# A rapid immunochromatographic assay for the detection of BK Polyomavirus in urine samples from hematopoietic stem cell transplant recipients

Etienne Brochot, Etienne Paubelle, Ophélie Fourdinier, Baptiste Demey, Aurélien Aubry, Virginie Morel, Antoine Touzé, Sandrine Castelain, François Helle

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Etienne Brochot, Etienne Paubelle, Ophélie Fourdinier, Baptiste Demey, Aurélien Aubry, et al. A rapid immunochromatographic assay for the detection of BK Polyomavirus in urine samples from hematopoietic stem cell transplant recipients. Journal of Virological Methods, 2026, 341, pp.115321. 10.1016/j.jviromet.2025.115321. hal-05411964

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Protocols

# A rapid immunochromatographic assay for the detection of BK Polyomavirus in urine samples from hematopoietic stem cell transplant recipients

Etienne Brochot<sup>a,b,*</sup>, Etienne Paubelle<sup>c,d</sup>, Ophélie Fourdinier<sup>b,e</sup>, Baptiste Demey<sup>a,b</sup>, Aurélien Aubry<sup>a,b</sup>, Virginie Morel<sup>a,b</sup>, Antoine Touzé<sup>f</sup>, Sandrine Castelain<sup>a,b</sup>, François Helle<sup>b</sup>

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## ARTICLE INFO

**Keywords:**

BKPyV

Hematopoietic stem cell transplant

Hemorrhagic cystitis

Antigenic assay

Point-of-care

## ABSTRACT

Hemorrhagic cystitis (HC) following hematopoietic stem cell transplantation (HSCT) is a significant event that can lengthen hospital stays and the need for care. The causes of HC can be multiple, but BK Polyomavirus (BKPyV) is the main protagonist in frequency. Currently, the clinical tool widely used to detect the presence of the virus in urine is PCR-based viral genome testing. We have developed a new rapid test for the antigenic detection of BKPyV in urine. The aim of this diagnostic study was to retrospectively evaluate the performance of this new assay as compared to BKPyV PCR on urine from HSCT patients with suspected HC. 49 samples from 49 different patients were evaluated, 20 of whom presented with HC. Of these 20 samples, 19 (95 %) were positive by the rapid antigen test. Of the 37 BKPyV PCR-positive samples, 31 were above 7 log₁₀ copies/mL, including the 20 patients with HC. Thus, the overall performance of the rapid test for HC is greatly improved compared with PCR, with specificity rising from 62.1 % to 86.2 % and positive predictive value from 64.5 % to 82.6 %. In conclusion, this new tool could be implemented as a point-of-care test to rapidly confirm or rule out a suspicion of BKPyV-HC after HSCT.

## 1. Introduction

BK polyomavirus (BKPyV) is a ubiquitous virus with a prevalence of around 90 % in the adult population worldwide (François et al., 2022). Four BKPyV genotypes further divided in subtypes exist with varying prevalence in different world regions (Morel et al., 2017). Some patients may experience a strong viral replication when they are immunode- pressed, especially after receiving a renal transplant or allogenic he- matopoietic stem cell transplantation (HSCT). In the case of HSCT, this may result in a Hemorrhagic cystitis (HC). Late-onset HC usually begins with neutrophil engraftment but can occur between 2 weeks and the first 6 months after HSCT. The first description of a correlation between BKPyV viruria and HC in patients receiving HSCT was made by Arthur and colleagues (Arthur et al., 1986). In terms of prevalence, BKPyV is the

main cause of HC.

Following an HSCT, BKPyV-HC remains a challenging complication, with an average prevalence of 13 % depending on the kind of transplant, procedure, and age of the patients (Chorão et al., 2025). BKPyV-HC usually manifests as microscopic hematuria (grade I), macroscopic he- maturia (grade II), macroscopic hematuria with clots (grade III) or macroscopic hematuria with clots, which impairs renal function due to urinary tract obstruction (grade IV) (Cesaro et al., 2018; Bedi et al., 1995). In their retrospective study of 323 stem cell transplant patients, Gilis and colleagues (Gilis et al., 2014) demonstrated that BKPyV-HC can lead to significantly longer hospitalization, more transfusions of packed red blood cells and more transfusions of platelets. In this way, BKPyV replication is often associated because of its high prevalence and uro- thelial tropism. For individuals who have had an HSCT, an elevated

