BK virus (BKV) quantification in urine samples of bone marrow transplanted patients is helpful for diagnosis of hemorrhagic cystitis, although wide individual variations exist

Abstract

Background: Hemorrhagic cystitis (HC) in allogeneic bone marrow transplanted (BMT) patients is associated with BK virus (BKV) reactivation manifested as BK viruria. However, since 77–90% of all adult BMT patients excrete BKV, viral reactivation alone cannot be responsible for HC. Recently, a significant overrepresentation of C→G mutations in the Sp1 binding site in the non-coding control region (NCCR) of BKV was shown to be present in HC patients and absent in non-HC patients. Objectives: We aimed to investigate if this mutation resulted in excessive BKV excretion in HC patients. Study design: A Real-Time PCR was developed and used to quantify BKV in urine samples from 21 patients with HC, with and without the mutations, as well as from patients without HC. Results: Quantification of BKV was successful in 18 of 21 urine patients (six with and six without C→G mutations) and six patients without HC. A mean of 3.0×10⁶ BKV copies/μl was detected in urine samples of HC patients with C→G mutations, compared to a mean of 1.5×10⁶ BKV copies/μl in HC patients without C→G mutations and a mean of 1.0×10⁶ BKV copies/μl in patients without HC. The obtained differences were however not statistically significant, due to one individual non-HC patient with an extremely high BKV copy number. Nevertheless, while 50% of the samples in the HC groups expressed 1×10⁶ copies/μl or more, only one of the samples in the non-HC group contained a virus quantity higher than 5×10⁵ copies. Conclusions: Although we could not confirm that the C→G mutations in the Sp1 site of BKV were responsible for an increased viral load in patients with HC, our data suggest that levels of BKV above 10⁴ copies/μl may indicate a risk for HC.

Introduction

BK virus (BKV), a human polyomavirus with a sero-prevalence of 60–90% (Walker and Padgett, 1983), infects humans at an early age and remains latent in kidneys, peripheral blood and brain (Chesters et al., 1983, Elsner and Dörries, 1992, Dorries et al., 1994). Although there is no clear evidence that primary infection is associated to any clinical illness, a few case reports on urinary tract disease have been described (Hashida et al., 1976, Saitoh et al., 1993). In allogeneic bone marrow transplanted (BMT) patients, BKV reactivation has been suggested to be linked to a late onset hemorrhagic cystitis (HC), a condition with an incidence of 5–34%, characterised by lower abdominal pain, dysuria, frequent micturition and hematuria (Arthur et al., 1986, Ilhan et al., 1997, Childs et al., 1998, Seber et al., 1999, Vogeli et al., 1999). Nevertheless, since BKV can be detected in urine samples of 77–90% of all adult BMT patients both with and without HC (Azzi et al., 1996, Bogdanovic et al., 1996), it is apparent that BKV reactivation alone is not sufficient to cause HC. Several studies have focused on investigating additional factors involved in the development of HC. Graft versus host disease (GVHD) has been implied (Ost et al., 1987), but has not been confirmed to be a co-factor (Bogdanovic et al., 1996). Primary BKV infection in BMT patients has also been rejected as a major cause of HC (Bogdanovic et al., 1998) and specific BK types have not been shown to induce HC (Jin et al., 1995). Recently, it has been shown that BMT patients with HC generally excrete more BKV in the urine than patients without HC (Azzi et al., 1999, Biel et al., 2000), suggesting that BKV reactivation indeed is strongly associated to HC. Furthermore, when we sequenced the non-coding control region (NCCR) of BKV in urine samples of BMT patients with and without HC, we found that 43% of BMT patients with HC, but none of the patients without HC had C→G mutations in the BKV NCCR Sp1 transcriptor factor binding site (Priftakis et al., 2001). Since the Sp1 transcription factor can be a positive regulator of BKV transcription (Cassill and Subramani, 1989, Sundsfjord et al., 1990), we wished to investigate if the detected Sp1 mutations could influence the replication activity of the virus and possibly also account for the increased BKV excretion newly reported in patients with HC (Azzi et al., 1999, Biel et al., 2000). For this purpose a BKV specific Real-Time PCR was developed and used to quantify BKV in urine samples of patients with HC (with and without C→G mutations in the BKV NCCR Sp1 site) and of patients without HC.