# Performance of an automated platform for CMV, EBV and BKPyV DNA detection and quantification

Yoann Fluhr, Karine Saune, Kevin Oliveira-Mendes, Estelle Raguin, Lila Poiteau, Arnaud del Bello, Nassim Kamar, Jacques Izopet, Florence Abravanel

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Yoann Fluhr, Karine Saune, Kevin Oliveira-Mendes, Estelle Raguin, Lila Poiteau, et al. Performance of an automated platform for CMV, EBV and BKPyV DNA detection and quantification. Journal of Clinical Virology Plus, 2025, 5, 10.1016/j.jcvp.2025.100228. hal-05523500

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# Performance of an automated platform for CMV, EBV and BKPyV DNA detection and quantification

Yoann Fluhr<sup>a</sup>, Karine Saune<sup>a,b</sup>, Kevin Oliveira-Mendes<sup>a</sup>, Estelle Raguin<sup>a</sup>, Lila Poiteau<sup>a</sup>, Arnaud Del Bello<sup>c</sup>, Nassim Kamar<sup>b,c</sup>, Jacques Izopet<sup>a,b</sup>, Florence Abravanel<sup>a,b,*</sup><sup>D</sup>

## ARTICLE INFO

**Keywords:**

CMV

EBV

BKPyV

Molecular quantification

## ABSTRACT

Quantification of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK polyomavirus (BKPyV) DNA is crucial for diagnosing related infections. The analytical performance of the Altostar® CMV, EBV and BKPyV DNA assays used in the AltoStar® AM16 instrument was assessed.

The Limit of detection was 215 international units/ml (IU/ml) for the CMV assay, 330 IU/ml for the EBV assay and 133 IU/ml for the BKPyV assay. A good concordance was found between the nominal value of the NIBSC standards and the AltoStar® results. Linearity was verified up to 5.7 log₁₀ IU/ml. Intra-assay and inter-assay reproducibility showed low disparity with a standard deviation < 0.27 log₁₀ IU/ml.

The concordance with our previous DNA assays on clinical samples for CMV detection was 95.5% (n = 22), 90.6% for EBV (n = 32) and 92% for BKV (n = 25). The quantitative results were highly correlated.

The Altostar® platform provides accurate quantification for clinical monitoring of CMV, EBV and BKPyV DNA.

## 1. Introduction

Individuals who have undergone transplantation and require ongoing immuno-suppression to maintain the functioning of the transplanted organ are vulnerable to viral infections or the reactivation of latent viruses. Among these infections, cytomegalovirus (CMV) infection is a serious cause of morbidity and mortality in hematopoietic stem cell and solid organ transplant recipients [1]. Typically emerging within the initial months post-transplant, CMV leads to noticeable clinical infections such as fever, pneumonia, gastrointestinal disorders, hepatitis, and retinitis, and necessitates CMV DNA detection in different clinical samples. Moreover, it contributes to both short-term and long-term graft malfunction. Congenital cytomegalovirus is the most common congenital viral infection with an estimated prevalence of 0.2%-5% worldwide [2]. In these cases, various biological samples may be of interest for CMV, such as amniotic fluid samples [3]. A urine or saliva sample is preferable to a blood sample to confirm a diagnosis of congenital CMV because the viral load is often significantly higher [3]. The Epstein-Barr virus (EBV) is a widely prevalent viral pathogen, with more than 90% of adults having antibodies against it. Following initial infection, the virus

establishes a lifelong presence within B lymphocytes. However, EBV-related post-transplant lymphoproliferative (PTLD) disorders are recognized as a significant cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation or who are solid organ recipients [4]. The introduction of new approaches for EBV disease/PTLD, including EBV monitoring by PCR, pre-emptive therapy and timely treatment with rituximab, has considerably improved outcomes. Primary BK polyomavirus (BKPyV) infections often occur during early childhood in a significant proportion of individuals, and usually manifest without noticeable symptoms. As an individual ages, the virus remains inactive (latent) within the kidneys. Instances of illness resulting from the BKPyV virus are largely observed in individuals undergoing immunosuppressive treatment following an organ transplant or those who have acquired immune deficiencies. BKPyV is estimated to contribute to a gradual deterioration of kidney function in approximately 1-10% of kidney transplant recipients [5]. To prevent these viral complications, clinicians need to monitor the viral load of these viruses in order to treat any reactivation or primary infection as quickly as possible.

