 42082542. https://www.nature.com/articles/s41598-026-49131-9 (Full text available as PDF file)

A hypothesis connecting dysgeusia due to defects in ATP-P2X3 signaling and fatigue in myalgic encephalomyelitis/chronic fatigue syndrome: lessons learned from long-COVID

Abstract:

Myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) is a neuroimmune disease characterized by debilitating post-exertional malaise (PEM), brain-fog/cognitive problems, and dysregulation of the autonomic nervous system. Currently, there are no objective biomarkers for ME/CFS despite decades of research.

Here, we compile evidence from literature that supports taste dysfunction, particularly alterations of taste perception mediated by Type II taste receptor cells, may be a critical underrecognized feature of ME/CFS. The impetus is drawn from the emerging evidence of clinicopathological similarities between long-COVID and ME/CFS. We discuss in parallel the mechanisms of cellular metabolism, inflammation, vascular dysfunction, and autonomic dysregulation in ME/CFS and long-COVID pathophysiology.

We postulate that mechanistically, dysregulation of ATP signaling through P2X2/P2X3 purinergic receptors underlies both gustatory impairment and core ME/CFS symptoms. Adopting information from the NIH-RECOVER shared resources, we present evidence that suggests chemosensory dysfunction as a potential indicator of progression/severity of PEM. We discuss standardized taste testing as a non-invasive screening tool complementary to molecular biomarkers for ME/CFS.

Notwithstanding, we acknowledge the limitations, confounding and contributing factors such as medications and deficiencies that may exacerbate or independently cause taste-related symptoms in ME/CFS.

In conclusion, we present a compelling case for the multi-factorial role of taste dysfunction in ME/CFS and suggest specific research priorities for investigating the relationship between chemosensory function and post-viral chronic illness.

Source: Srinivasan M, Joseph PV. A hypothesis connecting dysgeusia due to defects in ATP-P2X3 signaling and fatigue in myalgic encephalomyelitis/chronic fatigue syndrome: lessons learned from long-COVID. Front Med (Lausanne). 2026 Apr 8;13:1808646. doi: 10.3389/fmed.2026.1808646. PMID: 42040552; PMCID: PMC13107777. https://pmc.ncbi.nlm.nih.gov/articles/PMC13107777/ (Full text)

Proteomic signatures in cerebrospinal fluid and their clinical associations in patients with ME/CFS

Abstract:

This study evaluated the cerebrospinal fluid (CSF) proteomes from 31 patients diagnosed with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We quantified 902 proteins, each expressed in at least eleven samples, and systematically categorized clinical factors relevant to ME/CFS symptoms-including autonomic dysfunction, neuroinflammation and metabolic disturbances.

Differentially expressed protein and pathway analyses evaluated protein features associated with both postural orthostatic tachycardia syndrome (POTS) status and disease severity among the patients, while ratio-based analysis further explored associations with severity ratings.

Data are available via ProteomeXchange with identifier PXD076216. Neutrophil degranulation and platelet activation were enriched in patients with POTS, and several pathways, such as the complement cascade, coagulation-related pathways and IGFBP‑mediated insulin-like growth factor transport, were enriched in severe cases. Ratio-based analysis identified four biologically interpretable severity-associated protein ratios related to cellular stress, extracellular remodelling and immune-neuronal interaction.

Together, these findings provide insight into the biological processes associated with clinical heterogeneity in ME/CFS and generate hypotheses for future validation in larger independent cohorts.

Source: Bragée B, Li P, Meadows D, Widgren A, Sjögren P, Ghatan PH, Bertilson BC, Xiao W, Bergquist J. Proteomic signatures in cerebrospinal fluid and their clinical associations in patients with ME/CFS. Sci Rep. 2026 Apr 3. doi: 10.1038/s41598-026-46965-1. Epub ahead of print. PMID: 41932997.  https://www.nature.com/articles/s41598-026-46965-1 (Full text available as PDF file)

Authors’ Response to “Comment on ‘SMPDL3B as a novel biomarker and therapeutic target in myalgic encephalomyelitis’”

Letter:

We thank Chen and Yan for their thoughtful and positive comments on our recent publication and for their interest in the translational implications of SMPDL3B biology in myalgic encephalomyelitis (ME) [, ]. Their letter provides a welcome opportunity to clarify methodological points related to biomarker validation, in vitro pharmacological assays, and mechanistic interpretation []. We appreciate this constructive dialogue and address each issue below in a collegial and scientifically grounded manner.

