Establishment and application of a multiplex qPCR panel for multi-host surveillance of tick-Borne pathogens.

Tick-borne diseases pose significant public health threats. Non-specific symptoms and co-infections present diagnostic challenges for conventional methods. To address this, we developed the tick-borne pathogen (TBP) Panel, utilizing eight separate multiplex qPCR assays to simultaneously detect 25 TBPs (7 bacteria, 14 viruses, 4 parasites). Optimal Quantification Cycle (Cq) cut-offs were established using nested/semi-nested PCR amplicon sequencing, and digital PCR as reference standards. The Panel was then deployed for screening 2,333 ticks, 59 rodents, and 553 febrile patients. The Panel demonstrated robust analytical performance, achieving limits of detection (LOD99%) being 3.5-131 copies/reaction of plasmid, 102-103 copies/mL of blood and 103-104 copies/tick pool. This study established a tiered determination framework (Cq ≤ 36.6 as positive; Cq 36.6-39.6 requiring dual-replicate confirmation), yielding an overall AUC of 0.979, with 96.5% sensitivity and 96.5% specificity. Screening revealed a diverse pathogen landscape, identifying a total of 14 pathogens in 16.9% of the ticks (395/2,333), revealing a diverse geographical distribution. Five pathogens were identified in 7.9% of AFI patients (25/317), including Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Ehrlichia spp., which were found circulating in local ticks. In the FUO cohort, a single case of Rickettsia spp. was identified. Rodent surveillance identified four pathogens, predominantly Bartonella spp. (67.8%). In conclusion , the rigorously validated TBP Panel enables rapid etiological identification of undifferentiated febrile illnesses, serving as a high-throughput tool that bridges large-scale surveillance with clinical diagnostics to close critical gaps in tick-borne disease management.