<footnote><sup>a</sup> Department of Virology, Amiens University Medical Center, Amiens, France</footnote>

<footnote><sup>b</sup> Agents infectieux résistance et chimiothérapie Research Unit, UR4294, Jules Verne University of Picardie, Amiens, France</footnote>

<footnote><sup>c</sup> UR 4666, Jules Verne University of Picardie, Amiens, France</footnote>

<footnote><sup>d</sup> Department of Clinical Haematology and Cellular Therapy, Amiens University Hospital, Amiens, France</footnote>

<footnote><sup>e</sup> Department of Nephrology, Dialysis and Transplantation, Amiens University Medical Center, Amiens, France</footnote>

<footnote><sup>f</sup> Team "Biologie des Infections à Polyomavirus", UMR INRAE ISP 1282, University of Tours, Tours, France</footnote>

<footnote>* Corresponding author at: Laboratoire de Virologie, Centre de Biologie Humaine - CHU Amiens, Amiens cedex 1 F-80054, France.
E-mail address: etienne.brochot@u-picardie.fr (E. Brochot).</footnote>
<figure><img src="image_4.png" /><figcaption>Fig. 1. A: Protocol analysis flowchart. B: BKPyV antigen tests in cassette or strip format (1 highly positive, 1 negative and 1 positive test).</figcaption></figure>

BKPyV DNAuria rate of 7 log₁₀ genome copies/mL is linked to an increased risk of BKPyV-HC (Leung et al., 2001). As mentioned in the latest ECIL recommendations on BK polyomavirus-associated hemorrhagic cystitis, BKPyV-HC diagnosis requires the clinical and laboratory triad of cystitis, macrohematuria and high urine BKPyV loads but a significant role of other aetiologies must be excluded (Cesaro et al., 2018).

Therefore, identifying patients may be a crucial step in the preventive intervention for the development and management of HC. The only tool currently used to confirm or rule out the implication of BKPyV in hemorrhagic cystitis is urinary PCR (Priftakis et al., 2003). Two disadvantages of this technique seem to us to be disabling in this context. Firstly, the need to work in batch mode in most diagnostic laboratories, can take several days to obtain the results, limiting the speed of targeted therapeutic intervention. The second drawback is the very high sensitivity of the PCR technique and the high prevalence of the virus in patients' urothelium with many positive PCR results below the 7 log threshold (between 2 and 7 log₁₀ genome copies/mL), leading to a positive result for a large number of urinary samples, even in asymptomatic patients.

We have recently developed a new assay for the rapid and easy antigenic detection of all BKPyV subtypes in urine samples (Brochot et al., 2025). The aim of this project is to retrospectively evaluate the performance of this assay on urine from patients who have undergone HSCT, in order to detect the presence of BKPyV at a clinically relevant level in urine within a few minutes.

## 2. Materials and methods

### 2.1. Monoclonal antibody production and development of the lateral flow strip

The entire procedure for producing and selecting the monoclonal antibodies needed to manufacture the assay has been described previously (Brochot et al., 2025). Briefly, the selected F6 antibody is able to recognize all BK virus subtypes found worldwide. The F6 antibody was selected for its binding and affinity capacities on BKPyV-VP1 proteins. Due to the epitope repetition on viral capsid, it was used for capture and detection, and shows no cross-reactivity with the JC virus.

### 2.2. Lateral flow assay (LFA) procedure

To perform the LFA, 25 µL of urine samples were added to 25 µL Tris-HCl in order to buffer urinary pH. Pretreatment buffer (50 µL) was then added. The mixture was incubated at room temperature for 5 min before application to the sample window on the test strip. The result could be determined visually after 10 min of incubation at room temperature. BKPyV-positive samples result in the formation of a red-colored band on both the test and control lines, whereas negative samples produce a red-colored band only on the control line. The test is easy to read and was carried out by 2 independent readers.

