# Performance of a commercial assay for detecting JC
Polyomavirus DNA in human samples

Sébastien Lhomme, Isabelle da Silva, Oriane Cabanel, Estelle Raguin, Guillaume Martin-Blondel, Nassim Kamar, Jacques Izopet, Florence Abravanel

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Sébastien Lhomme, Isabelle da Silva, Oriane Cabanel, Estelle Raguin, Guillaume Martin-Blondel, et al.. Performance of a commercial assay for detecting JC Polyomavirus DNA in human samples. Journal of Clinical Virology, 2025, 180, pp.105849. 10.1016/j.jcv.2025.105849. hal-05514492

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# Performance of a commercial assay for detecting JC Polyomavirus DNA in human samples

Sébastien Lhomme<sup>a,b,*</sup>, Isabelle Da Silva<sup>a</sup>, Oriane Cabanel<sup>a</sup>, Estelle Raguin<sup>a</sup>, Guillaume Martin-Blondel<sup>b,c</sup>, Nassim Kamar<sup>b,d</sup>, Jacques Izopet<sup>a,b</sup>, Florence Abravanel<sup>a,b</sup>

## ARTICLE INFO

### Keywords:

JC Polyomavirus

Polyomaviridae

Altona

Automated assay

## ABSTRACT

**Introduction:** We evaluated the performance of the automated AltoStar JCV PCR platform for detecting and quantifying JC Polyomavirus (JCPyV) DNA in samples including urine, plasma and cerebrospinal fluid.

**Methods and results:** Using the NIBSC JCPyV DNA standard, the assay was linear from 1 to 6 log IU/mL standard and the limit of detection determined by Probit analysis corresponded to 9.3 [95 % CI: 7.0–16.5] IU/mL. Specificity was accessed by testing urines containing a high concentration of BK Polyomavirus; none tested positive for JCPyV. The intra-run and inter-run standard deviations were 0.02 IU/mL and 0.35 IU/mL, respectively. Lastly, clinical performance was determined by testing 45 samples quantified previously with a laboratory-developed test (LDT). The assays were concordant for 42/45 samples. One of the 3 samples that tested negative with the AltoStar assay had a low JCPyV DNA concentration. We were unable to re-test the 3 negative samples due to insufficient volume. A conservation problem could not be ruled out for the 3 samples with discordant results.

**Conclusions:** The AltoStar platform enables highly accurate testing for the detection and quantification of JCPyV DNA with very low limit of detection. This allowed us to shorten turnaround times and save time for technical staff.

## 1. Background

JC Polyomavirus (JCPyV) belongs to the *Polyomaviridae* family. It is a non-enveloped virus with circular double-stranded DNA with approximately 5100 base pairs. JCPyV is excreted in urine but also found in blood and cerebrospinal fluid (CSF) [1]. JCPyV is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the brain caused by the lytic infection of oligodendrocytes upon viral reactivation [1]. PML has mostly been described in patients living with human immunodeficiency virus (HIV), patients with haematological malignancies or chronic inflammatory diseases [2]. More recently, PML was also reported as an opportunistic infection occurring during the use of immunomodulatory therapies, including monoclonal antibodies [3].

To date, there are two pathways used for diagnosing PML proposed by the American Academy of Neurology. One is based on

histopathological evidence: demyelination, bizarre astrocytes and enlarged oligodendroglial nuclei coupled with techniques to reveal the presence of JCPyV whereas the second pathway requires the presence of clinical and radiological features consistent with the diagnosis, together with JCPyV DNA detection in CSF [4]. Therefore, detecting JCPyV DNA is crucial for the diagnosis of PML. Detecting JCPyV DNA with a very sensitive assay is of prime importance since the viral load in the CSF can be very low [5]. Recent published works also highlight the benefits of a quantitative test. A viral load higher than 2.7 log copies/mL in the CSF was strongly suggestive of PML [6]. The decrease in JCPyV DNA concentrations was associated with survival in the National Institutes of Health (NIH) natural history study cohort [7]. Lastly, quantitative assays are also useful for predicting the survival of patients receiving either recombinant human IL-7 (rhIL-7) or immune checkpoint inhibitors [8, 9].

<footnote><sup>a</sup> Laboratoire de Virologie, Centre Hospitalier Universitaire de Toulouse, 31300 Toulouse, France</footnote>

<footnote><sup>b</sup> Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), CNRS U5051, INSERM U1291, Université Toulouse III, Toulouse, France</footnote>

<footnote><sup>c</sup> Service des Maladies Infectieuses et Tropicales, Centre Hospitalier Universitaire de Toulouse, 31300 Toulouse, France</footnote>

<footnote><sup>d</sup> Service de Néphrologie et Transplantation d'Organes, Centre Hospitalier Universitaire de Toulouse, 31300 Toulouse, France</footnote>

<footnote>* Correspondence to: Laboratoire de Virologie, Institut Fédératif de Biologie, 330, Avenue de Grande Bretagne, 31300 Toulouse, France.</footnote>

<footnote>E-mail address: lhomme.s@chu-toulouse.fr (S. Lhomme).</footnote>
## 2. Objectives

We evaluated the performance of a commercial assay, the AltoStar® JCV PCR Kit 1.5, for detecting and quantifying JCPyV in urine, blood and CSF. Linearity, reproducibility, sensitivity, specificity and analytical performances in clinical specimens previously quantified with a laboratory-developed test (LDT) were all assessed.

