Age-specific alterations of the gut mycobiome in patients with myalgic encephalomyelitis/chronic fatigue syndrome and identification of potential diagnostic biomarkers

While bacterial dysbiosis and metabolic disruptions have been widely implicated in the pathogenesis of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), the contribution of the gut mycobiome remains largely unresolved, particularly in the context of host age. Here, high-throughput internal transcribed spacer (ITS) sequencing was applied to fecal samples from 118 individuals (59 ME/CFS patients and 59 healthy matched controls), stratified into three age cohorts: young (18–34 years), middle-aged (35–55 years), and elderly (56–85 years). ME/CFS patients demonstrated significant alterations in gut fungal community composition and diversity compared to controls, with age-specific patterns emerging upon stratified analysis. Reduced fungal amplicon sequence variant (ASV) richness was observed in ME/CFS patients within the young (P < 0.001) and middle-aged (P < 0.01) cohorts, while the elderly ME/CFS group unexpectedly exhibited increased alpha diversity. Principal coordinate analyses based on Bray–Curtis and Jaccard distances robustly differentiated ME/CFS and control groups across all age strata, with the magnitude of separation exceeding that observed in age-unstratified analysis. Age-dependent discriminatory taxa were identified, including Preussia, Endocarpon, Chlorocillium, and Verticillium in the young cohort; Preussia, Romagnesiella, Aspergillus, and Trichothecium in the middle-aged cohort; and Chaetomium, unclassified Ascomycota, and Chlorocillium in the elderly cohort. Classification models based on fungal genera achieved an overall accuracy of 65.2% (AUC = 0.786) without age stratification, but predictive performance was markedly enhanced, yielding accuracies of 87.5% (AUC = 1.00), 100% (AUC = 0.911), and 100% (AUC = 1.00) in the young, middle-aged, and elderly cohorts, respectively. Correlation analyses revealed that taxa positively associated with fatigue severity were consistently depleted in ME/CFS, whereas negatively associated genera were enriched, suggesting that these fungi function primarily as biomarkers of disease burden rather than as causal agents. These findings underscore the critical importance of age-specific analyses in mycobiome research and highlight gut fungal profiles as promising diagnostic biomarkers for ME/CFS when age is explicitly accounted for.