Correlation Between Urine and Serum BK Virus Levels After Renal Transplantation

Correlation Between Urine and Serum BK Virus Levels After Renal Transplantation

Correlation Between Urine and Serum BK Virus Levels After Renal Transplantation Y. Funahashi, M. Kato, T. Fujita, K. Tsuruta, S. Inoue, and M. Gotoh A...

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Correlation Between Urine and Serum BK Virus Levels After Renal Transplantation Y. Funahashi, M. Kato, T. Fujita, K. Tsuruta, S. Inoue, and M. Gotoh ABSTRACT Background. Quantification of the serum level of BK virus is used as a surrogate marker for the early onset of BK virus nephropathy. However, little is known about the diagnostic value of the urine level of BK virus for nephropathy or the relationship between the serum and urine viral load. We investigated the correlation between urine and serum BK virus levels after renal transplantation. Methods. From November 2008 to August 2013, a total of 270 renal transplant patients who were followed at our institution were included in this study. Urine and serum were collected simultaneously. BK virus levels were quantified in 894 urine and serum samples using a real-time polymerase chain reaction assay. Results. BK virus was detected in 178 urine samples and 36 serum samples. Among the BK virusepositive urine subjects, the positive predictive value for viral detection in the serum was 9% (13/147) when the urinary virus level was <107 copies/mL and 74% (23/31) when the urinary virus was 107 copies/mL. Serum BK viral levels were w2e3 log units lower than those in urine. Conclusions. BK virus was detected more frequently in serum when present in urine at 107 copies/mL after renal transplantation.

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OLYOMAVIRUS type BK infects >80% of adults [1e3]. After primary infection, BK virus remains latent in the renourinary tract, B cells, brain, spleen, etc [4]. With intense immunosuppression after renal transplantation, reactivation with asymptomatic viruria can progress to viremia, which can develop into BK virus nephropathy with interstitial nephritis and/or ureteral stenosis. This occurs in 1%e10% of recipients, leading to the loss of allograft function and a return to hemodialysis [5e11]. BK virus nephropathy is diagnosed by the immunohistochemical detection of BK viruseinfected tubular epithelial cells in an allograft biopsy specimen. In recent years, many studies have reported that quantification of the serum virus load by real-time polymerase chain reaction (PCR) is useful in identifying patients at risk of developing BK virus nephropathy and in following the course of BK virus nephropathy [5,6,11e13]. At the time of development of BK virus nephropathy, the urine viral load is beyond the upper threshold of that in generally used PCR systems (107 copies/mL). Therefore, little is known about the diagnostic value of urine BK virus for nephropathy or the relationship between the serum and ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 567e569 (2014)

urine viral load. We previously developed a real-time PCR system that enabled quantification of a wide range of viral load from 102 to 1011 copies/mL [14]. In the present study, we quantified the BK virus level in the serum and urine of renal transplant recipients and elucidated the relationship of the viral load between the 2 samples. MATERIALS AND METHODS The study protocol was approved by the Ethics Committee at Nagoya University Graduate School of Medicine before commencement. Each of the patients provided written informed consent to enroll in the study.

From the Department of Urology, Nagoya University Graduate School of Medicine, Nagoya, Japan. Supported by the Aichi Kidney Foundation. Address reprint requests to Yasuhito Funahashi, MD, PhD, Department of Urology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550; Japan. E-mail: [email protected] 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.154 567

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Patients and Samples From April 2005 to August 2013, a total of 270 renal transplant recipients who were followed in our institution were enrolled in this study. A total of 894 urine and serum samples each were collected simultaneously and kept at 20 C until viral extraction.

Viral DNA Extraction in Serum and Urine Samples Viral DNA was extracted from 140-mL urine samples with the use of an automatic DNA extraction machine, the QIAcube (Qiagen, Hilden, Germany), using QIAamp Viral RNA kits (Qiagen) and eluted in 60 mL water. Viral DNA was extracted from 200-mL serum samples with the use of QIAamp DNA blood kits (Qiagen) and eluted in 200 mL water.

Quantification of Viral DNA Viral DNA was quantified with the use of a Taqman real-time PCR assay that we established previously [14]. Briefly, the PCR assay was performed in a total reaction volume of 25 mL containing 5 mL DNA extract, 12.5 mL 2 Quantitect multiplex PCR master mix (Qiagen), forward and reverse primers, and the Taqman probe. A passive reference dye, Rox, was included in the reaction mixture. Amplification and real-time fluorescence detection were performed with the use of the Mx3000P real-time PCR system (Stratagene, La Jolla, California) and the following protocol: initial denaturation and polymerase activation for 15 minutes at 95 C, followed by 50 cycles at 94 C for 30 seconds and 60 C for 60 seconds. Real-time fluorescence measurements were recorded, and a threshold cycle (Ct) value for each sample was calculated by determining the point at which the fluorescence exceeded the threshold. Each real-time PCR assay contained a standard dilution series for DNA quantification, and all samples were analyzed in duplicate. Negative control samples were included with each run. The number of viral DNA copies was calculated from these standard curves and expressed as copies per 1 mL of urine or serum.

Statistical Analysis The chi-square test was used to test the positive predictive values between groups. Pearson correlation coefficient was used to compare the BK viral load in the urine and serum. A P value of <.05 was considered to be statistically significant. All statistical analyses were performed with the use of SPSS software.

