Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification

Abstract

With the growing importance of BK virus (BKV), effective and efficient screening for BKV replication in plasma and urine samples is very important for monitoring renal transplant and hematopoietic stem cell transplant recipients, who are at increased risk of BKV-associated diseases. However, recent assays proposed by many manufacturers have not been tested, and the available tests have not been standardized. The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene, GeneProof, and RealStar, on plasma and urine specimens from patients infected with various genotypes and to determine the correlations with the results from a reference laboratory. A qualitatively excellent global agreement (96.8%) was obtained. RealStar PCR tended to give a higher sensitivity, especially for subtype Ib1 samples. Comparison of 30 plasma samples and 53 urine samples showed a good agreement between the three assays, with Spearman's Rho correlation coefficient values falling between 0.92 and 0.98 (P < 0.001). Moreover, a perfect correlation was obtained for comparison of the assay performances with the AcroMetrix BKV panel (P < 0.001 for all comparisons). According to Bland-Altman analysis, more than 95% (240/249 comparisons) of sample comparisons were situated in the range of the mean ؎ 2 standard deviations (SD). The greatest variability between assays was observed for 10.2% of subtype Ib2 samples, with differences of >1 log 10 copies/ml. In conclusion, this study demonstrated the reliable and comparable performances of the R-gene, GeneProof, and RealStar real-time PCR systems for quantification of BKV in urine and plasma samples. All three real-time PCR assays are appropriate for screening of BKV replication in patients. BK virus (BKV) is a double-stranded DNA virus belonging to the Polyomaviridae family that causes chronic and usually asymptomatic infections in immunocompetent individuals. During initial infection, virions infect urothelial cells and establish latent infection. BKV reactivation in renal transplant recipients (RTR) is increasingly recognized as an opportunistic infection, particularly with the introduction of more potent immunosuppressive agents (1). Typically, viral particles are first detected in the urine, and this may be followed by viremia. High levels of BKV reactivation can lead to BKV-associated nephropathy (BKVAN), leading to graft failure in 20 to 80% of affected patients (2). In bone marrow transplant recipients, BKV reactivation may result in hemorrhagic cystitis. Molecular analyses of numerous isolates have led to the classification of the BKV genus into several subtypes (Ia, Ib1, Ib2, Ic, II, III, IVa, IVb, and IVc), based on phylogenetic analyses of full-genome viral DNA sequences (3, 4). The various genotypes have a specific geographic distribution in the population (5). Genotype I is widespread, genotype IV is predominant in East Asia, and genotypes II and III are rarely detected (6).Accurate monitoring of BKV DNA loads is essential for a s...

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