Impact of Genomic Sequence Variability on Quantitative PCR Assays for Diagnosis of Polyomavirus BK Infection

Abstract

Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed 184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in 81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as low as 10 copies/l. However, consistent detection of all BKV variants was possible only at concentrations of 10,000 copies/l or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV, genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical impression and the measured viral load.Polyomavirus BK (BKV) has become an important pathogen in kidney transplant patients. Immunosuppression given to prevent acute rejection triggers BK viruria in 10 to 60%, viremia in 5 to 30%, and biopsy-proven viral nephropathy in 1 to 10% of patients (8,10,11). Initial graft loss rates associated with BKV nephropathy were very high but have now dropped to Ͻ25%. This success has been attributed to intensive viral monitoring followed by preemptive reduction in immunosuppression (1,14). In a recent survey that had an overall response rate of 55.5%, 173 of 200 (86.5%) kidney transplant centers reported screening for BKV in blood by quantitative PCR, while 111 of 202 (55.5%) performed viral screening in urine (2). In the latter category, 90% of respondents preferred PCR screening to urine cytology. While cytology is a useful modality to screen for viral nephropathy in low-resource settings, it is less sensitive than quantitative PCR for detecting viral replication prior to the onset of clinical nephropathy. In addition, it cannot differentiate BKV infection from infection by the related polyomavirus JC, which causes significant graft dysfunction at a substantially lower frequency.Current quantitative PCR assays were developed several years ago using BKV Dun or similar genotype I strains as reference sequences for the design of primers and probes. However, our knowledge of BKV genomic diversity has expanded enormously (6,7,12,16). PCR-based diagnostic and treatment algorithms must be reevaluated to take into account newly discovered BKV single-nucleotide polymorphisms. One approach to define the potential extent of this probl...

BK Polyomavirus Genotypes Represent Distinct Serotypes with Distinct Entry Tropism

“…Our findings raise the interesting question of whether different BKV genotypes or subgenotypes exhibit altered tissue tropism or different levels of virulence in vivo. Although previous studies have surveyed BKV genotypes found in nephropathic lesions (15,16,(37)(38)(39), these studies have relied on BKV-I-biased PCR primers that underestimate the abundance of BKV genotypes II, III, and IV (40). The possibility that commonly used PCR primers do not robustly detect non-genotype I BKVs (particularly in the case of individuals coinfected with multiple BKV genotypes) is consistent with our observation that BKV-II seroprevalence (Fig.…”

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

“…We mostly evaluated patients with genotypes Ib1, Ib2, and IV, the most prevalent genotypes in our center and in Europe. It has been demonstrated clearly that some BKV quantification assays present decreased sensitivity for detection of less common subtypes of BKV, including genotypes II and Ic (12,13). It may therefore be useful to conduct further investigations with these rarer genotypes.…”

“…Most in-house quantitative PCR tests for BKV developed several years ago used the Dunlop strain (genotype I) as a reference sequence for the selection of primers and probes (13). Real-time PCR determination of VL is based on the primer amplification efficiency, the efficiency of hybridization of the primers and probes for the enzymatic cleavage, and finally the amplification efficiency relative to the target in the reference material.…”

“…However, it is very difficult to provide clinicians with accurate data, because most methods are in-house methods and no international standards have yet been established to allow comparisons between different tests. In addition, not all of the tests recently developed by manufacturers have been evaluated and compared, and measurements of BKV loads by real-time PCR assays have also been shown to vary according to BKV subtype (12,13).…”

“…However, it is very difficult to provide clinicians with accurate data, because most methods are in-house methods and no international standards have yet been established to allow comparisons between different tests. In addition, not all of the tests recently developed by manufacturers have been evaluated and compared, and measurements of BKV loads by real-time PCR assays have also been shown to vary according to BKV subtype (12,13).The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene (Argene, France), GeneProof (GeneProof, Czech Republic), and RealStar (Altona Diagnostics, Germany), on plasma and urine specimens from various patients infected with genotypes I and IV. The three PCR assays were also tested on the AcroMetrix BKV panel and by longitudinal monitoring of patients.…”