RESULTS

The mean patient age was 45 years (range, 8e77 y). A total of 190 patients were living-related renal transplant recipients. The median period of sample collection after transplantation was 32 months (range, 0e402 mo). The maintenance immunosuppressants included cyclosporine (n ¼ 115), tacrolimus (n ¼ 147), and mycophenolate mofetil (n ¼ 198). The kidney diseases making renal transplantation necessary included chronic glomerular nephropathy (n ¼ 119), IgA nephropathy (n ¼ 46), diabetic nephropathy (n ¼ 30), hypertension nephropathy (n ¼ 11), follicular glomerular sclerosis (n ¼ 10), and miscellaneous causes (n ¼ 54). BK virus was detected in 178 urine samples and 36 serum samples. The positive BK viral load ranged from 1.4  101 copies/mL to 1.9  1010 copies/mL in the urine and 7.1  101 copies/mL to 8.0  106 copies/mL in the serum. Among the

Fig 1. Correlation between serum and urine BK virus. When the urine BK virus level exceeded 107 copies/mL, BK virus was more frequently amplified in the serum. Serum BK viral levels were w2e3 log units lower than those in urine. A line indicates the correlation between the positive urine and serum BK viral loads.

subjects with BK virusepositive urine, the positive predictive value for viral detection in the serum was 9% (13/147) when the urinary virus level was <107 copies/mL and 74% (23/31) when the urinary virus level was 107 copies/mL (P < .001). Pearson correlation coefficient demonstrated a statistically significant correlation between urine- and serum-positive BK viral loads (P < .001; R2 ¼ 0.587). Serum BK viral levels were w2e3 log units lower than those in urine (Fig 1). DISCUSSION

BK virus is latently present in urothelial cells, even in normal populations; therefore, detection of BK virus DNA in the urine does not always imply the presence of a tubulointerstitial disorder. Latent BK virus in the urothelium of immunologically healthy humans becomes reactivated and proliferates under immunosuppressive conditions after renal transplantation, then brims over into the blood when the viral load increases in the kidney. Many studies have examined the relationship between BK virus nephropathy and the urine or serum BK viral level (Table 1). Viscount et al reported that a plasma BK virus DNA level of >1.6  104 copies/mL and a urine BK virus DNA level of >2.5  107 copies/mL were highly associated with concurrent nephropathy [11]. Hirsch et al reported that a plasma BK virus DNA level of >104 copies/mL is recommended for a presumed diagnosis of BK virus nephropathy, and the urine viral load in affected patients was >107 copies/mL [15]. The Kidney Disease: Improving Global Outcomes clinical practice guidelines also suggest the reduction of immunosuppression when the BK virus load in plasma is

URINE AND SERUM BKV

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Table 1. Proposed BK Viral Load for the Diagnosis of BK Virus Nephropathy Author

Serum BK virus

Boudreault et al 3.0  103 Randhawa et al 5  103 KDIGO guideline 104

Urine BK virus

N/A 107 N/A

Hirsch et al Chung et al Costa et al

104 1  104 1.6  104

107 1  1010 N/A

Viscount et al

1.6  104 2.5  107

Journal

J Clin Virol 2009 [17] J Clin Microbiol 2004 [18] Nat Rev Nephrol 2009 [16] Transplantation 2005 [15] Transpl Int 2012 [19] Nephrol Dial Transplant 2008 [20] Transplantation 2007 [11]

Serum and urine BK viral load are expressed as copies/mL.

persistently >104 copies/mL, to prevent disease progression to an irreversible phase [16]. These studies concluded that a presumed diagnosis of BK virus nephropathy may be made on the basis of surrogate markers of serum viral replication. Generally used PCR systems can quantify a BK virus level of <107 copies/mL, which urine viral load exceeds in the case of polyomavirus nephropathy. Therefore, little is known about the diagnostic value of the urine BK virus level for nephropathy or the relationship between serum and urine viral loads. We previously developed a real-time PCR system that enables quantification of a wide range of viral load from 102 to 1011 copies/mL [14]. Using this system, we revealed that serum BK viral levels were w2e3 log units lower than those in urine, and that the serum BK virus load of 104 copies/mL is comparable to a urine BK virus load of w107e108 copies/mL. Quantification of the urine viral load for the screening of BK virus nephropathy is superior to quantification of the serum viral load in several aspects. The BK virus appears in urine before it appears in serum in the clinical course of BK virus nephropathy; therefore, quantification of the urine viral load is more suitable to evaluation of the risk of development of nephropathy at an earlier stage. Urine decoy cells can be observed when the urine BK virus increases to w104e105 copies/mL [14], when BK virus is not amplified in the serum in many cases. Taken together, these findings indicate that screening for BK virus replication in urine can not only predict the risk of the onset of BK virus nephropathy but also allows for the follow-up of the clinical course of BK virus nephropathy. In conclusion, we investigated the correlation between urine and serum BK virus levels after renal transplantation. We found that serum BK viral levels were w2e3 log units lower than those in urine and that BK virus was detected more frequently in serum when present in urine at 107 copies/mL. ACKNOWLEDGMENTS This trial was completed in collaboration with the following urologists participating in the Tokai Urological Clinical Trial Group: Ryohei Hattori, Tokunori Komatsu, and Tsuneo Kinukawa.

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