## 3. Study design

### 3.1. Clinical samples and quality assurance reference samples

The National Institute for Biological Standards and Control (NIBSC) WHO reference standard (NIBSC code 14/114), genotype 1A, was used to determine the linearity and sensitivity of the assay. Anonymous samples (EDTA plasma (n = 4), urine (n = 10) and cerebrospinal fluid (n = 12)) collected from patients infected with JCPyV were tested. In addition, 19 samples from the Quality Control for Molecular Diagnosis (QCMD, Glasgow, Scotland) programme mimicking plasma were analysed; ten samples were genotype 1A and 9 were 1B. Ten urine samples which tested positive for BK polyomavirus (BKPyV) were used to conduct the specificity analysis.

### 3.2. Altostar assay

Nucleic acids were extracted with the Altostar AM16 instrument (Altona Diagnostics, Hamburg, Germany) and amplification was performed on the Light Cycler CFX96 (Bio-Rad) using the AltoStar JCV PCR Kit 1.5 according to the manufacturer's instructions (extracted sample volume: 500 µL; elution volume: 80 µL). This assay targets a region encoding the small t antigen conserved among all JCPyV genotypes. The required eluate volume for the PCR reaction using the AltoStar JCV PCR Kit 1.5 is 10 µL. The assay includes oligonucleotides for amplification and detection of the IC (Altostar Internal Control 1.5). The IC is automatically added at the beginning of the nucleic acid purification procedure on the Altostar AM16.

### 3.3. Quantitative JCPyV DNA PCR assay – comparison assay

The clinical samples were previously detected using a LDT targeting a conserved large T protein sequence adapted from Ryschkewitsch et al. [10]. No internal control was included. Briefly, nucleic acids were extracted with the MagNAPure96 instrument using the MagNAPure96 DNA and Viral NA Small Volume Kits (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions (extracted sample volume: 200 µL; elution volume: 100 µL). The primers used were JCV-T-3 and JCV-T-4 and the locked nucleic acid (LNA) probe was JCT-1.2. The 40 µL PCR reaction mix contained 15 µL ProbeMaster mix (Roche Diagnostics, Meylan, France), 15 µL extracted DNA, primers (500 nM), and probe (400 nM). The PCR was performed with the LightCycler480.

The standard curve was generated by using serial dilutions of the International Standard for JCPyV DNA (NIBSC code: 14/114) (ten fold dilution from $10^6$ to 10 IU/mL).

## 4. Results

### 4.1. Linearity

The linearity of the AltoStar JCV PCR Kit 1.5 was assessed with 10-fold serial dilution of the NIBSC standard (14/114). The regression analysis between the mean AltoStar assay value of each diluted sample and the expected values is presented in Fig. 1. The correlation coefficient was 0.98. The assay was linear from 1 to 6 log IU/mL.

<figure><img src="image_5.png" /><figcaption>Fig. 1. AltoStar JCPyV DNA assay linearity. Three replicates of each dilution were tested.</figcaption></figure>

### 4.2. Limit of detection and accuracy

The LOD for the AltoStar JCV PCR Kit 1.5 was determined by testing serial dilutions of the NIBSC standard. Each dilution was analysed using 5–10 replicates (Table 1). Probit analysis gave a limit of detection of 9.3 [95 % CI: 7.0–16.5] IU/mL. The bias ranged from –0.3 Log IU/mL for the lowest value (2.5 IU/mL) to 0.09 for the 6 log IU/mL.

### 4.3. Specificity

The specificity was assayed by testing urine samples containing a high DNA concentration of BKPyV, another member of the *Polyomaviridae* family frequently found in urine. All the BKPyV positive urine samples (median DNA concentration = 5.62 log IU/mL, range: 3.03–10.72 log IU/mL) tested negative for JCPyV DNA with the AltoStar JCV PCR Kit 1.5.

### 4.4. Reproducibility

The intra-run standard deviation (SD) was 0.02 log IU/mL for a sample of 3.5 IU/mL tested in 5 replicates over a single run. The inter-run SD was 0.35 log IU/mL for a sample (mean value 2.9 IU/mL) tested on 87 replicates in different runs. This inter-run value was influenced by the use of different batch of reagents.