### 2.3. Samples and patients

The urine samples used were kept at minus 80°C and came from patients who had undergone HSCT between July 01, 2020 and June 30, 2024, and for whom a BK virus PCR had been requested from the
<table><caption>**Table 1**

Characteristics of the study population.</caption><thead><tr><th></th><th>With BKPyV-HC<br>n = 20</th><th>Without BKPyV-HC<br>n = 29</th><th>p-value</th></tr></thead><tbody><tr><td>Recipient age<br>(mean ± SD)</td><td>50.4 ± 16.8</td><td>53.4 ± 16.9</td><td>NS</td></tr><tr><td>Recipient sex (M/F) (%/%)</td><td>17/3<br>(85/15)</td><td>18/11<br>(62/38)</td><td>NS</td></tr><tr><td>Previous HSCT, n (%)</td><td>0<br>20 (100)</td><td>1 (3)<br>29 (97)</td><td>NS</td></tr><tr><td>Type of transplant</td><td>0<br>9<br>11</td><td>4<br>13<br>12</td><td>NS</td></tr><tr><td>Geno-identical</td><td></td><td></td><td></td></tr><tr><td>Pheno-identical</td><td></td><td></td><td></td></tr><tr><td>Haplo-identical</td><td></td><td></td><td></td></tr><tr><td>Graft source</td><td></td><td></td><td>NS</td></tr><tr><td>Peripheral stem cells</td><td>19</td><td>25</td><td></td></tr><tr><td>BM</td><td>1</td><td>4</td><td></td></tr><tr><td>Conditionning, n (%)</td><td>2 (10)<br>18 (90)</td><td>2 (7)<br>27 (93)</td><td>NS</td></tr><tr><td>Myeloablative<br>Reduced intensity</td><td></td><td></td><td></td></tr><tr><td>HC grade</td><td></td><td></td><td></td></tr><tr><td>I</td><td>12</td><td>NA</td><td></td></tr><tr><td>II</td><td>6</td><td></td><td></td></tr><tr><td>III</td><td>2</td><td></td><td></td></tr><tr><td>IV</td><td>0</td><td></td><td></td></tr><tr><td>BKPyV subtype-subgroups (n,<br>(%))</td><td></td><td></td><td>NS</td></tr><tr><td>Ia/Ib1</td><td>5</td><td>3</td><td></td></tr><tr><td>Ib2</td><td>13</td><td>8</td><td></td></tr><tr><td>II</td><td>0</td><td>1</td><td></td></tr><tr><td>IV</td><td>2</td><td>2</td><td></td></tr></tbody></table>

NS: not significant.

NA: not applicable.

virology laboratory at Amiens University Hospital in a context of dysuria and mictional discomfort. The patient's clinical information for all available samples was then collected, and the presence or absence of post-transplant BK virus hemorrhagic cystitis was recorded in the physicians' letters and reports.

## 2.4. Quantification and genotyping of samples

Viral DNA was extracted from 200 µL of total urine using a NuclisENS easyMAG system (bioMérieux). The extracted DNA was recovered in 50 µL elution buffer and frozen at -80°C before use. BKPyV DNA was amplified using a quantitative real-time PCR assay kit (RealStar® BKV PCR Kit), as previously described (Descamps et al., 2015). The detection limit of this method is 1.85 log10 genome copies/mL BKPyV genotype was determined by sequencing the BKPyV virus typing and grouping region, as described previously (Morel et al., 2017).

## 2.5. Ethics

This retrospective study using patient samples was approved by the local institutional review board (reference: PI2022_843_0079). The study was conducted in accordance with the French national reference methodology (MR-004) and was recorded in Amiens University Medical Center's registry of data-processing activities, in accordance with Article 30 of the European Union's General Data Protection Regulation.

# 3. Results

## 3.1. Study design and patient characteristics

In order to evaluate the diagnostic performance of the rapid anti-genic test for HC diagnosis in comparison with the commonly used BKPyV PCR technique, we retrospectively analyzed all frozen preserved urine samples from July 01, 2020, over a 4-year period (Fig. 1A). These

