Development and evaluation of the digital PCR-based method for clinical monitoring of viral loads during severe fever with thrombocytopenia syndrome virus infection

Abstract

Background

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) represents a novel bunyavirus that poses significant public health challenges. As a key prognostic indicator of clinical outcome, the viral load determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is relatively inaccurate and incomparable across different studies. Digital PCR (dPCR) has recently proved to be a more ideal tool for viral load assessment.

Objective

To develop a dPCR-based S-segment-specific method for SFTSV viral load monitoring and evaluate its performance in clinical samples.

Study design

Specific dPCR was developed using primers/probes for the N region in the S segment of the SFTSV genome. The performance of dPCR was confirmed using serial dilutions of viral cultures, and dPCR viral load quantification was compared with the result of RT-qPCR in 166 suspected SFTS patients.

Results

DPCR demonstrated superior sensitivity with a detection limit of 190.5 copies/mL, high linearity, and good reproducibility. Six false negative samples were detected by dPCR among the 28 RT-qPCR negative samples. The correlation between RT-qPCR and dPCR was low at a low viral load level. Both dPCR and RT-qPCR were important risk factors for severity and mortality by the multivariate logistic regression analysis The accurate viral load based on dPCR has a strong predictive ability for patient outcomes and shows significant correlation with multiple host response markers.

Conclusion

The results suggest that dPCR is a highly sensitive alternative to the measurement of SFTSV and should be considered for clinical utilization in patients with suspected SFTS.