Analytical Performance Disclosure in JCV PCR: An Overdue Standard

Analytical Performance Disclosure in JCV PCR: An Overdue Standard | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 1 September 2025 V1 Latest version Share on Analytical Performance Disclosure in JCV PCR: An Overdue Standard Author : Jagannadha Avasarala 0000-0002-2322-503X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175670486.65949584/v1 228 views 130 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract One of the most devastating central nervous system infections, progressive multifocal leukoencephalopathy (PML), is caused by the JC virus (JCV). It often presents in immunosuppressed patients, specifically in those treated with natalizumab, rituximab, or other immunomodulatory agents. A brain biopsy is rarely required for establishing a diagnosis and a clinical diagnosis is also possible, the latter based on evolving symptoms and MRI data. The cornerstone of diagnosis of PML is the detection of JCV DNA in the cerebrospinal fluid (CSF) by polymerase chain reaction (PCR). However, an alarming and under-recognized vulnerability exists in the way JCV PCR results are reported - most laboratories provide a binary result (”positive” or ”negative”) while omitting critical assay performance metrics such as the LOQ and LOD data. This practice creates a false sense of diagnostic certainty and may lead to missed or delayed diagnoses of early-stage PML, where viral loads are often low. This systemic reporting failure is best understood as a diagnostic shortcoming with consequences for timely care. I argue for mandatory inclusion of LOQ and LOD across all JCV PCR reports to restore diagnostic transparency and prevent diagnostic delay. Since most clinical laboratories do not disclose the analytical sensitivity of their assays, a negative JCV PCR result may be falsely reassuring. Unless clinicians recognize this possibility, diagnosis of PML can be delayed—or missed entirely. Because limits of quantitation (LOQ) and detection (LOD) are pivotal for establishing PML, laboratories that fail to report them risk misleading clinicians, potentially provoking legal consequences. Transparent disclosure of assay performance is essential for accurate decision-making and timely intervention. Analytical Performance Disclosure in JCV PCR: An Overdue Standard Jagannadha Avasarala, MD, PhD, FAAN, Department of Neurology, 740 S Limestone, Lexington, KY 40536 Address for correspondence: Jagannadha Avasarala, MD, PhD, FAAN Professor of Medicine and Director, Multiple Sclerosis and Neuroimmunological Disorders University of Kentucky Medical Center, 740 S Limestone, Lexington, KY 40536 WC: 1425 Abstract One of the most devastating central nervous system infections, progressive multifocal leukoencephalopathy (PML), is caused by the JC virus (JCV). It often presents in immunosuppressed patients, specifically in those treated with natalizumab, rituximab, or other immunomodulatory agents. A brain biopsy is rarely required for establishing a diagnosis and a clinical diagnosis is also possible, the latter based on evolving symptoms and MRI data. The cornerstone of diagnosis of PML is the detection of JCV DNA in the cerebrospinal fluid (CSF) by polymerase chain reaction (PCR). However, an alarming and under-recognized vulnerability exists in the way JCV PCR results are reported - most laboratories provide a binary result (”positive” or ”negative”) while omitting critical assay performance metrics such as the LOQ and LOD data. This practice creates a false sense of diagnostic certainty and may lead to missed or delayed diagnoses of early-stage PML, where viral loads are often low. This systemic reporting failure is best understood as a diagnostic shortcoming with consequences for timely care. I argue for mandatory inclusion of LOQ and LOD across all JCV PCR reports to restore diagnostic transparency and prevent diagnostic delay. Since most clinical laboratories do not disclose the analytical sensitivity of their assays, a negative JCV PCR result may be falsely reassuring. Unless clinicians recognize this possibility, diagnosis of PML can be delayed—or missed entirely. Because limits of quantitation (LOQ) and detection (LOD) are pivotal for establishing PML, laboratories that fail to report them risk misleading clinicians, potentially provoking legal consequences. Transparent disclosure of assay performance is essential for accurate decision-making and timely intervention. Case Consider a clinical vignette: a patient on rituximab who presents with progressive neurologic symptoms and MRI findings concerning for PML. A CSF sample sent to a commercial laboratory for JCV PCR assay has an LOD of 30 copies/mL returns the results as negative. The treating physician, concerned about PML based on clinical evidence, orders a repeat CSF PCR through a different lab that uses a more sensitive assay with an LOD of 10 copies/mL. The result