Comparative evaluation of laboratory developed real-time PCR assays and RealStar® BKV PCR Kit for quantitative detection of BK polyomavirus
Introduction
The BK polyomavirus, also called BK virus (BKV) is ubiquitous and highly prevalent in humans but is thought to cause disease mainly in immunocompromised individuals (Nickeleit and Singh, 2015). BKV is a non-enveloped, double-stranded DNA virus of the polyomaviridae family, which sub-clinically infects 82%–90% of the general population worldwide (Mandell et al., 2010, Pinto and Dobson, 2014). The primary infection is usually asymptomatic and occurs in early childhood followed by latent infection in the kidneys as well as other anatomical sites. Reactivation of BKV and subsequent viral shedding in urine occurs in 0–20% of asymptomatic immunocompetent individuals and in 20–60% of immunocompromised patients (Dalianis and Hirsch, 2013). BKV reactivation is associated with significant morbidity in immunocompromised patients such as polyomavirus nephropathy (PVN) in renal allograft recipients and early and late onset of hemorrhagic cystitis (HC) in BMT recipients (Han et al., 2014, Nickeleit and Singh, 2015). The risk of developing these conditions has increased as a consequence of the introduction of new potent immunosuppressants such as tacrolimus and mycophenolate in kidney transplantation and new myeloablative conditioning regimens for patients undergoing hematopoietic stem cell transplantation (HSCT) (Borni-Duval et al., 2013, Dalianis and Ljungman, 2011).
The BKV predilection for the genitourinary tract is of particular concern in renal transplant patients where virus reactivation is associated with nephropathy that occurs in up to 10% of kidney allograph recipients (Hirsch et al., 2005, Nickeleit and Singh, 2015). Hemorrhagic cystitis occurs in 9–31% of all stem cell transplantation (SCT) recipients and is associated with urinary tract obstruction, renal dysfunction and renal failure, and longer hospital stays (Han et al., 2014).
The diagnostic challenges in predicting the risk of the development of PVN and HC are significant (Nickeleit and Singh, 2015). BKV can be detected in urine by polymerase chain reaction (PCR), or by urine cytology to observe the presence of decoy cells, which is believed to be indicative of BK virus infection. BKV PCR on both urine and plasma has been proven superior to cytology for screening BK virus associated nephropathy with much higher sensitivity, specificity, and positive and negative predictive values (Bechert et al., 2010). Renal biopsy to observe viral replication in the tubular epithelial cells with large intranuclear inclusions is also used in the diagnosis of nephropathy, but a false-negative result in up to 30% can be observed (Mandell et al., 2010, Pinto and Dobson, 2014). Current clinical screening recommendations include PCR based BKV screening in urine and plasma as it is known that most cases of clinically significant PVN are preceded by a period of up to months of asymptomatic viruria followed by viremia (Nickeleit and Singh, 2015, Randhawa and Brennan, 2006). This approach is useful to rule out BKV induced nephropathy as the negative predictive value of BKV PCR in urine and blood approaches 90%. In contrast, the positive predictive value only ranges between 25 and 75%. To improve the positive predictive value of PCR based BKV assays, threshold levels of 104 copies/ml for BKV viremia have been advocated (Drachenberg et al., 2004, Nickeleit and Singh, 2015). It may pose a problem in that not all BKV PCR assays are equally sensitive and specific (Nickeleit and Singh, 2015).
In fact, variability in viral load PCR results from different laboratories has been reported which may be partly due to polymorphisms within viral genes of different subtypes of BKV but also due to the of lack of standardization of BKV PCR assays (Buller, 2010, Hoffman et al., 2008). Nevertheless, regular monitoring of BK viral load in blood and urine in transplant patients is recommended for the early detection of BKV replication for pre-emptive therapy and to monitor the course of BKV infection (Nickeleit and Singh, 2015, Randhawa and Brennan, 2006). In this study, we have compared the performance of three laboratory-developed PCR assays that target the highly conserved regions of the BKV genome, based on most recent sequence information, with a commercially available, RealStar® BKV PCR Kit.
Section snippets
Specimens
One hundred and sixteen serum specimens submitted to the Microbiology and Virology laboratory of BC Children’s Hospital between February 2014 to October 2014 were maintained at −80 °C following initial testing for BK virus. To maintain patient anonymity, each sample was coded and all patient identifiers were removed to ensure that personnel involved in this study were unaware of any patient information. Ethics approval was not considered necessary, because studies that involve the retrospective
Results
In this study, serum specimens submitted for quantitative detection of BK virus were first analysed by RealStar® BKV PCR assay, followed by laboratory developed, Taqman assays using three sets of primers and probes (Table 1): (i) VP1 assay, based on a study described by Mitui et al. (Mitui et al., 2013) designed to target the VP1 gene (ii) VP1MOD assay, based on a modification of assay described by Mitui et al. (Mitui et al., 2013) and (iii) BKLTA assay, a newly designed, minor groove binding
Discussion
Molecular diagnostic assays for infectious diseases, in particular real-time PCR, have become an indispensable part of clinical microbiology laboratories. Because of their higher sensitivity and faster turn-around time, PCR assays for many infectious pathogens are now considered as the gold standard, replacing traditional culture methods (Nolte and Caliendo, 2010). Moreover, PCR assays have enabled laboratories to perform testing for an infectious agent that is either difficult to culture or
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