Development of a BK Virus Real-Time Quantitative Assay Using the biomérieux Analyte-Specific Reagents in Plasma Specimens

Transcription

1 Development of a BK Virus Real-Time Quantitative Assay Using the biomérieux Analyte-Specific Reagents in Plasma Specimens Hanna Rennert, PhD, 1,2 Helen Fernandes, PhD, 1,2 Zahid Gilani, 2 and John Sipley, PhD 2 From the Department of Pathology and Laboratory Medicine, 1 Weill Cornell Medical College, New York, NY, and 2 NewYork-Presbyterian Hospital, New York, NY. Key Words: BK virus quantitation; Viral loads; Real-time PCR test; Renal nephropathy Am J Clin Pathol December 2015;144: ABSTRACT Objectives: Viral load testing for BK virus (BKV) has become the standard of care for diagnosing BKV infection and monitoring therapy in kidney transplant patients. However, there are currently no US Food and Drug Administration approved assays and no standardization among available tests. Methods: This study evaluated the performance of the analyte-specific reagent (ASR) BKV primers r-gene and probe r-gene reagents (biomérieux, Marcy l Étoile, France) soon to become available on the US market for accuracy, linearity, precision, analytical sensitivity, specificity, and correlation with the Qiagen (Germantown, MD) BKV ASR test using commercial material and patient plasma samples. Results: The assay was linear from 204 to 3.92 million ( log 10 ) DNA copies/ml (coefficient of determination; R 2 = 0.999). A dilution series demonstrated limits of detection and quantitation of 2.14 log 10 and 2.30 log 10 copies/ml (95% hit rate detection), respectively. Interrun precision was highly reproducible, with coefficients of variance ranging from 2.2% to 6.0%. A comparison of 34 matched samples showed a good agreement (R 2 = 0.87) between the biomérieux BKV laboratory test and the Qiagen BKV ASR assay results, with an average negative bias ( 0.28 log 10 copies/ml). Conclusions: The laboratory-developed test with biomérieux BKV reagents is a reliable and sensitive assay for BKV DNA quantitation compared with the Qiagen ASR test. BK virus (BKV) is a common polyomavirus that establishes subclinical and persistent infection predominantly in the kidney in up to 90% of adults, typically without adverse effects. 1 Renal transplant recipients are at risk of developing BKV-associated nephropathy (BKVAN), which frequently leads to graft failure in 15% to 80% of patients, 2-4 while BKV reactivation in bone marrow transplant patients may result in hemorrhagic cystitis. 5 Disseminated disease due to BKV has also been reported in patients with AIDS, chronic lymphocytic leukemia, and congenital immunodeficiency, in whom immunosuppression could not be reduced. 6 The pathology associated with BKV is thought to result from destruction of infected urothelial cells undergoing active viral replication. 2 BKVAN results in a decline in urinary tract function, and 30% to 60% of the patients experience irreversible graft failure. 4,7 In some instances, antiviral therapy with cidofovir and leflunomide may be used, 8,9 and the patient should be monitored closely for the onset of rejection. Polymerase chain reaction (PCR) to detect and quantitate BKV DNA in plasma has been promoted as a noninvasive way to identify patients at risk of BK nephropathy and to monitor response to therapy. 1,3,4,10,11 Detection of BKV in plasma has a stronger correlation with disease than BKV in urine, since urine specimens may have very high viral loads that may overlap between symptomatic and asymptomatic patients. Although the viral load cutoff associated with nephropathy is not well established, a small number of studies 4 have shown that plasma BKV loads more than 10 4 copies/ ml have a predictive value greater than 80%, while the absence of viremia and/or viruria rules out the diagnosis Am J Clin Pathol 2015;144:

