Detection and quantitation of BK virus DNA by real-time polymerase chain reaction in the LT-ag gene in adult renal transplant recipients

https://doi.org/10.1016/j.jviromet.2005.06.025Get rights and content

Abstract

Determination of polyomavirus BK (BKV) load in urine and plasma has been advocated for monitoring adult renal transplant recipients suffering from BKV-related nephropathy. An “in-house” real-time quantitative PCR assay was developed using the BKV-1/BKV-3 primers set in the large tumor antigen (LT-ag) region to quantitate BK virus loads in plasma and urine in renal transplant patients. This assay was adapted to routine virology laboratory by evaluating two extraction procedures of nucleic acids from urine and plasma, one manual and the other using an automatic extractor, and by evaluating the Light Cycler versus Taqman apparatus. Both the manual and automatic extraction procedures and real-time PCR apparatus were equivalent. The Light Cycler and Taqman instruments allow similarly rapid, accurate, reproducible and specific quantitative detection of the three major BKV subtypes, with a detection limit of 10 BKV DNA copies/ml, and a range from 100 to 107 copies/ml. Of 855 renal transplant patients, 128 (15%) had BKV DNA in both plasma and urine samples with a mean viral load of 5.1 log/ml in plasma and 6.8 log/ml in urine and in 5 (4%) BKV-associated tubulo-interstitial nephropathy; 332 (39%) BKV DNA was found only in the urine, not in the plasma, without further development of nephropathy and 395 patients had no BKV in plasma and urine. These observations emphasize the usefulness of real-time PCR to assess the BKV load by routine testing, and confirm the need to combine both plasma and urine determinations of the BKV DNA load in order to identify renal transplant patient at high risk for BKV-associated nephropathy.

Introduction

The ubiquitous papovavirus BK (BKV) harbors primary tropism for renal epithelium and urothelium. Classically, BKV infection depicts asymptomatic primary-infection followed by latent persistence (Hirsch and Steiger, 2003). Asymptomatic reactivation and urinary shedding may be observed in pregnant women and immunocompromised patients, such as transplant patients (Chan et al., 1994). In addition, symptomatic BKV reactivations have been reported in patients suffering from hemorrhagic cystitis after bone marrow transplantation (Arthur et al., 1988) and from tubulo-interstitial nephropathy after renal transplantation (Rosen et al., 1983, Coleman et al., 1978, Mathur et al., 1997, Smith et al., 1998, Binet et al., 1999, Drachenberg et al., 1999, Howell et al., 1999, Randhawa et al., 1999, Nickeleit et al., 2000). BKV-associated tubulo-interstitial nephropathy has been described recently as an emerging infection in renal transplant patients, leading to graft loss in some patients which may be difficult to differentiate from allograft rejection (Randhawa et al., 1999). The identification of renal transplant patients at risk of developing BKV-associated nephropathy may be achieved by the detection of BKV-infected urothelial cells (“decoy cells”) in urine, with high negative predictive value of 98% (Drachenberg et al., 1999) but poor positive predictive value of less than 30% concerning the possibility of nephropathy development (Nickeleit et al., 2000). Polyoma BKV load in plasma was higher in patients with BKV-associated nephropathy than in those without nephropathy (Hirsch, 2002b). Finally, quantitation of BKV load in blood and urine has been suggested for the control of both asymptomatic and symptomatic BKV reactivation in transplant patients by modulating the immunosuppressive treatment (Limaye et al., 2001, Leung et al., 2002, Vats et al., 2003, Randhawa et al., 2004).

In the present study, routine detection and quantitation of BKV DNA in plasma and urine from renal transplant patients are described, using an “in house” BKV-specific real-time PCR.

Section snippets

Plasmids, primers and probe for quantitation

A 140-bp DNA fragment derived from the human albumin gene was generated from human DNA by conventional PCR with phosphorylated primers 5′-AAACTCATGGGAGCTGCTGGT-3′ and 5′-GCTGTCATCTCTTGTGGGCTG-3′. The PCR product was cloned into pcDNA3.1/HisC (Invitrogen, Carlsbad, CA, USA), leading to pcDNA3.1/HisC-alb. A 179-bp PCR product encompassing nucleotides 4376–4539 of the LT-ag gene was generated with primers BKV-F 5′-CGGAATTCCGGGTGTAGATCAGAGGGAAAGTCT-3′ and BKV-R 5′-CGGGATCCCGGAGTCCTGGAGTTCCTT-3′

Detection limit, specifity and reproducibility

The detection limit, specifity and intra- and inter-assay reproducibility of BKV real-time PCR using the BKV-1 and BKV-3 primers sets and corresponding BKV-TMS probe were assessed in parallel using the Light Cycler instrument and the ABI 7700 sequence detector system.

Ten-fold serial dilutions of the pcDNA-BKV plasmid from 107 to 101 copies/ml were used to generate a standard curve. Each dilution was detected in triplicate by both real time procedures. The point corresponding to 10 copies per

Discussion

Traditional methods for detecting and identifying polyomaviruses include serologic methods, virus isolation by cell culture and electron microscopy (Giraldo et al., 1982, Knowles et al., 1989, Nilsen et al., 1991, Haysom et al., 2004). Recently, several studies have shown molecular diagnosis of BKV to be an effective tool for detecting polyomaviruses in a range of clinical samples (Arthur et al., 1989, De Santis and Azzi, 1996). Thus, molecular detection specific of BKV DNA reported previously

Acknowledgment

The study was supported by the PHRC no P021104 from the Direction de la Recherche Clinique, Assistance Publique-Hôpitaux de Paris, France.

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