Irene Giovannelli1, Nunziata Ciccone2, Guendalina Vaggelli2, Nunzia Della Malva2, Francesca Torricelli3, Gian Maria Rossolini4, Simone Giannecchini5. 1. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. 2. Clinical Microbiology and Virology unit, Careggi University Hospital, University of Florence, Florence, Italy. 3. Genetics Diagnostic unit, Careggi University Hospital, Florence, Italy. 4. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Clinical Microbiology and Virology unit, Careggi University Hospital, University of Florence, Florence, Italy; Department of Medical Biotechnologies, University of Siena, Siena, Italy. 5. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address: simone.giannecchini@unifi.it.
Abstract
BACKGROUND: Quantitative PCR (qPCR) is the standard molecular method for detection of polyomavirus JC (JCPyV) DNA reactivation in serum and cerebrospinal fluid (CSF) in patients at risk of progressive multifocal leukoencephalopathy (PML). Recently, digital PCR has shown potential benefits over qPCR in viral diagnostics. OBJECTIVE: To evaluate the performance of droplet digital PCR (ddPCR) assay in assessing JCPyV-DNA status in clinical samples of patients at risk for PML. STUDY DESIGN: JCPyV specific ddPCR was developed with primers/probes targeting Large T and the noncoding control region used in qPCR. The ddPCR accuracy of JCPyV-DNA quantification was investigated using serial dilutions of genomic JCPyV-DNA. The ddPCR JCPyV-DNA quantification and qPCR confirmation were performed on 150 CSF and 100 serum clinical samples. RESULTS: Using genomic JCPyV-DNA, ddPCR was highly sensitive, repeatable and reproducible for both molecular targets. Using clinical samples, JCPyV-DNA was detected in 13% of CSF and in 50% of serum samples with limit of detection of 30 copies/ml. Among the 19 JCPyV-DNA-positive CSF detected using the ddPCR, 15 also tested positive with the qPCR. Among the 50 JCPyV-DNA-positive serum identified with ddPCR, 41 tested positive with qPCR. All the ddPCR-negative samples were negative when assessed using qPCR. Additionally, the mean JCPyV-DNA viral load obtained with ddPCR in all samples was not significantly different from that of qPCR. CONCLUSION: The results demonstrate that ddPCR is a highly sensitive alternative for measuring JCPyV-DNA that should be considered in clinical diagnostic testing of JCPyV-DNA in patients at risk of PML and other associated diseases.
BACKGROUND: Quantitative PCR (qPCR) is the standard molecular method for detection of polyomavirus JC (JCPyV) DNA reactivation in serum and cerebrospinal fluid (CSF) in patients at risk of progressive multifocal leukoencephalopathy (PML). Recently, digital PCR has shown potential benefits over qPCR in viral diagnostics. OBJECTIVE: To evaluate the performance of droplet digital PCR (ddPCR) assay in assessing JCPyV-DNA status in clinical samples of patients at risk for PML. STUDY DESIGN:JCPyV specific ddPCR was developed with primers/probes targeting Large T and the noncoding control region used in qPCR. The ddPCR accuracy of JCPyV-DNA quantification was investigated using serial dilutions of genomic JCPyV-DNA. The ddPCR JCPyV-DNA quantification and qPCR confirmation were performed on 150 CSF and 100 serum clinical samples. RESULTS: Using genomic JCPyV-DNA, ddPCR was highly sensitive, repeatable and reproducible for both molecular targets. Using clinical samples, JCPyV-DNA was detected in 13% of CSF and in 50% of serum samples with limit of detection of 30 copies/ml. Among the 19 JCPyV-DNA-positive CSF detected using the ddPCR, 15 also tested positive with the qPCR. Among the 50 JCPyV-DNA-positive serum identified with ddPCR, 41 tested positive with qPCR. All the ddPCR-negative samples were negative when assessed using qPCR. Additionally, the mean JCPyV-DNA viral load obtained with ddPCR in all samples was not significantly different from that of qPCR. CONCLUSION: The results demonstrate that ddPCR is a highly sensitive alternative for measuring JCPyV-DNA that should be considered in clinical diagnostic testing of JCPyV-DNA in patients at risk of PML and other associated diseases.