2 Rennert et al / BK Virus Quantitation by Real-Time PCR of BKVAN. 10,12 Viral loads of more than 10 7 copies/ml in urine are more likely associated with disease. 11,13,14 Quantitative measurements of BKV DNA have also shown that decreasing virus levels over time correlate with response to treatment and improved outcome. 11,14 Moreover, screening guidelines from the Infectious Diseases Society of America (2005) currently recommend that renal transplant patients be screened for BK viral load (BKVL) in the urine every 3 months and in plasma every 1 to 3 months up to 2 years posttransplant or when allograft dysfunction occurs, or when allograft biopsy is performed. 2 Virus levels are commonly quantitated by direct measurement of BKV DNA in plasma using real-time PCR amplification technologies. 1,15-17 However, most assays are laboratory-developed tests (LDTs), and there are no international standards to compare among the different tests. In this study, we evaluated the performance of an LDT using the BKV primers r-gene and BK virus probe r-gene and an unassayed DNA Internal Control r-gene (biomérieux, Marcy l Étoile, France). This combination is coupled with an automated extraction and PCR setup. We show that this assay is comparable to the Qiagen (Germantown, MD) BKV analyte-specific reagent (ASR) assay, providing additional testing options for clinical laboratories. Materials and Methods Samples and Performance Evaluation Panels A total of 68 consecutive plasma samples from kidney transplant patients submitted to our clinical laboratory for BKV testing were prospectively collected during Thirty-four samples were negative and 34 positive, allowing a broad dynamic range of BKV quantitation. Samples were deidentified and stored at 70 C, until further analysis. No clinical information was available on these patients. All samples had undergone BKVL testing by NewYork Presbyterian Hospital Clinical Laboratories (New York, NY) before validation. The study was approved by the Institutional Review Board Committee at Weill Cornell Medical College. Assay linear range, precision, and reproducibility acceptability criteria were based on New York State Department of Health Clinical Lab Evaluation Program Guidance in the Microbiology Molecular Checklist (February 2011; The OptiQuant BK Linearity Panel (Acrometrix, Benicia, CA) was used to evaluate accuracy, reproducibility, linearity, and sensitivity. The six-member panel, formulated with intact BKV (subtype Ia) particles in a defibrinated, delipidized human plasma matrix, spanned the clinically significant range ( to BKV copies/ml) for BK-infected individuals, allowing characterization of the linear range. For determining the limit of detection (LOD) and the limit of quantitation (LOQ), the copies/ml OptiQuant BKV panel member was further diluted to six concentrations ranging from 200 copies/ml to 6.25 copies/ ml in BKV-negative plasma (Acrometrix) and analyzed in 12 replicates. Panel members were quantitated by the manufacturer s real-time PCR assay (Luminex, Austin, TX), targeting a 62 base pair (bp) region in the VP3 gene and calibrated against a commercial purified BKV DNA. The between-run precision was determined with replicates using OptiQuant BKV panel samples (range, 275 copies/ml to 5.9 million copies/ml) measured on 3 different days. Crossreactivity (specificity) was determined using high viral load samples titered in-house (cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) or by Viracor (IBT Laboratories, Lee s Summit, MO) (human herpesvirus [HHV] 7). Herpes simplex virus (HSV) 1, HSV-2, HHV-6, HHV-8, and JC viruses (JC MAD1 strain, quantitated viral DNA catalog ) were obtained from Advanced Biotechnologies (Columbia, MD) and varicella zoster virus from the Centers for Disease Control and Prevention (Atlanta, GA). Adenovirus was from Acrometrix. biomérieux BKV r-gene Primers/Probe LDT The LDT assay used in this study comprised two ASRs a BKV primers r-gene ( ) and BKV probe r-gene ( ) (biomérieux) and an unassayed DNA Internal Control r-gene ( ) (biomérieux, Ivry sur Seine, France). BKV DNA was extracted from 200 µl plasma in the presence of 10 µl BKV internal control (IC) r-gene, using