Read the rest of this letter HERE>>

Source: Rostami-Afshari B, Elremaly W, Franco A, Moreau A. Authors’ Response to “Comment on ‘SMPDL3B as a novel biomarker and therapeutic target in myalgic encephalomyelitis'”. J Transl Med. 2026 Jan 16;24(1):75. doi: 10.1186/s12967-025-07583-z. PMID: 41546078; PMCID: PMC12809929. https://pmc.ncbi.nlm.nih.gov/articles/PMC12809929/ (Full text)

People with ME/CFS have a consistent faulty cellular structure, new research confirms

Press Release:

A faulty ion channel function is a consistent biological feature of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), providing long-awaited validation for hundreds of thousands of Australians living with the debilitating illness.

The new Griffith University research found a crucial cellular structure responsible for calcium transport, the TRPM3 ion channel, was faulty in immune cells from people with ME/CFS.

The paper “Large-scale investigation confirms TRPM3 ion channel dysfunction in ME/CFS” has been published in Frontiers in Medicine.

Director and senior author, Professor Sonya Marshall-Gradisnik from Griffith’s National Center for Neuroimmunology and Emerging Diseases (NCNED), said the TRPM3 played an essential role in calcium transport into cells, regulating responses properly in the body, immune function, and maintaining normal cellular balance.

“When it fails, cells cannot function properly as calcium signaling is essential for healthy immune cell activity,” Professor Marshall-Gradisnik said.

“Our findings provide clear and definitive scientific evidence that TRPM3 ion channels are not working properly in people with ME/CFS.”

Read the rest of this press release HERE>>

Large-scale investigation confirms TRPM3 ion channel dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Introduction: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease hallmarked by multiple systemic symptoms, such as neurocognitive, respiratory, immunological, gastrointestinal, and cardiovascular impairment, which worsen following physical and mental exertion. ME/CFS is characterized by an elusive pathomechanism, profound impact on quality of life, and an absence of diagnostic tests or evidence-based treatments. Transient Receptor Potential Melastatin 3 (TRPM3) ion channel has been suggested as a potential biomarker and target for therapeutics in people with ME/CFS, supported by a series of publications reporting genetic and protein changes. This study aimed to undertake a multi-site, large-scale investigation to determine the consistency of TRPM3 ion channel dysfunction in people with ME/CFS.

Methods: TRPM3 ion channel activity was assessed in two distinct laboratory sites by independent investigators using whole-cell patch-clamp recordings performed in isolated natural killer (NK) cells from 36 ME/CFS participants, characterized according to the Canadian Consensus Criteria, and 42 healthy controls. The Mann–Whitney U test was used to compare endogenous TRPM3-like currents between cohorts. The effect of location was determined using a covariance analysis, while antagonist sensitivity was determined using Fisher’s Exact test.

Results: Electrophysiological experiments revealed a significant reduction in TRPM3 function in NK cells from individuals diagnosed with ME/CFS compared with controls in all parameters analyzed. Importantly, there was no significant effect of the laboratory sites on the results of this investigation, which confirms TRPM3 as a consistent biomarker for ME/CFS.

Conclusion: The current large-sample-size study confirmed previous results regarding TRPM3 ion channel dysfunction in NK cells in ME/CFS, demonstrating involvement of TRPM3 in the pathomechanism of this condition. Therefore, this multiple-site investigation offers strong evidence demonstrating TRPM3 as a potential biomarker for the diagnosis of ME/CFS, given the accumulating evidence.

Source: Sasso Etianne Martini , Er Teagan S. , Eaton-Fitch Natalie , Hool Livia , Muraki Katsuhiko , Marshall-Gradisnik Sonya. Large-scale investigation confirms TRPM3 ion channel dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Frontiers in Medicine, Volume 12 – 2026. https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2025.1703924 10.3389/fmed.2025.1703924 ISSN=2296-858X https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2025.1703924/full (Full text)