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

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 10(0) to 10(7) 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.     

A comparative study of BK and JC virus infections in organ transplant recipients

JC virus (JCV) rarely causes kidney disease, whereas BK virus (BKV) is a known cause of viral nephropathy. Existing studies on prevalence of JCV in healthy and transplanted subjects have reported only qualitative detection of viral DNA. We used quantitative PCR (qPCR) to assess JC viral load in transplant recipients and non-immunosuppressed controls, and compared JCV loads to BKV loads. JC viruria was seen in 8/23 (34.7%) controls, 23/103 (22.3%) renal, and 10/44 (22.7%) liver transplant patients. No patient developed JC viremia. BK viruria was seen in 2/23 (8.7%) controls, 36/103 (34.9%) renal, and 7/44 (15.9%) liver transplant patients. BK viremia was seen only in the kidney (8/103 = 7.7%) patients. The mean BKV urinary load was higher in kidney compared to liver patients and controls (4.22E + 07 vs. 2.88E + 05 vs. 4.39E + 02 copies/ml), whereas JC viral load was similar for all three patient groups (1.55E + 06 vs. 2.66E + 06 vs. 2.13E + 06 copies/ml). JCV viral loads were surprisingly high in all patient categories studied, but did not result in viremia or viral nephropathy. Although both BKV and JCV are widely latent in patients accepted for transplantation, concurrent reactivation of both viruses was infrequent. BKV viremia was seen in kidney but not liver recipients. The mechanisms underlying these notable phenomena remain to be investigated.     

A prospective longitudinal study of BK virus infection in 120 Czech renal transplant recipients

Polyomavirus BK (BKV) is a common human polyomavirus that rarely causes clinical symptoms in immunocompetent individuals. However, BK virus reactivation occurs in 20-40% of kidney transplant patients and 1-10% of cases present with BK virus-associated nephropathy (BKVN) and reduced kidney allograft survival. In this study, 120 consecutive renal allograft recipients were monitored for BK virus replication by real-time PCR (qPCR) in the blood and urine during the first year post-transplantation and risk factors for BK viremia, viruria, and polyoma BKV-associated nephropathy were evaluated. Receiver operating characteristic curve analysis was used to determine the cutoff points for assessing the risk of developing BKVN. In total, 1,243 samples were tested. BK-DNAuria >10(7) copies/ml and BK-DNAemia >10(4) copies/ml were found in 25.8% and 5% of the samples screened, respectively, during the 12 month follow-up period. BKVN was confirmed histologically in 3/120 patients and viremic patients were treated with dialysis for longer time periods and had higher levels of panel [corrected] reactive antibodies. Patients with viruria were also treated longer with dialysis and had impaired graft function 12 months post-transplantation. Patients with sustained viruria exhibited more acute rejection episodes than patients with transient viruria. Using receiver operating characteristic curve analysis, the cutoff point for viremia and viruria was redefined to 10(3) copies/ml serum for BK viremia and a cutoff point of 6.7 × 10(7) copies/ml in urine. In conclusion, polyoma BK viremia and viruria are frequent findings in kidney transplant recipients that warrant intensive monitoring as a means of preventing graft failure [corrected].     

Distribution of BK polyomavirus genotypes in Tunisian renal transplant recipients

BK polyomavirus (BKV) is a ubiquitous virus in humans that remains latent in the urogenital tract after a primary infection during childhood. The virus, which is reactivated frequently and excreted in urine, can cause nephropathy in renal transplant recipients. BKV sequences are classified into four subtypes (I-IV). Subtype I and IV are divided further into four and six subgroups, respectively. To characterize the subtypes of BKV prevalent in Tunisia, the presence of the virus was investigated by real-time PCR in urine samples from 77 renal transplant recipients. For subtype identification, a DNA fragment in the VP1 coding region, amplified by nested PCR from positive samples, was sequenced and a phylogenetic analysis was performed. In the studied population, subtype I (75.5%), II (14.5%), and IV (2.5%) were identified with a clear predominance of subtype Ib-2 (73%) as observed in European population. This study suggests that in North Africa, the BKV genotype distribution is similar to that of Europe and different from that of sub-Saharan Africa.     