<footnote><sup>a</sup> CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, National Reference Center for Hepatitis E, 31300 Toulouse, France</footnote>

<footnote><sup>b</sup> Inserm UMR 1291, CNRS UMR5051, Université Toulouse III, Toulouse, France</footnote>

<footnote><sup>c</sup> CHU Toulouse, Hôpital Rangueil, Department of Nephrology and Organ Transplantation, Toulouse, France</footnote>

<footnote>* Corresponding author.
E-mail address: abravanel.f@chu-toulouse.fr (F. Abravanel).</footnote>
## 2. Objectives

Viral load quantification is a technique that is now effectively automated in laboratories. In France, medical laboratories must validate the performance of analytical assays according to the ISO15189 standard. In this study, we assessed the performance of the Altostar® CMV PCR, EBV PCR and BKPyV PCR kits used in the Altostar® AM16 instrument for CMV, EBV and BKPyV DNA detection and quantification developed by Altona Diagnostics. The intra-assay and inter-assay reproducibility, linearity, accuracy and limit of detection were investigated. The correlation with the previous assays used in our lab was also assessed.

## 3. Materials and methods

First, we assessed the analytical performance of the Altostar® assays. Then, clinical performance was determined by comparison with the previous assays used in our lab.

### 3.1. Altostar instruments and assays

The Altostar® AM16 automation system is an automated system for DNA extraction and purification and PCR setup (Altona Diagnostics, Hamburg, Germany). Analysis takes place in 3 steps: a purification run on the AltoStar® AM16 using the AltoStar® Whole Blood Pretreatment Buffer 1.5 for whole blood samples and AltoStar® Purification Kit 1.5, a PCR configuration run on the AltoStar® AM16 using the AltoStar® PCR Kit 1.5, and to finish, a real-time PCR run on a CFX96TM DW Dx. AltoStar® CMV PCR Kit 1.5, EBV PCR Kit 1.5, and BKPyV PCR Kit 1.5 are in vitro diagnostic tests based on real-time PCR technology, for virus-specific DNA detection and quantification. The kits also contain an internal control and quantification standards. They have been calibrated in accordance with the 1st WHO standard for CMV, EBV and BKPyV DNA (NIBSC Code: 09/162 for CMV; NIBSC Code: 09/260 for EBV and NIBSC Code: 14/212 for BKPyV). We performed the tests according to the manufacturer's instructions.

### 3.2. Limit of detection, accuracy and range of linearity

We used the WHO International Standards provided by the NIBSC. Serial dilutions of the WHO international standards were prepared in negative plasma and tested. Analytical sensitivity (limit of detection: LoD) is defined as the concentration in international units/ml (IU/ml) of DNA molecules specific for CMV, EBV or BKPyV that can be detected with a positivity rate ≥ 95 %.

### 3.3. Precision

Intra-assay reproducibility was assessed by testing 5 replicates of whole blood samples with positive pools for CMV (mean CMV DNA=4.6 log₁₀ IU/ml), BKPyV (mean BKPyV DNA=3 log₁₀ IU/ml), or EBV (mean EBV DNA=3.6 log₁₀ IU/ml). Inter-assay reproducibility was assessed by quantifying 50 replicates of positive whole blood samples with positive pools for CMV, BKPyV or EBV (one per day).

### 3.4. Method comparison

Samples were collected between July and September 2022 and were stored frozen at -20°. They were then tested in October 2022 to validate the performance. The Altostar® results were compared with the previous assays used in the laboratory.

Viral DNA extraction was run on the MagNA Pure 96 instrument using the Small Volume 2.0 kit (Roche Diagnostics, Mannheim, Germany) (200 µl of sample eluted in 100 µl). The EBV DNA quantification was performed with the Real Star EBV PCR KIT 1.2 (Altona Diagnostics) for EBV. CMV and BKPyV DNA quantification were compared with our

home brew polymerase chain reaction (PCR) assays as previously described [6]. For CMV, we compared 18 whole blood samples, 1 urine sample, 1 saliva sample, 1 feces sample, 1 amniotic fluid sample, 1 bronchoalveolar lavage sample and 2 intestinal biopsies. For EBV, we used 25 whole blood samples, one bronchoalveolar lavage sample, one bone marrow sample and three throat swabs. For BKPyV, we used 16 whole blood samples and 9 urine samples. All pairwise assay comparisons used log₁₀ transformed results that were within the linear range for both tests and Bland-Altman plot including the 95 % limits of agreement and the 95 % confidence intervals. There were no additional procedures in this non-interventional study. Biological material and clinical data were obtained only for standard viral diagnosis following the physician's orders (no specific sampling, no modified sampling protocol, no questions in addition to the national standardized questionnaire). The patients were included in the Nephrogen cohort (Ethical approval was obtained from the institutional review board at Toulouse University Hospital (RC31/21/0154).