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** BKPyV DNAuria (log₁₀ genome copies/mL) of 49 samples according to antigenic BKPyV test result (LFA: lateral flow assay) and according to presence or absence of hemorrhagic cystitis. The dotted line corresponds to the threshold of 7 log₁₀ genome copies/mL. HC: Hemorrhagic cystitis.</figcaption></figure>

samples had to be from hematopoietic stem cell transplant patients with a request for urinary BKPyV PCR in a context of dysuria and mictional discomfort for suspected HC. The first sample available for each patient, taken at least 7 days post-transplant, was analyzed. During this period, 215 patients had undergone HSCT, and BKPyV genome testing was requested for 57 of them. For these 57 patients, 49 urine samples were still available (median time from HSCT being 33 days (IQR 25–56 days), 37/49 (75.5 %) had a BKPyV PCR above the LOD and 20/49 (41 %) had documentation of hemorrhagic cystitis. The rapid antigen detection test was performed for these 49 samples according to the procedure described in materials and methods. We obtained 23 positive results (47 %) using this technique, including 19/20 patients (95 %) with documentation of HC at least day 7 post-transplant. A visualization of LFA in strip or cassette format is shown in Fig. 1B. The characteristics of the 49 patients are given in Table 1. Comparing patients with and without HC, we found no significant differences in patient demographics, graft type or BKPyV viral subtypes.

## 3.2. Performance of the BKPyV LFA in HC diagnosis

The amount of viral DNA found in the urine samples (DNAuria in log₁₀ genome copies/mL) of the 49 patients are shown in Fig. 2, according to the result of the LFA and the presence or absence of HC. The mean urine BKPyV DNA was 4.23 ± 3.9 and 9.20 ± 0.6 log₁₀ genome copies/mL for patients with a negative and positive LFA, respectively. 31 samples (63.4 %) had a PCR greater than 7 log₁₀ genome copies/mL, i.e. the 20 patients with HC but also 11 patients without any sign of HC documented in letters or transmissions from physicians. The performance of the LFA in HC diagnosis was therefore assessed in comparison with positive urinary PCR (above the limit of detection) or PCR above 7 log₁₀ genome copies/mL (Table 2). The sensitivity of the LFA for HC diagnosis was 95 % (19/20) as compared to 100 % for the PCR. One patient in the cohort presented a negative rapid test with HC. Viral PCR was 8.01 log₁₀ genome copies/mL. However, the specificity for detecting HC was much higher for the LFA (86.2 % (25/29) versus 62.1 % (18/29) for PCR above 7 log₁₀ genome copies/mL). Thus, the Youden index (sensitivity + specificity – 1) was in favor of LFA (0.81 as compared to 0.62 and 0.41 for the PCR above 7 log₁₀ genome copies/mL and the positive PCR, respectively). We also calculated the agreement and the Kappa index (89.8 % and 0.794 ± 0.087 respectively for LFA). Similarly, these two values were largely in favor of the LFA, when compared to PCR above 7 log₁₀ genome copies/mL thresholds (77.6 % and 0.572 ± 0.103 for agreement and Kappa index respectively) (Table 2). The closer the Kappa index value is to 1, the greater the concordance of results. A value between 0.61 and 0.8 is considered to be a substantial agreement (Landis and Koch, 1977). In addition, whereas the negative predictive values were similar for LFA and PCR (96.2 % (25/26) and
<table><caption>**Table 2**
Test performance for detecting hemorrhagic cystitis.</caption><thead><tr><th></th><th>Sensitivity</th><th>Specificity</th><th>Youden's index</th><th>PPV</th><th>NPV</th><th>Agreement (n, %)</th><th>Kappa ± SE</th></tr></thead><tbody><tr><td>Quantitative PCR<br/>> LOD</td><td>100</td><td>41.4</td><td>0.41</td><td>54.1</td><td>100</td><td>32 (65.3 %)</td><td>0.366 ± 0.096</td></tr><tr><td>Quantitative PCR<br/>> 7 log<sub>10</sub> genome copies/mL</td><td>100</td><td>62.1</td><td>0.62</td><td>64.5</td><td>100</td><td>38 (77.6 %)</td><td>0.572 ± 0.103</td></tr><tr><td>LFA</td><td>95</td><td>86.2</td><td>0.81</td><td>82.6</td><td>96.2</td><td>44 (89.8 %)</td><td>0.794 ± 0.087</td></tr></tbody></table>

LOD: limit of detection.

LFA: lateral flow assay.

PPV: positive predictive value.

NPV: negative predictive value.

100% (18/18), respectively), the positive predictive value of the LFA (82.6% (19/23)) was much higher than that of the PCR above 7 log₁₀ genome copies/mL threshold (64.5% (20/31)). Overall, our results show that the LFA developed could be very useful to clinicians and/or diagnostic laboratories for rapid screening of HC after HSCT either to rule out the main viral cause of hemorrhagic cystitis or for rapid management of BKPyV.

