Monocyte TLR2/4 as predictive biomarkers for progression from dengue warning signs to severe disease.

Background: The World Health Organization (WHO) classification system, comprising dengue fever (DF), dengue with warning signs (DWS), and severe dengue (SD), has been widely adopted globally; however, clinical management remains challenging. Current warning sign criteria lack specificity, frequently failing to distinguish patients progressing to severe disease from those with transient illness, thereby contributing to delayed interventions and missed therapeutic windows. Although Toll-like receptors (TLRs) mediate innate antiviral immunity and are implicated in severe dengue pathogenesis, their expression dynamics during the critical DWS transition window, a phase in which intervention could prevent progression to severe disease, remain entirely unknown. Methods: An integrated biomarker discovery study was conducted, comprising bioinformatics analysis of publicly available single-cell RNA sequencing (scRNA-seq) data (GSE220969) and experimental validation in an independent clinical cohort of 45 dengue patients and 10 healthy controls (H) in Guangdong Province, China. Phase-specific immune signatures were identified from the scRNA-seq data. TLRs expression was quantified by multiparameter flow cytometry. Diagnostic performance was assessed using ROC curve analysis, and correlations with clinical laboratory parameters were evaluated using Spearman's rank correlation. Results: Bioinformatics analysis of 23 cell clusters revealed that TLR2/4/7/8 expression peaked specifically during the DWS phase, predominantly in monocytes. Flow cytometry confirmed elevated surface TLR2 (AUC 0.874) and TLR4 (AUC 0.763) in DWS versus other phases, with discriminatory capacity to distinguish DWS from SD (TLR2: AUC 0.833; TLR4: AUC 0.810). Moreover, correlation analysis of TLRs expression with clinical indicators demonstrated that the expression levels of TLR2 and TLR4 were associated with the development of SD. Conclusions: Elevated monocyte TLR2/4 expression during DWS identifies patients at risk for progression to severe dengue before clinical decompensation. These mechanism-informed biomarkers provide a foundation for point-of-care risk stratification in endemic regions.