Polyomavirus BK DNA quantification assay to evaluate viral load in renal transplant recipients

Merlino, Chiara; Bergallo, Massimiliano; Gribaudo, Giorgio; Gregori, Gabriella; Segoloni, Giuseppe; Giacchino, F; Ponzi, A. Negro; Cavallo, Rossana

doi:10.1016/s1386-6532(03)00012-x

Cited by 27 publications

(26 citation statements)

References 35 publications

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“…The detection limits, as well as the intra-and interassay variabilities, were consistent with those reported for other nonmultiplexed PyV-targeted qPCR assays (4,9,14,28,30). Using clinical samples, we found that the triplex qPCR assay compared very favorably to the conventional nested triplex assay described by Fedele et al (15), with specificities of Ͼ93% ( Table 4).…”

Section: Vol 46 2008 Triplex Qpcr Assay For Jc and Bk Polyomavirusesupporting

confidence: 88%

“…For example, reactivation of BKPyV is associated with hemorrhagic cystitis in bone marrow transplant recipients (22,24,25) and can also cause transplant organ failure in renal transplantation patients receiving immunosuppressive therapy (7,17,23,26,30,33). The reactivation of JCPyV is associated with the development of progressive multifocal leukoencephalopathy, a fatal condition associated with AIDS-related immunosuppression (13,20,35).…”

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confidence: 99%

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Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

2008

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We have developed a triplex TaqMan-based quantitative PCR assay for the human polyomaviruses JC (JCPyV) and BK (BKPyV). The assay simultaneously detects and quantifies both JCPyV and BKPyV in human clinical samples, and it includes an internal amplification control consisting of murine polyomavirus (MuPyV) plasmid DNA. We developed the assay for the Roche LightCycler 480 platform with the reporter dyes VIC, 6-FAM, and Cy5 for MuPyV, BKPyV, and JCPyV, respectively. The assay had a high specificity for BKPyV and JCPyV when either viral genome was present alone or in mixed samples over a range of 10 1 to 10 7 copy numbers per reaction. The analytical sensitivity was 50 copies for BKPyV and 10 copies for JCPyV. The use of the MuPyV internal control ensured monitoring of the quality of the extraction and of PCR inhibition, even in samples such as cerebrospinal fluid and plasma in which controls based on host genes cannot be effectively used. In addition, we developed a similar assay using a different dye configuration (6-FAM, VIC, and NED) that could be used on an ABI 7500 Fast platform. This assay had sensitivities similar to those of the LightCycler 480 configuration for BKPyV and JCPyV when either viral genome was present alone, but the sensitivity of detection of BKPyV was greatly decreased when an excess of JCPyV (>100-fold) was present in the sample. This internally controlled combined assay offers greater convenience and cost-effectiveness compared to separate assays for each virus and can also detect unexpected PyV activations by testing for both viruses in all samples.

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Section: Vol 46 2008 Triplex Qpcr Assay For Jc and Bk Polyomavirusesupporting

confidence: 88%

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confidence: 99%

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

2008

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“…Measurement of BKV loads in the urine and plasma of RTR is a powerful tool for identifying patients at risk of developing BKVAN and for monitoring the virologic response to therapy by such patients (13,19,26). In the present study, we described a novel rt-QPCR using the LightCycler probing system for detection and quantitation of BKV viruria and viremia.…”

Section: Discussionmentioning

confidence: 99%

“…Few studies have directly examined the association between BKV viremia and viruria (5,12,17,19,26). High levels of viruria are probably predictive of a greater risk of BKV viremia and BKVAN.…”

Section: Discussionmentioning

confidence: 99%

“…Following primary infection, the virus remains latent in the urogenital tract (30). BKV reactivation with urinary shedding of infected urothelial cells occurs in 10 to 60% of renal transplant recipients (RTR) (19,25). Although early studies found that only sporadic cases of graft dysfunction were associated with viral activation (1, 14, 15), more-recent studies demonstrate BKV-associated nephropathy (BKVAN) in as many as 8% of renal allograft recipients, with as many as 50% of patients experiencing graft loss over the next 2 to 3 years of follow-up (8, 9, 11, 20-24, 27, 28, 29).…”