the QIAcube/QIAamp DNA extraction reagents (Qiagen), and eluted in a final volume of 50 μl. The BKV viral load was then measured by a quantitative TaqMan realtime PCR on the Rotor-Gene Q thermal cycler (Qiagen). These primers and probes amplify a 158-bp specific sequence located in the BKV small T antigen (StAg), covering all available BKV sequences, including the rare strains of types II, III, and IV (biomérieux, Marcy l Étoile, France, unpublished communications, 2014). A plasmid DNA containing the whole BKV genome plasmid, diluted 10-fold to produce a 4-point standard curve ranging from 5,000 to 5 copies/μl, was used for BKV quantitation (biomérieux). PCR amplification was performed in a final volume of 25 µl reaction comprising 15 μl PCR master mix and 10 μl IC-containing BKV DNA. The two primers sets (BKV genome and IC) were amplified by quantitative PCR (qpcr) using the Rotor-Gene Q instrument (Qiagen) with the following amplification conditions: 95 C for 15 minutes, followed by 45 cycles of 95 C for 10 seconds and 60 C for 40 seconds. The cycle threshold (Ct) for BKV and the IC were calculated by the system software, and the resulting Ct was 910 Am J Clin Pathol 2015;144:

3 Observed used to quantitate the BKV copy number by interpolation against standard curve values generated using quantitation standards. Qiagen BKV ASR Assay The laboratory-developed Qiagen BKV ASR test was performed using the BKV primer and probe ASRs as previously described. 18 Briefly, nucleic acid was extracted from 200 μl plasma in the presence of a BKV positive control, using the QIAcube automated isolation system and the Total Nucleic Acid Isolation kit (Qiagen). The DNA was eluted into a final volume of 50 μl, of which 15 μl was added to the master mix containing primers, probes, Taq polymerase, magnesium chloride, and buffers. The BKV viral load was then measured by qpcr using primers that amplify the conserved VP2 and VP3 region of the BKV genome and the Rotor-Gene Q system (Qiagen). Amplification conditions were 95 C for 10 minutes, followed by 45 cycles of 95 C for 15 seconds, 65 C for 30 seconds, and 72 C for 20 seconds with 10 cycles of touchdown during the annealing step (1 C). Statistical Methods 3.36 The correlation of log 10 BKV loads in plasma and Acrometrix validation specimens was determined by the least squares regression model using Excel 2003 (Microsoft, Redmond, WA). A log 10 difference of 1.0 was used as the cutoff for the determination of concordance between the two assays as previously described. 1,2,19 The between-run Expected Figure 1 Linear range of the biomérieux (Marcy l Étoile, France) BK virus (BKV) test using reference samples. The linearity and dynamic range were measured using the Acrometrix (Benicia, CA) OptiQuant BKV panel, ranging from 2.3 to 6.6 log 10 copies/ml. The linearity was determined by plotting the log of the mean measured titers against the log of the mean input titers. The results demonstrate a linear response over 4 logs of detection with an R 2 value of y = x ; R 2 = Table 1 Dilution Series for Limit of Detection Determination No. precision of the qpcr method for BKV quantitation was expressed by coefficient of variation (CV). Agreement between the biomérieux BKV r-gene LDT and the Qiagen BKV Rotor Gene PCR was determined using a Bland- Altman plot of all samples (34 samples). The LOD (95% hit rate detection) was determined by probit analysis (MiniTab 16 Statistical Software; MiniTab, College State, PA). The LOQ was defined as the lowest dilution with an SD within approximately 0.2 log ,20 Results Input DNA, Input DNA, Positive No. of Samples Total Testing of a commercial linearity panel across the range of detection from 204 to 392 million ( log 10 ) DNA copies/ml demonstrated an excellent agreement between the expected and observed viral load with a coefficient of determination (R 2 ) of Figure 1. The assay was linear with a slope approximating 1 (1.1) across six logs of detection. A high-titer specimen to measure the