### 3.5. Statistical analyses

All Statistical analyses were performed with the XLStat software. The calculation of the limit of detection (LoD) was performed using a probit analysis (including 95 % confidence intervals). Bland-Altman and Passing-Bablok tests were done to compare quantitative values obtained for blood or urine samples only.

## 4. Results

### 4.1. Limit of detection and accuracy

We used the international standards provided by the NIBSC to assess the limit of detection by probit analysis (Table 1). The LOD determined was 215 IU/ml (95 %CI: 0–6539 IU/ml) for the CMV PCR assay, 330 IU/ml (95 % CI: 0–1031 IU/ml) for the EBV PCR assay and 133 IU/ml (95 % CI: 87 – 178 IU/ml) for the BKPyV PCR assay. Accuracy for each Altostar® PCR assay is presented in Table 2.

### 4.2. Linearity

The linearity was verified from 2.7 to 5.7 log₁₀ UI/ml for CMV using NIBSC 09/162, from 2.5 to 5.7 log₁₀ UI/ml for BKPyV and with NIBSC 09/260 for EBV and from 1.0 to 5.0 log₁₀ UI/ml with NIBSC 14/212 for EBV (Fig. 1).

### 4.3. Precision

For the Altostar® CMV DNA assay, the intra-run standard deviations for the Altostar® CMV DNA, EBV DNA and BKPyV assays was 0.03 IU/

<table><caption>**Table 1**
Limit of detection of the Altostar assays.</caption><thead><tr><th>Virus</th><th>NIBSC WHO standard concentration (IU/ml)</th><th>Number of Positive replicates</th></tr></thead><tbody><tr><td rowspan="5">CMV</td><td>1000</td><td>2/2</td></tr><tr><td>500</td><td>5/5</td></tr><tr><td>300</td><td>5/5</td></tr><tr><td>250</td><td>5/5</td></tr><tr><td>200</td><td>2/5</td></tr><tr><td rowspan="5">EBV</td><td>1000</td><td>2/2</td></tr><tr><td>500</td><td>5/5</td></tr><tr><td>400</td><td>5/5</td></tr><tr><td>300</td><td>2/5</td></tr><tr><td>200</td><td>0/5</td></tr><tr><td rowspan="4">BKPyV</td><td>250</td><td>5/5</td></tr><tr><td>100</td><td>4/5</td></tr><tr><td>50</td><td>3/5</td></tr><tr><td>20</td><td>1/5</td></tr></tbody></table>
<table><caption>**Table 2**
Accuracy results determined with WHO reference standards.</caption><thead><tr><th>Virus</th><th>Nominal log<sub>10</sub> titer</th><th>Observed log<sub>10</sub> IU/mL</th><th>Difference observed with nominal log<sub>10</sub> IU/mL (bias)</th><th>Standard Deviation log<sub>10</sub> IU/mL</th></tr></thead><tbody><tr><td rowspan="4">CMV</td><td>5.7</td><td>5.44</td><td>0.26</td><td>0.02</td></tr><tr><td>4.7</td><td>4.60</td><td>0.10</td><td>0.28</td></tr><tr><td>3.7</td><td>3.64</td><td>0.06</td><td>0.16</td></tr><tr><td>2.7</td><td>2.1</td><td>0.6</td><td>0.23</td></tr><tr><td rowspan="4">EBV</td><td>5.7</td><td>5.44</td><td>0.25</td><td>0.11</td></tr><tr><td>4.7</td><td>4.42</td><td>0.27</td><td>0.05</td></tr><tr><td>3.7</td><td>3.31</td><td>0.39</td><td>0.08</td></tr><tr><td>2.6</td><td>2.21</td><td>0.39</td><td>0.05</td></tr><tr><td rowspan="3">BKPyV</td><td>5</td><td>4.74</td><td>0.26</td><td>0.04</td></tr><tr><td>4</td><td>3.44</td><td>0.56</td><td>0.05</td></tr><tr><td>3</td><td>2.74</td><td>0.26</td><td>0.15</td></tr></tbody></table>

ml, 0.014 IU/ml and 0.01 IU/ml respectively for a sample tested in 5 replicates in a single run. The coefficient of variation was 1 %, 4 % and 0.3 % respectively. The inter-run standard deviations were 0.25 IU/ml, 0.27 IU/ml, 0.20 IU/ml for a sample tested in 50 replicates in different runs using the Altostar® CMV DNA, EBV DNA and BKPyV assays respectively. The coefficient of variation was 5.4 %, 6.9 % and 7 % respectively.