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Monitoring of Polyomavirus BK Virus Viruria and Viremia in Renal Allograft Recipients by Use of a Quantitative Real-Time PCR Assay: One-Year Prospective Study

et al. 2007

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We have developed a real-time quantitative PCR (rt-QPCR) assay to detect and kinetically monitor BK virus viruria and viremia in renal transplant recipients (RTRs). A total of 607 urine and 223 plasma samples were collected from 203 individuals including those with BK virus-associated nephropathy (BKVAN) (n ‫؍‬ 8), those undergoing routine posttransplant surveillance (SV) (n ‫؍‬ 155), those with nontransplant chronic kidney disease (NT-CKD) (n ‫؍‬ 20), and healthy living kidney donors (LD) (n ‫؍‬ 20). The rt-QPCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.4 to 11 log 10 copies/ml) and very good precision (coefficient of variation, ϳ5.9%). There was a significant difference in the prevalences of viruria and viremia between the BKVAN (100% and 100%) and SV (23% and 3.9%) groups (P < 0.001). No viruria or viremia was detected in LD or in NT-CKD patients. The median (range) peak levels of BK virus viruria and viremia, in log 10 copies/ml, were 10.26 (9.04 to 10.83) and 4.83 (3.65 to 5.86) for the BKVAN group versus 0 (0 to 10.83) and 0 (0 to 5.65) for the SV group, respectively (P < 0.001). When the BK virus load in the urine was <7.0 log 10 copies/ml, no BK virus viremia was detected. When the BK virus load in the urine reached 7.0, 8.0, 9.0, and >10.0 log 10 copies/ml, the corresponding detection of BK virus viremia increased to 20, 33, 50, and 100%, respectively. We propose monitoring of BK virus viruria in RTRs, with plasma BK virus load testing reserved for those with viruria levels of >7.0 log 10 copies/ml. BK virus (BKV) is a polyomavirus with a circular DNA genome of approximately 5,300 bp. BKV causes ubiquitous infection in early childhood, with seroprevalence in adults ranging from 60 to 100% (31). Following primary infection, the virus remains latent in the urogenital tract (30). BKV reactivation with urinary shedding of infected urothelial cells occurs in 10 to 60% of renal transplant recipients (RTR) (19,25). Although early studies found that only sporadic cases of graft dysfunction were associated with viral activation (1, 14, 15), more-recent studies demonstrate BKV-associated nephropathy (BKVAN) in as many as 8% of renal allograft recipients, with as many as 50% of patients experiencing graft loss over the next 2 to 3 years of follow-up (8, 9, 11, 20-24, 27, 28, 29). Accumulated data suggest that prospective monitoring of patients at risk for BKVAN may identify those with active infection before renal function deteriorates (3,8,13,18,22). Early identification provides the opportunity for intervention with reduction of the immunosuppression in an effort to control BKV replication and prevent BKVAN (4, 16).Progression to BKVAN occurs without clinical signs or symptoms except for increasing serum creatinine concentrations. The "gold standard" for diagnosis of BKVAN continues to be renal biopsy with demonstration of viral cytopathic tubulointerstitial changes (7, 23). Sometimes, however, the biopsy result can be falsely negative due to the focal nature of the diseas...

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BK virus‐associated nephropathy in kidney transplant recipients

Bruyn

Limaye

2004

Reviews in Medical Virology

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Polyomavirus BK has emerged as an important cause of renal allograft infection leading to allograft dysfunction and loss in kidney transplant recipients. Significant progress has been made, particularly in the area of diagnostic methods for BK virus, thereby facilitating diagnosis, screening and monitoring of infection. This review outlines current concepts on the epidemiology, pathogenesis, diagnosis and therapy of BK virus nephropathy. The precise risk factors that are important for induction and progression of infection to invasive disease, the most effective diagnostic strategies, and the efficacy of current therapeutic approaches, all remain to be defined fully. It is to be hoped that these deficiencies will stimulate research to address these important questions.

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Polyomavirus BK DNA quantification assay to evaluate viral load in renal transplant recipients

Cited by 27 publications

References 35 publications

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

Monitoring of Polyomavirus BK Virus Viruria and Viremia in Renal Allograft Recipients by Use of a Quantitative Real-Time PCR Assay: One-Year Prospective Study

BK virus‐associated nephropathy in kidney transplant recipients

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