linear range beyond 6.60 log 10 was not available for inclusion in the analyses. When a larger number of interassay dilutions were tested to assess the LOD Table 1, the concentration at which BKV DNA was detected in at least 95% of the replicates was log 10 copies/ml (95% confidence interval [CI], log 10 copies/ml), corresponding to 140 copies/ml (95% CI, copies/ml) Figure 2. The LOQ was 2.30 log 10 copies/ml (200 copies/ml) based on an SD less than or equal to 0.2 log 10 copies/ml (Table 1). Interassay precision analysis using the Acrometrix OptiQuant BKV panel samples ( BKV log 10 copies/ml) measured on 3 different days exhibited excellent reproducibility throughout the linear range of detection with low interassay variation. The commercial validation specimens yielded BKV viral load means (SD, CV) ranging from 2.44 log 10 copies/ml (0.15, 6.0%) and 6.77 log 10 copies/ml (0.15, 2.2%) for the low and high panel members, respectively Table 2. A high degree of intra-assay precision was also observed Table 3. Of the 68 patient samples tested by the Qiagen BKV assay, 34 were positive (range, log 10 copies/ml) Am J Clin Pathol 2015;144:

4 Rennert et al / BK Virus Quantitation by Real-Time PCR Percentage Concentrations in Log Figure 2 Probit analysis. The limit of detection (LOD) was determined using serial dilutions of the 500-copy/mL OptiQuant BKV panel member in EDTA plasma (Acrometrix, Benicia, CA) to six concentrations from 200 to 6.25 copies/ ml ( log 10 copies/ml) assayed in replicates of 12. LOD 95% = log copies/ml; LOD 95% = 140 copies/ml. and 34 were negative in both assays. There was good agreement in the performance of the biomérieux BKV LDT compared with the in-house Qiagen BKV assay viral load test, demonstrating an excellent correlation with a coefficient of determination (R 2 ) of Figure 3. A Bland-Altman plot demonstrated that the average difference (bias) of 0.28 log was within approximately twofold, indicating that on average, the biomérieux BKV LDT yielded viral load values that were twofold higher than the values obtained with the Qiagen BKV ASR assay. Fifty percent of the positive samples were within 0.3 log of the Qiagen assay, while 68% of the positive samples were within 0.5 log. All results were within 1 log of the Qiagen method Figure 4, and agreement between the assays is demonstrated by the 95% CI (±1.96 SD) for the mean difference, including zero. 21 The greatest difference from the Qiagen results was a single result of 0.8 log 10 copies/ml observed at the low value of 1.73 log 10 copies/ml. There were no biological false-positive results with any other pathogens potentially found in human plasma due to shared targets with the viruses tested as described under Materials and Methods. Discussion Quantitative testing for BKV plays an important clinical role in the monitoring of immunocompromised patients, especially in the setting of posttransplantation BKV nephropathy. 14,22-24 However, the marked variability among laboratory-developed PCR tests and the lack of standardized reference material make the interpretation of clinical management guidelines and BKV testing results difficult. 4 In this study, we used a validation panel comprising commercial and patient samples to evaluate the analytical characteristics of a new LDT using the biomérieux BKV ASR reagents. To our knowledge, this is the first study validating the use of these reagents. A study validating the European Conformity marked BKVs r-gene kit was recently performed by Sueur et al 25 in Europe. Performance characteristics results indicate that the new BKV assay has a dynamic range of at least six logs (R 2 = 0.999), Table 2 Intra-Assay Reproducibility of the BK Virus ASR Test Using Reference Material Nominal, No. of Samples Tested Mean Viral Load, Difference Expected-Observed SD, ASR, analyte-specific reagent; CV, coefficient of variation. % CV: Intra-assay Table 3 Inter-Assay Precision of the BK Virus ASR Test Using Reference Material Nominal, No. of Samples Tested Mean Viral Load, SD, % CV: Inter-assay ASR, analyte-specific reagent; CV, coefficient of variation. 912 Am J Clin Pathol 2015;144:

5 biomérieux BK PCR Qiagen BK ASR Difference Expected Observed SD SD 1.50 Average Figure 3 Correlation between the biomérieux (Marcy l Étoile, France) and the Qiagen (Germantown, MD) BK virus (BKV) viral loads test results. Linear regression of the 34 positive BKV viral load samples tested in parallel yielded a coefficient of determination (R 2 value) of 0.87 with the biomérieux assay, indicating a high degree of accuracy throughout the assay range. ASR, analyte-specific reagent; PCR, polymerase chain reaction. a lower detection limit of 2.0 log 10 copies/ml, and an intra-assay CV of less than 6.0% for all concentrations, confirming a high degree of assay precision across the entire range of BKV concentrations tested. Moreover, there was complete agreement between this test and the laboratory BKVL ASR assay with an R 2 value of 0.874, indicating a high degree of accuracy throughout the assay range. A wide dynamic range is significant for identification and monitoring of patients with high levels of viremia at risk of developing BKVAN, while still effectively monitoring for prospective BKV reactivation at low viral loads. 13,26 Several clinical studies reported median BKVL in plasma ranging from 2.84 to 4.1 log 10 copies/ml (range, ) for patients with BKVAN. 13,14,27 Moreover, serial determination of BK viremia is considered the best tool for predicting disease evolution during follow-up, allowing reduction of immunosuppression. 28 The LOQ is particularly important for the identification of patients who should be monitored for BKV reactivation. However, the relationship between cutoff levels of BKV viremia and prediction of BKVAN is still under investigation and varies widely. 26,27 Recently, the American Society of Transplantation (AST) defined a BKVL of 4 log 10 copies/ ml or more as presumptive BKVAN, recommending reduction in immunosuppression. 29 Randhawa et al 13 suggested predictive values of active BKVAN of 3.7 log 10 copies/ml of BKV in viremia, while in a separate study, a reduction Figure 4 Bland-Altman plot of 34 matched patient plasma samples tested in the biomérieux (Marcy l Étoile, France) test vs the Qiagen (Germantown, MD) BK virus (BKV) assay. The gray dashed line represents the overall bias of 0.28 log 10 (copies/ml) of the biomérieux BKV test. The 95% (1.96 SD) limit of agreement is 0.80 log 10 (gray lines). y = x ; R 2 = in immunosuppression was done upon detection of more than 2.7 log 10 copies/ml. 30 Although there is no accepted standard LOQ for BKV, we have determined the LOQ as the lowest concentration that could be reliably quantitated with an SD of 0.2 log 10 copies/ml or less to be 2.30 log 10 copies/ml, which is in agreement with the LOQ reported in the literature 11,24,25 and our currently used Qiagen BKV ASR assay. 18 The biomérieux BKV LDT also showed excellent precision in BKVL quantitation with overall CV values (2.2%-6.0%) comparable to the values reported in plasma samples in other, similar studies. 11,14,31 There was good agreement between the laboratory test and the bio- Mérieux LTD assay, with an overall negative bias of 0.28 log 10 copies/ml, indicating that the laboratory Qiagen assay gives a lower result compared with the biomérieux BKV LDT. A positive bias was more likely to be observed with the lowest viral load. The reasons for this bias in the assay are unknown. In this study, there was no cross-reactivity with either JC virus (JCV) or other pathogens that may affect the detection of BKV. Since BKV shares 75% sequence homology with JCV and is highly polymorphic, careful design of primers is vital. 1 Hoffman et al 19 have observed substantial discordance among assays primarily attributed to primer and probe mismatch due to subtype-associated polymorphisms, particularly for the less common BKV subtypes. Moreover, in a recent study, Hassan et al 26 have shown that using the AST guideline of BKVL cutoff, the referral laboratory BKV Am J Clin Pathol 2015;144:

