Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples

Background: Quantitative PCR (qPCR) is the standard molecular method for detection of polyomavirusJC (JCPyV) DNA reactivation in serum and cerebrospinal fluid (CSF) in patients at risk of progressivemultifocal leukoencephalopathy (PML). Recently, digital PCR has shown potential benefits over qPCR inviral diagnostics.Objective: To evaluate the performance of droplet digital PCR (ddPCR) assay in assessing JCPyV-DNA statusin clinical samples of patients at risk for PML.Study design: JCPyV specific ddPCR was developed with primers/probes targeting Large T and the noncod-ing control region used in qPCR. The ddPCR accuracy of JCPyV-DNA quantification was investigated usingserial dilutions of genomic JCPyV-DNA. The ddPCR JCPyV-DNA quantification and qPCR confirmation wereperformed on 150 CSF and 100 serum clinical samples.Results: Using genomic JCPyV-DNA, ddPCR was highly sensitive, repeatable and reproducible for bothmolecular targets. Using clinical samples, JCPyV-DNA was detected in 13% of CSF and in 50% of serumsamples with limit of detection of 30 copies/ml. Among the 19 JCPyV-DNA-positive CSF detected usingthe ddPCR, 15 also tested positive with the qPCR. Among the 50 JCPyV-DNA-positive serum identifiedwith ddPCR, 41 tested positive with qPCR. All the ddPCR-negative samples were negative when assessedusing qPCR. Additionally, the mean JCPyV-DNA viral load obtained with ddPCR in all samples was notsignificantly different from that of qPCR.Conclusion: The results demonstrate that ddPCR is a highly sensitive alternative for measuring JCPyV-DNAthat should be considered in clinical diagnostic testing of JCPyV-DNA in patients at risk of PML and otherassociated diseases.