is positive. This is an example that LOD sensitivity varies between different laboratories and unless a clinician is aware of this possibility, the outcome for patients can be catastrophic. The initial ‘negative’ test reflects an assay limitation rather than true viral absence. Such examples underscore how undisclosed assay thresholds generate diagnostic failure and imperil patient outcomes. Why LOQ matters In viral diseases, copy number directly impacts patient care by informing diagnosis, prognosis, and therapeutic strategies across acute and chronic infections. Higher viral loads are typically associated with increased disease severity and longer hospital stays 1-2 . Quantitative viral load testing enables risk stratification, guiding escalation of care and allocation of resources. In chronic infections, viral load is integral for monitoring treatment response, guiding preemptive therapy, and determining duration of antiviral therapy 3 .Viral load quantification is also used to estimate infectivity and transmission risk, influencing infection control measures. Higher viral copy numbers are associated with increased disease aggressiveness, severe clinical presentations, greater tissue damage, enhanced immune evasion, and higher fatality rates. Therefore, every JCV PCR assay must report its LOQ to ensure clinicians can interpret results in line with established standards across other infectious diseases. Despite the clinical utility of LOQ, limitations persist due to inter assay variability, lack of universally accepted thresholds, and need for further standardization in sample processing and reporting. Large-scale prospective studies are needed to refine risk stratification and optimize integration of viral load data into clinical algorithms. However, the copy number results bring therapeutic relevance to the diagnosis. Why LOD matters Most clinicians interpret PCR results as binary outcomes, unaware of underlying analytic thresholds. If a patient’s viral load is below an assay’s LOD, the virus may not be detected even if present. In early PML—when intervention has the greatest chance of preventing irreversible damage—viral loads often fall below 100 copies/mL. In such cases, the difference between detection thresholds of 10, 30, or 75 copies/mL is not just academic since it determines whether a diagnosis is made or missed. Variability and omission in JCV assay reporting practices dent diagnostic clarity and clinical confidence. Unless a physician is aware of inherent differences in assay reporting, a diagnosis of PML can be missed, and represents a preventable diagnostic failure. Assay Variability Across Labs: Qualitative, quantitative or ultrasensitive data? Commercial laboratories vary widely in their analytic capabilities and more importantly, their disclosure of results. For example, Quest, LabCorp and ARUP laboratories publish only qualitative LOD data but no LOQ data. Viracor Eurofins provides an LOQ of around 72 copies/mL, and an LOD of about 25 copies/mL. The Mayo clinic assay has an LOD around 10,000 copies/mL and no LOQ on the catalog page (Table 1, and supplement 1). The National Institutes of Health (NIH) deploys ultra-sensitive techniques as part of clinical research protocols detecting down to university laboratory reports an LOD of 10 copies/mL. This is not a complete or exhaustive list but highlights the main thrust of this essay. Discrepancies in conveying results between laboratories can be dangerous. In natalizumab-associated PML, multiple studies have shown that CSF JCV DNA levels can be exceedingly low in early disease—often copies/mL 4,5 . Warnke et al. demonstrated that even seropositive patients can have very low copy numbers at clinical onset 4 . Ryschkewitsch and colleagues validated multiplex qPCR techniques capable of detecting such low titers 5 . Nakamichi et al. showed how ultrafiltration of CSF prior to PCR significantly improves yield in suspected PML 6 . Droplet digital PCR (ddPCR) represents another promising advance, achieving detection down to single digit copy levels 7 . Among related techniques, real-time PCR allows for absolute quantification of the viral genome, providing information on the viral load which is not possible using PCR-Southern hybridization 8 . No regulatory or mandatory requirement to standardize reporting Despite these advances, there is no regulatory requirement under CLIA for laboratories to disclose LOD or LOQ values to clinicians. Often, these thresholds are buried in validation documents or withheld under claims of proprietary assay design. The result is a clinical blind spot and providers may erroneously assume that ‘not detected’ means absence of disease, which is not necessarily true. This becomes a critical point of failure in high-risk patients where early diagnosis can mean the difference between stabilization and catastrophic progression. The failure to mandate transparency transforms a