6 Rennert et al / BK Virus Quantitation by Real-Time PCR test misdiagnosed BKVAN compared with the LDT, resulting in renal graft loss. BKV isolates are classified into four subtypes (I-IV), with subtype I exceedingly the most prevalent throughout the world among individual patients with BKV infection. 18,32 Probe and primer design have been also described as significant sources of BKVL discrepancies among different assays. Genomic regions encoding the VP1 capsid protein and the large T antigen have been shown to have sequence divergence, 33,34 while the StAg gene used in this assay is described to be displaying only a few single-nucleotide polymorphisms. 35 Here we demonstrate that the new LTD is highly comparable to the Qiagen ASR assay designed to amplify a consensus region of VP2/VP3 genes, indicating that the new test is highly accurate. Correct quantitation of BKVL samples was also obtained by Sueur et al 25 using the r-gene kit. Given BKV genetic heterogeneity and the lack of US Food and Drug Administration approved assays, an important advantage of the study is that it provides information regarding standardized reagents for BKV testing produced under good manufacturing practices, offering additional testing options for clinical laboratories. Since PCR tests vary considerably among laboratories, the availability of such reagents is expected to greatly improve assay performance and generation of guidelines for clinical management. Finally, although correlation of results with the Qiagen ASR assay was excellent, possible limitations of the study include the relatively small number of patients and lack of a high-titer specimen to measure the linear range beyond 7.0 log 10 copies/ml. In summary, the LDT with biomérieux BKV reagents is a highly sensitive, specific, and reliable assay for measuring BKV loads in plasma and identifying patients at risk of BKVAN. Corresponding author: Hanna Rennert, PhD, Dept of Pathology and Laboratory Medicine, Weill Cornell Medical College, 525 E 68th St, F544B, New York, NY 10065; har2006@med.cornell.edu. This study was funded in part by biomérieux with reagents. Acknowledgment: We thank Stephen G. Jenkins, PhD, for his detailed review of our article. References 1. Bechert CJ, Schnadig VJ, Payne DA, et al. Monitoring of BK viral load in renal allograft recipients by real-time PCR assays. Am J Clin Pathol. 2010;133: Hirsch HH, Brennan DC, Drachenberg CB, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation. 2005;79: Bohl DL, Storch GA, Ryschkewitsch C, et al. Donor origin of BK virus in renal transplantation and role of HLA C7 in susceptibility to sustained BK viremia. Am J Transplant. 2005;5: Cimbaluk D, Pitelka L, Kluskens L, et al. Update on human polyomavirus BK nephropathy. Diagn Cytopathol. 2009;37: Cesaro S, Facchin C, Tridello G, et al. A prospective study of BK-virus-associated haemorrhagic cystitis in paediatric patients undergoing allogeneic haematopoietic stem cell transplantation. Bone Marrow Transplant. 2008;41: Degener AM, Pietropaolo V, Di Taranto C, et al. Detection of JC and BK viral genome in specimens of HIV-1 infected subjects. New Microbiol. 1997;20: Hariharan S. BK virus nephritis after renal transplantation. Kidney Int. 2006;69: Bernhoff E, Tylden GD, Kjerpeseth LJ, et al. Leflunomide inhibition of BK virus replication in renal tubular epithelial cells. J Virol. 2010;84: Bennett WM, Meyer L, Ridenour J, et al. Surveillance and modification of immunosuppression minimizes BK virus nephropathy. Am J Nephrol. 2010;32: Bonvoisin C, Weekers L, Xhignesse P, et al. Polyomavirus in renal transplantation: a hot problem. Transplantation. 2008;85(suppl):S42-S Herman J, Van Ranst M, Snoeck R, et al. Polyomavirus infection in pediatric renal transplant recipients: evaluation using a quantitative real-time PCR technique. Pediatr Transplant. 