Long-COVID: Ultrasensitive multiplex protein biomarker profiling reveals subclinical neuroinflammatory and innate immune dysregulation without correlation to symptom severity

Abstract Routine laboratory tests are consistently normal in long-COVID, yet patients suffer persistent and debilitating symptoms. Whether subclinical inflammatory and neurological dysregulation underlies this discordance remains unclear. Ultra-sensitive multiplex proteomics may detect low-abundance biomolecular signals invisible to conventional methods, and alternative mechanisms such as central sensitization may be operative. In this single-centre, age- and sex-matched case–control study, plasma from 48 Long-COVID patients and 48 recovered controls was analysed using the NULISAseq™ CNS Disease Panel 120, quantifying 123 biomolecules with attomolar sensitivity. The median interval from SARS-CoV-2 infection was 65.5 weeks in patients and 72.5 weeks in controls. Differentially expressed proteins were identified using Mann–Whitney U test with Benjamini–Hochberg correction. Principal Component Analysis (PCA), hierarchical cluster analysis, and Ingenuity Pathway Analysis (IPA) were used to characterize biological subgroups and activated pathways. Despite no separation between patients and controls on conventional PCA, 19 proteins were significantly dysregulated, with CCL22, IL-6R, and CST3 being prominently upregulated. IPA revealed activation of the Pathogen-Induced Cytokine Storm, Neuroinflammation, and IL-10 Signaling pathways. Hierarchical clustering identified two biologically distinct patient subgroups—one characterized by metabolic reprogramming and mitochondrial dysfunction, the other by neuroinflammatory pathway activation—yet neither cluster differed in fatigue, depression, cognitive symptoms, or any other patient-reported outcome measure. Long-COVID harbors subclinical innate immune activation and neuroinflammatory dysregulation detectable only at attomolar sensitivity, with no direct mapping to clinical symptom profiles. This biological-clinical dissociation suggests that additional mechanisms—including central sensitization or epigenetic dysregulation—may translate peripheral immune signals into the symptom burden experienced by patients.