Droplet digital PCR-based nucleic acid amplification test for improved molecular and clinical diagnosis of severe fever with thrombocytopenia syndrome patients.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV) and is characterized by high mortality and non-specific clinical manifestations, which make early and accurate molecular diagnosis essential. This study aims to investigate whether reverse transcription droplet digital PCR (RT-ddPCR) provides improved diagnosis of SFTS compared to existing molecular diagnostic platforms. Detection limits of RT-ddPCR, reverse transcription quantitative PCR (RT-qPCR), and reverse transcription nested PCR (RT-nested PCR) were determined using RNA extracted from metrologically validated SFTSV nucleic acid reference material. Then, diagnostic performance metrics of each PCR method were compared using RNA from 29 SFTS patients and 29 disease controls with scrub typhus. Statistical analyses of key laboratory parameters were performed to identify markers significantly correlated with SFTSV load, as defined by RNA copy number, in early SFTS patients. RT-ddPCR showed the lowest detection limit of 3.82 copies/μL among the three PCR methods. The sensitivity of RT-ddPCR was 82.75% in clinical samples, which was higher than that of RT-qPCR at 72.41%. The specificity of RT-ddPCR was 100% compared to 89.66% for RT-nested PCR. Overall, RT-ddPCR demonstrated the highest diagnostic performance among the three PCR methods, with an accuracy of 91.38% and an area under the curve value of 0.91. A strong correlation between viral RNA loads and laboratory parameters was observed in the early stage of SFTS, particularly with high-sensitivity C-reactive protein, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and activated partial thromboplastin time. Our findings demonstrate that a novel molecular diagnosis method using RT-ddPCR shows improved diagnostic performance for detecting SFTSV RNA from SFTS patients and potentially contributes to accurate early diagnosis and enhanced clinical diagnosis.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is a high-mortality tick-borne infection caused by SFTS virus (SFTSV), and reliable molecular diagnosis is essential for patient care in endemic regions. Existing PCR-based assays for SFTS show limited sensitivity and specificity, particularly in patients with suspected or early-stage disease. Here, we describe the development and analytical validation of a reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection and quantification of SFTSV RNA from patient sera. By using metrologically validated viral nucleic acid reference materials and clinical samples, this assay demonstrates significantly improved sensitivity and overall diagnostic performance compared with conventional reverse transcription quantitative PCR and reverse transcription nested PCR. RT-ddPCR can provide clinical microbiology laboratories with a useful tool to enhance early and reliable molecular diagnosis of SFTS.