2004;8: Nickeleit V, Mihatsch MJ. Polyomavirus nephropathy in native kidneys and renal allografts: an update on an escalating threat. Transpl Int. 2006;19: Randhawa P, Ho A, Shapiro R, et al. Correlates of quantitative measurement of BK polyomavirus (BKV) DNA with clinical course of BKV infection in renal transplant patients. J Clin Microbiol. 2004;42: Pang XL, Doucette K, LeBlanc B, et al. Monitoring of polyomavirus BK virus viruria and viremia in renal allograft recipients by use of a quantitative real-time PCR assay: one-year prospective study. J Clin Microbiol. 2007;45: Si-Mohamed A, Goff JL, Desire N, et al. Detection and quantitation of BK virus DNA by real-time polymerase chain reaction in the LT-ag gene in adult renal transplant recipients. J Virol Methods. 2006;131: Gu Z, Pan J, Bandowski MJ, et al. Quantitative real-time polymerase chain reaction detection of BK virus using labeled primers. Arch Pathol Lab Med. 2010;134: Burrel S, Brunet C, Hamm N, et al. Validation of the QIAsymphony RGQ system for DNA quantitation of different BK virus genotypes in whole blood samples. J Virol Methods. 2014;196: Rennert H, Jenkins SG, Azurin C, et al. Evaluation of a BK virus viral load assay using the QIAGEN Artus BK Virus RG PCR test. J Clin Virol. 2012;54: Hoffman NG, Cook L, Atienza EE, et al. Marked variability of BK virus load measurement using quantitative realtime PCR among commonly used assays. J Clin Microbiol. 2008;46: Premkumar DR, Ma XZ, Maitra RK, et al. The nef gene from a long-term HIV type 1 nonprogressor. AIDS Res Hum Retroviruses. 1996;12: Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23: Merlino C, Bergallo M, Daniele R, et al. BKV-DNA and JCV-DNA co-quantification assay to evaluate viral load in urine and serum. Mol Biotechnol. 2005;30: Am J Clin Pathol 2015;144:

7 23. Hirsch HH, Drachenberg CB, Steiger J, et al. Polyomavirusassociated nephropathy in renal transplantation: critical issues of screening and management. Adv Exp Med Biol. 2006;577: Randhawa P, Shapiro R, Vats A. Quantitation of DNA of polyomaviruses BK and JC in human kidneys. J Infect Dis. 2005;192: Sueur C, Solis M, Meddeb M, et al. Toward standardization of BK virus monitoring: evaluation of the BK virus R-gene kit for quantification of BK viral load in urine, whole-blood, and plasma specimens. J Clin Microbiol. 2014;52: Hassan S, Mittal C, Amer S, et al. Currently recommended BK virus (BKV) plasma viral load cutoff of >/=4 log10/ml underestimates the diagnosis of BKV-associated nephropathy: a single transplant center experience. Transpl Infect Dis. 2014;16: Pollara CP, Corbellini S, Chiappini S, et al. Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN. New Microbiol. 2011;34: Costa C, Bergallo M, Astegiano S, et al. Monitoring of BK virus replication in the first year following renal transplantation. Nephrol Dial Transplant. 2008;23: Hirsch HH, Randhawa P. BK virus in solid organ transplant recipients. Am J Transplant. 2009;9(suppl 4):S136-S Almeras C, Vetromile F, Garrigue V, et al. Monthly screening for BK viremia is an effective strategy to prevent BK virus nephropathy in renal transplant recipients. Transpl Infect Dis. 2011;13: Rubio L, Pinczewski J, Drachenberg CB, et al. A multiplex real-time PCR method for quantification of BK and JC polyomaviruses in renal transplant patients. Diagn Mol Pathol. 2010;19: Huang G, Chen LZ, Qiu J, et al. Prospective study of polyomavirus BK replication and nephropathy in renal transplant recipients in China: a single-center analysis of incidence, reduction in immunosuppression and clinical course. Clin Transplant. 2010;24: Randhawa PS, Vats A, Zygmunt D, et al. Quantitation of viral DNA in renal allograft tissue from patients with BK virus nephropathy. Transplantation. 2002;74: Boldorini R, Allegrini S, Miglio U, et al. Genomic mutations of viral protein 1 and BK virus nephropathy in kidney transplant recipients. J Med Virol. 2009;81: Iwaki KK, Qazi SH, Garcia-Gomez J, et al. Development of a real-time quantitative PCR assay for detection of a stable genomic region of BK virus. Virol J. 2010;7:295. Am J Clin Pathol 2015;144: