Abstract
BACKGROUND: Acute undifferentiated febrile illness (AUFI) is a leading cause of morbidity and mortality worldwide, particularly in tropical and subtropical regions. The causative pathogen is often unidentified due to the wide range of potential etiologies and non-specific clinical features. A diagnostic approach capable of detecting multiple pathogens simultaneously could improve clinical management and support disease surveillance efforts. METHODS: We evaluated a real-time PCR–based TaqMan Array Card (TAC) for detecting 21 AUFI-associated pathogens, comprising eight viruses, one parasite, and twelve bacteria. Analytical performance was assessed using DNA control plasmids and extracted or synthesized nucleic acids from reference strains to determine the limit of detection (LOD), cross-reactivity, intra- and inter-assay reproducibility. Clinical performance was evaluated with 185 archived clinical samples and compared against pathogen-specific real-time PCR assays. RESULTS: The TAC assay detected all 21 AUFI pathogen, with LODs ranging from 10 to 100 copies/µL for plasmid controls and 2 to 2,000 copies/µL for extracted nucleic acids. Based on clinical specimens, the overall accuracy, sensitivity, and specificity for detecting pathogens causing AUFI were 92.97% (95% CI, 88.28–96.21%), 96.43% (95% CI, 91.11–99.02%), and 87.67% (95% CI, 77.88–94.20%), respectively. After Ct cut-off optimization, the accuracy increased to 95.14% (95% CI, 90.97–97.75%) and the specificity to 93.15% (95% CI, 84.74–97.74%). CONCLUSIONS: The AUFI-customized TAC demonstrated satisfactory analytical and clinical performance for simultaneous multi-pathogen detection, with improved accuracy using pathogen-specific Ct cut-offs. The assay shows potential utility for diagnostic support and surveillance of acute febrile illness in Thailand and Southeast Asia. However, limited low-titer clinical samples and sporadic late amplification for certain bacterial targets warrant confirmatory testing and further prospective validation before broader implementation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-026-12639-6.