laboratory detail into a diagnostic systems error. Clinical and Regulatory Consequences PML is a time-sensitive diagnosis. Earlier detection improves prognosis, and delayed recognition can lead to irreversible disability or death. An MRI may suggest PML, but confirmation still relies on PCR assay results. A brain biopsy and/or a clinical diagnosis are possible options but PCR testing has become the la référence absolue for diagnosis. Research has demonstrated both the feasibility and clinical importance of ultrasensitive detection methods but their availability limits use. Techniques such as CSF ultrafiltration and droplet digital PCR are operational and impactful but unless reporting of LOQ and LOD becomes standard of care practice, the diagnosis of PML can hang in the balance. Mandatory disclosure of analytic sensitivity is therefore both a diagnostic and regulatory imperative. Recommendations and future steps All laboratories offering JCV PCR testing must disclose assay LOQ and LOD on every result report. At a minimum, every laboratory must report their LOD cut-offs. Referring clinicians need this information to contextualize results and make informed decisions about repeat testing, imaging, or biopsy. National pathology societies and regulatory agencies should require transparency in diagnostic test performance for high-stakes infections like PML. What is currently framed as a laboratory reporting detail has a direct effect on patient outcomes. Diagnostic failure is preventable when transparency is followed or regulated. Until such disclosure is standardized, neurologists, oncologists, rheumatologists, and transplant physicians should proactively inquire about LOQ and LOD when interpreting negative JCV PCR results. Virologists and molecular pathologists must ensure their reports reflect the clinical realities of diseases they assay. A ”negative” result without sensitivity data is a diagnostic hazard. Beyond transparency in reporting, laboratories and regulatory bodies must recognize the potential medicolegal implications of current practices. Failure to disclose LOD and LOQ data may expose both laboratories and clinicians to litigation when diagnoses are missed due to false-negative results. In high-risk settings such as natalizumab, rituximab, or post-transplant immunosuppression interpreting a ‘negative’ JCV PCR without contextual performance data can be equated to clinical negligence. Lastly, neurology and infectious disease societies should issue joint guidance statements, updating criteria to clarify when repeat CSF testing or referral to a reference laboratory is warranted. References 1 Gullett JC, Nolte FS. Quantitative viral load testing in the clinical laboratory. Clin Chem. 2015;61(1):72-8. doi:10.1373/clinchem.2014.223289 2 Razonable RR, Hayden RT. Clinical utility of viral load testing for human viral infections. Clin Microbiol Rev. 2013;26(4):703-27. doi:10.1128/CMR.00015-13 3 Cardeñoso Domingo L, Roy Vallejo E, Zurita Cruz ND, et al. Clinical applications of viral load monitoring in chronic viral infections. J Med Virol. 2022;94(11):5260-70. doi:10.1002/jmv.27989 4 Warnke C, Stettner M, Lehmensiek V, et al. JC virus antibody index for diagnosis of natalizumab-associated PML. Ann Neurol. 2014;76(6):792-801. doi:10.1002/ana.24153 5 Ryschkewitsch CF, Jensen PN, Major EO. Multiplex qPCR assay for ultra-sensitive detection of JCV DNA. J Clin Virol. 2013;57(3):243-8. doi:10.1016/j.jcv.2013.03.009 6 Nakamichi K, Miyazaki D, Harada Y, et al. Improving detection of JC virus by ultrafiltration of cerebrospinal fluid before polymerase chain reaction for the diagnosis of progressive multifocal leukoencephalopathy. BMC Neurol. 2019;19:252. doi:10.1186/s12883-019-1476-2 7 Giovannelli I, Ciccone N, Vaggelli G, et al. Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples. J Clin Virol. 2016;82:70-5. doi:10.1016/j.jcv.2016.07.008 8 Ryschkewitsch C, Bergmann C, Gaeta AA, et al. Comparison of PCR-southern hybridization and quantitative real-time PCR for JCV detection. J Virol Methods. 2004;121(2):217-21. doi:10.1016/j.jviromet.2004.06.021 Supplementary Material File (table 1.docx) Download 481.74 KB Information & Authors Information Version history V1 Version 1 01 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords immune system infection jc virus nervous system pathogenesis reactivation virulence virus classification Authors Affiliations Jagannadha Avasarala 0000-0002-2322-503X [email protected] University of Kentucky Albert B Chandler Hospital View all articles by this author Metrics & Citations Metrics Article Usage 228 views 130 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jagannadha Avasarala. Analytical Performance Disclosure in JCV PCR: An Overdue Standard. Authorea . 01 September 2025. DOI: https://doi.org/10.22541/au.175670486.65949584/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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