### 4.5. Analytical performances in clinical and quality control specimens

The overall agreement for positive samples was 93.3 % (42/45). One CSF which tested positive with the LDT was negative with Altostar. It corresponded to a sample with a concentration of 10 IU/mL. In addition, 2 QCMD (366 IU/mL and 2420 IU/mL, both genotype 1B) samples tested negative with Altostar. The mean difference between the Altostar and the LDT results was –1.637 log IU/mL (Fig. 2).

<caption>**Table 1**</caption>

Analytical sensitivity of the Altostar JCPyV assay using the NIBSC reference standard (#14/114) and accuracy.

<table><thead><tr><th colspan="2">Sample concentration</th><th colspan="3">Altostar</th></tr><tr><th>IU/mL</th><th>Log IU/mL</th><th>No. of samples detected/No. tested</th><th>Mean Log IU/mL</th><th>Bias</th></tr></thead><tbody><tr><td>10<sup>6</sup></td><td>6</td><td>5/5</td><td>5.91</td><td>0.09</td></tr><tr><td>10<sup>5</sup></td><td>5</td><td>5/5</td><td>4.94</td><td>0.06</td></tr><tr><td>10<sup>4</sup></td><td>4</td><td>5/5</td><td>3.89</td><td>0.11</td></tr><tr><td>10<sup>3</sup></td><td>3</td><td>5/5</td><td>2.94</td><td>0.06</td></tr><tr><td>10<sup>2</sup></td><td>2</td><td>5/5</td><td>2.16</td><td>-0.16</td></tr><tr><td>10</td><td>1</td><td>9/10</td><td>1.12</td><td>-0.12</td></tr><tr><td>5</td><td>0.7</td><td>8/10</td><td>1.03</td><td>-0.33</td></tr><tr><td>2.5</td><td>0.4</td><td>3/10</td><td>0.7</td><td>-0.30</td></tr><tr><td>1</td><td>0</td><td>0/10</td><td>n.a</td><td>n.a</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>Fig. 2. Bland-Altman plot for bias analysis between Altostar and laboratory-developed test (LDT) results.</figcaption></figure>

## 5. Discussion

The analytical evaluation of the AltoStar JCPyV PCR Kit demonstrated satisfying results for sensitivity, specificity, linearity and accuracy. The automated extraction and PCR set-up process frees up time for technical staff and avoids sample inversion.

The mean bias between the LDT and Altostar assay was -1.637 log IU/mL. During external quality control, overestimation occurred frequently with our LDT. Since concordance between the theoretical values and concentrations obtained with the Altostar assay was high, quantification with this particular assay is more accurate. A recent study found that a positive JCPyV DNA concentration ≥2.7 log copies/mL was strongly indicative of PML with a positive predictive value of 92.3 % [6]. However, detection of JCPyV DNA in the CSF of PML cases remains inconstant: a recent analysis showed that PCR were positive in 152/174 cases of PML, corresponding to a clinical sensitivity rate of 87.4 % (95 % CI: 81.6–91.5 %) [6]. The negative results could at least be partly explained by a lack of sensitivity of the assay, a pitfall overcome by the Altostar assay. The announced LOD was 3.6 IU/mL (95 % confidence interval: 2.5–6.6 IU/mL), which is close to the one found in our study. The assay developed by Ryschkewitsch has a sensitivity of 10 copies/mL [10]. Correspondence between copies/mL and IU/mL is yet to be determined with the help of the WHO standard to compare the assays.

We found an inter-run SD of 0.35 log IU/mL for a mean target value of 2.9 IU/mL. A recent analysis suggests that variation as low as 0.25 log copies/mL by Day 60 was more commonly associated with survival in the NIH natural history study cohort [7]; such a cut-off value should take into consideration the inter-run SD. In medical laboratories, an inter-run SD <0.5 Log IU/mL is required. Lastly, the agreement was 42/45 for positive samples. Re-testing the 3 negative samples was not possible due to insufficient volume. A conservation problem could not be ruled out. More samples and different genotypes should be tested. Testing brain biopsies would be also interesting but this material is rarely collected due to invasiveness of the sampling method.

To conclude, the Altostar assay is a valuable tool for the diagnosis and follow-up of patients infected with JCPyV. No assay has yet been recommended by expert societies for the clinical management of

patients but the use of a commercial assay calibrated to the WHO reference standard could be helpful for comparing the results of different cohorts that usually contain a small number of cases.

## CRediT authorship contribution statement

**Sébastien Lhomme**: Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization. **Isabelle Da Silva**: Methodology, Formal analysis, Data curation. **Oriane Cabanel**: Investigation, Formal analysis. **Estelle Raguin**: Investigation, Formal analysis. **Guil-laume Martin-Blondel**: Writing – review & editing, Investigation. **Nassim Kamar**: Writing – review & editing, Investigation. **Jacques Izopet**: Writing – review & editing, Validation. **Florence Abravanel**: Writing – review & editing, Validation, Supervision.

## Funding

This research did not receive any specific grants from funding agencies in the public, commercial or not-for-profit sectors.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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