## 4. Discussion

In this study, we evaluated the performance of the first rapid antigenic screening test for urinary BKPyV in the context of BKPyV-HC. No other causes of hemorrhagic cystitis were identified in the patient cohort. Compared with the commonly used PCR technique, we were able to demonstrate greatly increased specificity and positive predictive value, while retaining very good sensitivity. The test is easy, quick to perform, less expensive than qPCR and can be used as a point-of-care test to confirm or exclude BKPyV as a cause of HC, enabling rapid therapeutic intervention. We analyzed PCR turnaround times for these 49 samples in our center, and found that the average turnaround time was 74 h (± 30 h), i.e. over 3 days (data not shown). In diagnostic laboratories, it is not possible to carry out PCRs for the different viruses and matrices every day, and this requires serial work, lengthening turnaround times and therapeutic intervention. Depending on the activity of the diagnostic laboratory, this turnaround time can vary widely. Compared with the few minutes for carrying out an antigen test, this new tool can save precious time in patient management, enabling rapid intervention to limit the consequences of HC, or, in the event of a negative result, to rapidly investigate another etiology.

PCR technology remains an expensive technique (reagents, equipment and personnel) for screening purposes only, to determine the cause of HC. Clinicians may also be perturbed by a weakly positive urinary BKPyV PCR (< 7 log₁₀ copies/mL), probably reflecting a long history of viral replication, with a significant presence of viral genome in urothelial cells, which may exfoliate and produce a positive PCR signal without effective viral replication at the time of genome detection. We also reported 7 out of 31 patients with a PCR > 7 log₁₀ genome copies/mL and a negative LFA without any sign of haemorrhagic cystitis, which may suggest that high DNAuria are not indicative of replication. Leuzinger et al. on plasma samples. PCR detects in large majority BKPyV genome which is largely free and not encapsidated and thus does not objectively indicate viral replication (Leuzinger et al., 2019).

One patient in the cohort presented a negative rapid test with HC. Viral PCR was 8.01 log₁₀ genome copies/mL. We sequenced using ultra deep sequencing technology the BKPyV VP1 region, the antigenic recognition site of the monoclonal antibody produced, and did not identify any mutations that could impact this recognition. The explanation could be poor storage of this sample or multiple freezing and thawing cycles and we cannot rule out another etiology, viral or non-viral, with an elevated BKPyV PCR, as demonstrated in this study and many others.

The limitations of our study are the retrospective and mono-centric nature, as well as the limited number of samples. However, the

frequency of HC, the high prevalence of the viral genome detected by PCR in patients' urine and the unpredictability of cystitis required a multi-centric prospective study of sufficient duration to obtain sufficient data. Certainly, the use of fresh samples could enhance even further the performance of this assay.

In conclusion, our results suggest potential diagnostic value of a new rapid antigenic screening tool for BKPyV in the urine of HSCT patients, enabling rapid orientation and therapeutic action in cases of suspected HC. These results need to be confirmed in a larger and prospective study. In the future, this new test could be implemented as a triage tool in the diagnostic process, either in diagnostic laboratories or as point of care test.

## CRediT authorship contribution statement

**Etienne Brochot:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. **Etienne Paubelle:** Writing – original draft, Methodology, Data curation. **Fourdinier Ophelie:** Writing – original draft, Data curation. **Baptiste Demey:** Writing – original draft, Validation, Investigation. **Aurélien Aubry:** Writing – original draft, Formal analysis. **Virginie Morel:** Writing – original draft, Validation. **Antoine Touzé:** Writing – review & editing, Writing – original draft. **Sandrine Castelain:** Writing – review & editing, Writing – original draft. **François Helle:** Writing – review & editing, Writing – original draft, Conceptualization.

## Funding

Etienne Brochot and Antoine Touzé reports financial support was provided by C-Valo (Tours, France).

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Brochot Etienne, Antoine Touze has patent #NEW ANTIBODY TARGETING THE VP-1 PROTEIN, FRAGMENTS THEREOF, AND USES OF SAME FOR DETECTING INFECTION WITH THE BK POLYOMAVIRUS issued to US2022396612 (A1). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data Availability

Data will be made available on request.

## References

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