Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy

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.

EBNA1 Inhibitors Block Proliferation of Spontaneous Lymphoblastoid Cell Lines From Patients With Multiple Sclerosis and Healthy Controls

“…After emulsification, PCR amplification was performed within each droplet using a GeneAmp 9700 thermocycler (Applied Biosystems, Grand Island, NY) with the cycling conditions as previously described. 36 , 37 After PCR amplification, a QX200 droplet reader (Bio-Rad, Hercules, CA) was used to quantify droplets. Thresholds were established manually for each experiment, based on negative controls, which included a no template control and a negative sample (EBV − cell lines, Ramos or BJAB).…”

Liquid Biopsy in Neurological Diseases

“…Another viral pathogen related to demyelinating diseases is the human John Cunningham virus (JCV), causing progressive multifocal leukoencephalopathy (PML) [66], which mainly affects MS patients due to the reactivation of a latent JCV infection in the brain as a severe adverse effect originating from the immunosuppressive treatment [67]. The viral DNA can be detected in CSF by PCR assays, helping to arrive at an accurate diagnosis in combination with typical brain MRI findings [68].…”

Progressive multifokale Leukenzephalopathie: Diagnostik und Therapieoptionen

“…Eine sichere Diagnosestellung der PML setzt den Nachweis von HPyV-2 im Liquor oder im Gehirngewebe der Betroffenen voraus. Die Anwendung der quantitativen Polymerasekettenreaktion (qPCR) ist der Goldstandard zur Detektion des Virus in Liquorproben, wobei die Sensitivität von Labor zu Labor variieren kann [16,17]. ▶ Tab.…”