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Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive
Nyater Ngouth1, Maria Chiara Monaco1, Lorenzo Walker2
1Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
Abstract:
Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.
Insights
Droplet digital PCR (ddPCR) offers increased sensitivity for detecting JC viral DNA in cerebrospinal fluid compared to quantitative PCR (qPCR), aiding in progressive multifocal leukoencephalopathy (PML) diagnosis. This advanced ddPCR method accurately quantifies viral loads, improving diagnostic capabilities for PML patients.
Area of Science:
- Virology
- Neuroscience
- Molecular Diagnostics
Background:
- Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by JC polyomavirus (JCPyV) lytic infection of oligodendrocytes.
- Detection of JCPyV DNA in cerebrospinal fluid (CSF) via PCR is crucial for diagnosing PML, often obviating the need for brain biopsy.
- Quantitative PCR (qPCR) is the current gold standard for viral load quantification, but its sensitivity can be limiting.
Purpose of the Study:
- To compare the diagnostic sensitivity and quantification accuracy of droplet digital PCR (ddPCR) against the gold standard qPCR for JCPyV DNA.
- To characterize a novel triplex ddPCR assay for detecting JCPyV variants and a cellular reference gene.
- To evaluate the utility of ddPCR in analyzing serum samples from PML patients and multiple sclerosis (MS) cohorts.
Main Methods:
- Analyzed 62 CSF samples from 31 PML patients using both qPCR and ddPCR to quantify JCPyV DNA.
- Validated ddPCR by comparing results with qPCR data from 42 international laboratories using standardized plasma samples.
- Developed and employed a triplex ddPCR assay to detect JCPyV prototype/archetype variants and a housekeeping gene in CSF and serum samples.
Main Results:
- ddPCR detected JCPyV DNA in 8% of CSF samples (5/62) that were negative by qPCR.
- For 15% of CSF samples (9/62) below qPCR's limit of quantification (<250 copies/mL), ddPCR provided exact viral load quantification.
- JCPyV DNA was detected in serum from PML patients but not in healthy controls or non-natalizumab-treated MS patients; it was also detected in natalizumab-treated MS patients who seroconverted.
Conclusions:
- ddPCR demonstrates significantly increased sensitivity and accurate quantification capabilities for JCPyV DNA compared to qPCR.
- The ddPCR assay shows strong correlation with qPCR, offering improved detection of viral DNA in clinical samples.
- Further research is warranted to explore the potential of multiplex ddPCR for assessing PML risk in natalizumab-treated MS patients.
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