{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T21:24:05Z","timestamp":1792445045353,"version":"4.5.0"},"reference-count":34,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,6,8]],"date-time":"2022-06-08T00:00:00Z","timestamp":1654646400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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 &lt;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.<\/jats:p>","DOI":"10.3390\/v14061246","type":"journal-article","created":{"date-parts":[[2022,6,13]],"date-time":"2022-06-13T06:31:59Z","timestamp":1655101919000},"page":"1246","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy"],"prefix":"10.3390","volume":"14","author":[{"given":"Nyater","family":"Ngouth","sequence":"first","affiliation":[{"name":"Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Chiara","family":"Monaco","sequence":"additional","affiliation":[{"name":"Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorenzo","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sydney","family":"Corey","sequence":"additional","affiliation":[{"name":"Neuroimmunology Clinic, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ijeoma","family":"Ikpeama","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary","family":"Fahle","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irene","family":"Cortese","sequence":"additional","affiliation":[{"name":"Neuroimmunology Clinic, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanchita","family":"Das","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven","family":"Jacobson","sequence":"additional","affiliation":[{"name":"Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1016\/S0140-6736(71)91777-6","article-title":"Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy","volume":"1","author":"Padgett","year":"1971","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1128\/CMR.05031-11","article-title":"Molecular biology, epidemiology, and pathogenesis of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain","volume":"25","author":"Ferenczy","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1038\/s41582-020-00427-y","article-title":"Progressive multifocal leukoencephalopathy and the spectrum of JC virus-related disease","volume":"17","author":"Cortese","year":"2021","journal-title":"Nat. Rev. Neurol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1016\/S1474-4422(18)30040-1","article-title":"Pathogenesis of progressive multifocal leukoencephalopathy and risks associated with treatments for multiple sclerosis: A decade of lessons learned","volume":"17","author":"Major","year":"2018","journal-title":"Lancet Neurol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"576","DOI":"10.1002\/ana.20431","article-title":"JC virus granule cell neuronopathy: A novel clinical syndrome distinct from progressive multifocal leukoencephalopathy","volume":"57","author":"Koralnik","year":"2005","journal-title":"Ann. Neurol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1002\/ana.21619","article-title":"Fulminant JC virus encephalopathy with productive infection of cortical pyramidal neurons","volume":"65","author":"Wuthrich","year":"2009","journal-title":"Ann. Neurol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1128\/jvi.51.2.458-469.1984","article-title":"Human polyomavirus JC virus genome","volume":"51","author":"Frisque","year":"1984","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1080\/13550280152537102","article-title":"A classification scheme for human polyomavirus JCV variants based on the nucleotide sequence of the noncoding regulatory region","volume":"7","author":"Jensen","year":"2001","journal-title":"J. Neurovirol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.jcv.2013.03.009","article-title":"Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants","volume":"57","author":"Ryschkewitsch","year":"2013","journal-title":"J. Clin. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1016\/j.tibtech.2018.12.002","article-title":"The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time","volume":"37","author":"Taylor","year":"2019","journal-title":"Trends Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1128\/JCM.02620-12","article-title":"Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus","volume":"51","author":"Hayden","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/j.jcv.2004.05.016","article-title":"Quantitation of HTLV-I and II proviral load using real-time quantitative PCR with SYBR Green chemistry","volume":"31","author":"Lee","year":"2004","journal-title":"J. Clin. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1093\/cid\/ciw370","article-title":"Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples","volume":"63","author":"Preiksaitis","year":"2016","journal-title":"Clin. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1128\/JCM.02044-16","article-title":"Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards","volume":"55","author":"Hayden","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1021\/ac202578x","article-title":"Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification","volume":"84","author":"Pinheiro","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"8604","DOI":"10.1021\/ac202028g","article-title":"High-throughput droplet digital PCR system for absolute quantitation of DNA copy number","volume":"83","author":"Hindson","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1007\/978-1-4939-7778-9_6","article-title":"Using Droplet Digital PCR to Detect Coinfection of Human Herpesviruses 6A and 6B (HHV-6A and HHV-6B) in Clinical Samples","volume":"1768","author":"Vellucci","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.3389\/fonc.2019.01417","article-title":"Precision Medicine in Lymphoma by Innovative Instrumental Platforms","volume":"9","author":"Arrigoni","year":"2019","journal-title":"Front. Oncol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.tube.2019.07.001","article-title":"Droplet digital PCR applications in the tuberculosis world","volume":"117","author":"Nyaruaba","year":"2019","journal-title":"Tuberculosis"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Leibovitch, E.C., Brunetto, G.S., Caruso, B., Fenton, K., Ohayon, J., Reich, D.S., and Jacobson, S. (2014). Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0092328"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1186\/s40478-016-0342-3","article-title":"BK virus encephalopathy and sclerosing vasculopathy in a patient with hypohidrotic ectodermal dysplasia and immunodeficiency","volume":"4","author":"Darbinyan","year":"2016","journal-title":"Acta Neuropathol. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.jcv.2013.02.002","article-title":"A second-generation ELISA (STRATIFY JCV\u2122 DxSelect\u2122) for detection of JC virus antibodies in human serum and plasma to support progressive multifocal leukoencephalopathy risk stratification","volume":"57","author":"Lee","year":"2013","journal-title":"J. Clin. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e1452","DOI":"10.1212\/WNL.0000000000008243","article-title":"Risk of natalizumab-associated PML in patients with MS is reduced with extended interval dosing","volume":"93","author":"Ryerson","year":"2019","journal-title":"Neurology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1007\/s13365-014-0249-3","article-title":"Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM\/TSP patients and identification of viral mutations","volume":"20","author":"Brunetto","year":"2014","journal-title":"J. Neurovirol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/s13027-016-0081-x","article-title":"Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls","volume":"11","author":"Lin","year":"2016","journal-title":"Infect. Agent Cancer"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1177\/1352458518765654","article-title":"Prevalence of salivary human herpesviruses in pediatric multiple sclerosis cases and controls","volume":"25","author":"Leibovitch","year":"2019","journal-title":"Mult. Scler. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.jcv.2016.07.008","article-title":"Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples","volume":"82","author":"Giovannelli","year":"2016","journal-title":"J. Clin. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1080\/22221751.2020.1772679","article-title":"Analytical comparisons of SARS-CoV-2 detection by qRT-PCR and ddPCR with multiple primer\/probe sets","volume":"9","author":"Liu","year":"2020","journal-title":"Emerg. Microbes Infect."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1080\/22221751.2020.1772678","article-title":"ddPCR: A more accurate tool for SARS-CoV-2 detection in low viral load specimens","volume":"9","author":"Suo","year":"2020","journal-title":"Emerg. Microbes Infect."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"358","DOI":"10.4014\/jmb.2009.09006","article-title":"Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets","volume":"31","author":"Park","year":"2021","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1002\/ana.22107","article-title":"Assessment of JC virus DNA in blood and urine from natalizumab-treated patients","volume":"68","author":"Rudick","year":"2010","journal-title":"Ann. Neurol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"102008","DOI":"10.1016\/j.msard.2020.102008","article-title":"Which is the best PML risk stratification strategy in natalizumab-treated patients affected by multiple sclerosis?","volume":"41","author":"Prezioso","year":"2020","journal-title":"Mult. Scler. Relat. Disord."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2240","DOI":"10.1056\/NEJMc1214233","article-title":"JC viremia in natalizumab-treated patients with multiple sclerosis","volume":"368","author":"Major","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1086\/597126","article-title":"Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors","volume":"199","author":"Egli","year":"2009","journal-title":"J. Infect. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/6\/1246\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:25:56Z","timestamp":1760138756000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/6\/1246"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,8]]},"references-count":34,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["v14061246"],"URL":"https:\/\/doi.org\/10.3390\/v14061246","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,8]]}}}