### 4.4. Comparison between Altostar® assays and previous assays

The concordance between the previous assay and the Altostar® assay for the 22 CMV DNA samples was 95.5 %, (21/22). The only whole blood sample that was negative with Altona but positive with the previous home brew assay was quantified at 1.8 log₁₀ IU/mL by the former method, which is below the detection limit of the Altostar assay. Similarly, the 32 EBV DNA samples were tested with a concordance of 90.6 %, (29/32). Of the three discordant samples, 2 were whole blood and 1 was bone marrow. All three samples were positive with the Altostar® assay but negative with the previous MagNA system, suggesting a higher sensitivity for the Altostar® assay. The 25 samples were tested for BKPyV DNA with a concordance of 92 % (23/25). The two discordant samples were urine, and were negative with the Altona and positive with the old method (1.3 and 3.3 log₁₀ IU/ml).

Fig. 2 illustrates the mean differences between the Altostar assays and the previous assays. The mean bias between the Altostar results and

previous results was 0.21 log₁₀ IU/ml for CMV, 0.20 log₁₀ IU/ml for EBV and 0.98 log₁₀ IU/ml for BKPyV. Note that the Altostar® BKPyV DNA measurements are expressed in IU/ml while our previous assay was quantified using a plasmid and was expressed in copies/ml, which could explain this high bias between the 2 assays. The Altostar® results were correlated with the previous assays used in our lab (Fig. 2).

To assess the equivalence between BKPyV DNA quantification expressed in UI/ml or in copies/ml, we have tested 2 dilutions of the WHO international standard (10,000 and 1000 UI/ml) in five replicates with both assays. The ratio of the values obtained with both assays was calculated. The conversion factor to transform the DNA titers from UI/ml to copies/ml is 3.4.

## 5. Discussion

The aim of the current study was to evaluate the Altostar® PCRs assays for CMV, EBV and BKPyV, an automated platform for the routine diagnosis of these viral infections. The evaluation indicated that this system is suitable for CMV, EBV and BKPyV monitoring in different clinical settings.

The evaluation of the AltoStar® CMV PCR Kit 1.5, AltoStar® EBV PCR Kit 1.5 and AltoStar® BKPyV PCR Kit 1.5 showed highly satisfying results for sensitivity, linearity, and precision. For each assay, the detection limit was in line with the Altona Diagnostics leaflet. The performance was consistent with other automated molecular platforms from different manufacturers [7–12]. Automated molecular platforms, have excellent linearity, sensitivity, reproducibility and precision. These assays can be adapted to several matrix such as urine, amniotic fluid and breast milk specimens). An advantage of these fully automated platforms compared with the Altostar® system is the more rapid turnaround time. However, it is not possible to perform several PCR with the same nucleic acid extract. Moreover, the nucleic acid extract cannot be recovered for additional viral detection if necessary.

Our evaluation agrees with previous studies on the Altostar® kits for EBV and CMV DNA quantification [13,14]. One study found that Altostar® EBV assay performance was similar to the RealTime EBV assay (Abbott) [14] and another found that Altostar® CMV assay agreed well with the Artus® CMV-QS-RGQ (Qiagen). Moreover, this platform has been previously evaluated for other viral detection and quantification in blood such as human herpes virus 6 (HHV-6) and Adenovirus (ADV) [15,

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Linearity of the Altostar® assays.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Bland-Altman analysis (A, B and C) and Passing-Bablok regression (D, E and F) comparing BKV, CMV and EBV DNA concentrations between Altostar® and previous assays used in the lab.</figcaption></figure>

Note: for Bland-Altman plots: the blue line represents the mean difference, and dashed lines represent the 95 % confidence intervals. For Passing-Bablok regression: the red line is the regression line; the dotted line is the line of identity, and solid lines represent the 95 % confidence intervals.

