Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays

Abstract

BK virus (BKV) is the infectious cause of polyomavirus-associated nephropathy. Screening guidelines for renal-transplant recipients define levels of viremia and viruria that are actionable for additional testing or intervention. However, standardized real-time PCR primers, probes, and standards are unavailable, and the extent of agreement among published assays is unknown. We compared seven TaqMan real-time PCR primer/ probe sets (three designed at this institution, three described in the literature, and one purchased) in conjunction with two different standards to prospectively measure BKV titers in 251 urine specimens submitted to our clinical laboratory. We observed substantial disagreement among assays attributable both to features of primer and probe design and to choice of reference material. The most significant source of error among individual specimens was primer or probe mismatch due to subtype-associated polymorphisms, primarily among subtype III and IV isolates. In contrast, measurement of the most abundant subtypes (Ia, V, and VI) were typically uniform among all seven assays. Finally, we describe and validate a new clinical assay designed to reliably measure all subtypes encountered in our study population (Ia, Ic, III, IV, and VI). Consideration of available BKV sequence information in conjunction with details of subtype distribution allowed us to develop a redesigned assay with markedly improved performance. These results suggest that both accurate BKV measurement and the uniform application of BKV screening guidelines could be significantly improved by the use of standardized reference materials and PCR primers and probes.BK virus (BKV) is a small, nearly ubiquitous DNA virus in the family Polyomaviridae that causes chronic, usually asymptomatic infection in immunocompetent individuals. Since the advent of potent antirejection therapy, BKV has emerged as the primary infectious cause of polyomavirus-associated nephropathy (PVAN), affecting 1 to 10% of renal-transplant recipients, and with frequencies of graft loss between 10% and 80% (7).Quantitative assays for BKV in urine and serum are used as both screening and diagnostic tests for PVAN in renal-transplant recipients. Prospective studies of renal-allograft patients have demonstrated that high levels of BKV viruria precede sustained viremia, which in turn precedes evidence of allograft dysfunction (2). Furthermore, screening blood or urine for high levels of BKV replication in conjunction with interventions, such as reduction of immunosuppression (2) or treatment with cidofovir (12), triggered at defined levels of viremia or viruria has been associated with improved clinical outcomes.Renal-transplant programs have instituted BKV screening protocols, and quantitative BKV testing is increasingly performed in molecular virology laboratories. In 2005, an expert panel recommended the use of either urine cytology or nucleic-acid-based testing to screen renal-transplant recipients during the first 2 years after transplantation who present with evidenc...

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.…”

Quantitative Nucleic Acid Amplification Methods for Viral Infections

“…Mini-Reviews standard of care for most renal transplant programs, there is no FDA-cleared test, and a number of laboratorydeveloped tests are deployed in clinical laboratories with marked variability in BKV viral load measurements among commonly used tests (21 ). This interassay variability complicates uniform application of BKV screening guidelines.…”

Topics in Transplantation Medicine for General Nephrologists

“…Interlaboratory variability in the PCR assay makes the diagnosis challenging (38), and there is no consensus regarding the level of plasma BK PCR that should be treated. We have little idea as to the natural history of patients with low-level viremia and normal allograft function.…”