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.