[16]. With the Altostar® AM 16 system, up to 4 qPCR assays can be set up and performed from a single nucleic acid eluate, and because all DNA viruses, such as CMV, EBV, BKPyV, ADV, parvovirus B19 and HHV-6, share common cycling parameters, this combination of semi-automation and compatible qPCR reagents allows efficient virologic testing post-transplantation. This is only possible with the ELITech ELITE InGenius automated platform but not with other automated assays run on the Panther, the Alinity m or the Cobas instruments.

The results observed for the WHO standards indicated a negative bias, particularly at lower titers. This may have clinical implications at low viral load. This limitation was reported by Kraft et al. They recommend, when monitoring patients posttransplant, that trends in viral load may be more useful than any single viral load value, unless the viral load is very high [17].

The viral DNA quantification results for the previous methods developed in our lab and the AltoStar® tests were correlated. The discrepant samples had low viral loads (<3 log₁₀ IU/ml), and the evaluation was retrospective. Degradation of viral DNA during storage is possible. Fortunately, these low viral loads are not considered to require therapeutic intervention. However, they can be a warning to clinicians to monitor their patients more regularly. Note that, for the DNA BKPyV quantification, previous results were expressed in copies/mL and AltoStar® results are expressed in IU/mL. To ensure that this change of unit did not confuse prescribers, physicians were informed in advance and it was specified in the report. The Altostar® results are expressed in international units/ml with good accuracy based on our results with the WHO reference standards. Therefore, it should provide an opportunity for increased standardization and commutability of test results across laboratories.

The Altostar® AM16 system automatically performs DNA extraction and purification and the PCR setup with the Altostar® PCR mix to run on a CFX96 instrument. However, interestingly, the Altostar® purification kit is validated and CE-IVD marked for DNA and RNA extraction from whole blood, plasma, urine, stool, cerebrospinal fluid and swabs in a viral transport medium. We have also validated that it is suitable for saliva, amniotic fluid, bronchoalveolar lavage and bone marrow samples (data not shown). The Altostar® CMV PCR kit is therefore suitable for several clinical situations: diagnosis of congenital cytomegalovirus, meningoencephalitis or pneumonia due to CMV [3]

Moreover, efficient extraction of nucleic acids from whole blood samples requires pretreatment with a lysis buffer, which is usually performed manually due to the heterogeneity of this sample material. This manual step is labor-intensive and can be error-prone. The Altostar® AM16 instrument can directly aspirate and dispense whole blood samples. Whole blood is transferred to a secondary tube and mixed with a pretreatment buffer.

Our study presents some limitations. The number of available stored positive samples for each parameter with the required volume was low. With regard to linearity, we did not test very high viral DNA concentrations (≥ 6 log₁₀ IU/ml). Although the package inserts indicate that the linearity zone is verified up to these values, we did not have patients with viral loads that were high enough. The Altostar® system also works in batches of samples. It is not possible to run samples throughout the day, but the number of samples remains high (96 samples) and offers the possibility of running a large series of analyses per day.

In conclusion, the results of this study demonstrate the validity and accuracy of the AltoStar® CMV, EBV and BKPyV assays in determining viral load in clinical specimens. It provides a semi-automated option for the clinical monitoring of CMV, EBV and BKPyV DNA in different clinical samples. Automation, ease of use and the ability to study a large number of matrices make it a powerful tool for clinical virologists and patient management.

## Funding

Altona provided the reagents but did not participate in the study design or the data interpretation.

## Authorship contribution statement

Yoann Fluhr: co-writing the original draft, data curation. Karine Saune: methodology, data curation. Kevin Oliveira-Mendes, Lila Poteau, Estelle Raguin: performed the analysis. Arnaud Del Bello, Nassim Kamar, Jacques Izopet: revision and edition. Florence Abravanel: conceptualization, data curation, co-writing the original draft.
CRediT authorship contribution statement

**Yoann Fluhr:** Writing – original draft, Data curation. **Karine Saune:** Writing – review & editing, Methodology, Data curation. **Kevin Oliveira-Mendes:** Formal analysis. **Estelle Raguin:** Formal analysis. **Lila Poiteau:** Formal analysis. **Arnaud Del Bello:** Writing – review & editing. **Nassim Kamar:** Writing – review & editing. **Jacques Izopet:** Writing – original draft. **Florence Abravanel:** Writing – original draft, Data curation, Conceptualization.

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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