Evaluation of a BK virus real-time PCR assay designed using novel bioinformatics technology

Monitoring of active polyomavirus BK (BKV) infections by quantitative real-time PCR is becoming a progressively more routine practice in the care of renal transplant patients due to the potential for these infections to injure transplanted kidneys. Quantitative BKV results from a previously validated, laboratory-developed real-time PCR assay based on commercially available MGB Alert® reagents were compared to results obtained from the same urine and plasma specimens using a commercially designed real-time PCR assay by IntelligentMDx. When compared qualitatively, the two assays performed identically with the exception of one urine specimen in which BKV DNA was detected near the lower limit of quantification by the MGB Alert® assay. A quantitative comparison of the results showed an average 0.55 log₁₀ copies/mL difference between the two assays. These findings suggest that despite small differences, the IntelligentMDx assay could be adopted for clinical BKV monitoring of renal transplant patients.     

Rare subtypes of BK virus are viable and frequently detected in renal transplant recipients with BK virus-associated nephropathy

BK virus-associated nephropathy (BKVN) occurs in up to 5% of kidney transplants and is a significant cause of graft loss. Four major subtypes of BKV have been described, with the vast majority of individuals persistently infected with BKV Type I (> 80% of the population). Sequencing of BKV isolates subcloned from BKVN patients revealed a high percentage of variants in the urine (40%) in the VP1 subtyping region. In vitro analysis of several viral variants revealed that all variants recovered from the urine of BKVN patients produced infectious viral particles and were replication competent in cell culture while some of the variants induced cytopathic changes in infected cells when compared to the major BKV subtype, VP1 Type I. These results suggest that rare BKV VP1 variants are more frequently associated with disease and that some variants could be more cytopathic than others in renal transplant recipients.     

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

BACKGROUND: Several studies have disclosed a correlation between polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients and its quantification in urine and serum is therefore required to assess the role of BKV infection in nephropathy. OBJECTIVE: This paper describes a urine and serum BKV-DNA quantification protocol devised to evaluate the viral load. STUDY DESIGN: Screening of samples containing > or =10(3)/ml viral genome copies by a semi-quantitative polymerase chain reaction (PCR) assay is followed by precise quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Generation of the competitor construct relied on the different sizes of wild-type and competitor amplicons. RESULTS AND CONCLUSIONS: Screening by semi-quantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and -expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. The results obtained in BKV-DNA quantification in urine and serum samples from 51 renal transplant recipients (22 on treatment with tacrolimus (FK506) and 29 on cyclosporine A (Cy A)) are interesting: BKV-DNA findings (43.1%) in urine samples are in agreement with the BKV urinary shedding reported in literature (5-45%). With regard to immunosuppressive treatment, the percentage of activation of the infection (revealed by BKV-DNA detection in urine samples) in the two groups of therapy is similar (40.9% vs 44.8%). The observation that the viral load in urine is dissociated with that of serum suggests that both parameters should be investigated in evaluation of the pathogenetic role of BKV reactivation in renal transplant recipients. Moreover, our BKV-DNA quantification protocol could be used to monitor viral load in urine and serum samples from renal transplant recipients so as to detect those at risk of nephropathy and monitor their response to immunosuppression reduction therapy if it occurs.     

Impact of genomic sequence variability on quantitative PCR assays for diagnosis of polyomavirus BK infection

Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed 184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in 81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as low as 10 copies/μl. However, consistent detection of all BKV variants was possible only at concentrations of 10,000 copies/μl or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV, genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical impression and the measured viral load.     

Update on human polyomavirus BK nephropathy

Polyomavirus BK (BKV) has ebeen identified as the main cause of polyomavirus‐associated nephropathy, a major cause of renal allograft failure. Although BKV‐associated nephropathy develops in only 2% to 5% of renal transplant recipients, its prognosis when present is very poor, with irreversible graft failure developing in 45% of affected patients. While the use of urine cytology for the detection of decoy cells has been in use for decades, other diagnostic modalities to detect BKV have emerged, including tissue biopsy, polymerase chain reaction, viral culture, and serology. Currently, there is no consensus regarding the laboratory technique best suited for clinical monitoring. This review article will discuss essential and clinical features of polyomavirus, followed by a discussion pertaining to the various diagnostic modalities that contribute to detecting polyomavirus‐associated nephropathy. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Quantitation of BK virus DNA for diagnosis of BK virus-associated nephropathy in renal transplant recipients

Quantitative measurement of BK virus DNA (Q-BKDNA) has been used for the early diagnosis and monitoring of BK virus-associated nephropathy (BKVAN). This study was designed to determine the BKDNA cutoff for the diagnosis of BKVAN. Between June 2005 and February 2007, 64 renal transplant recipients taken renal biopsies due to renal impairment submitted plasma and urine for Q-BKDNA. Eight BKVAN patients (12.5%) had median viral loads of 6.0 log10 copies/mL in plasma and 7.3 log10 copies/mL in urine. Among 56 non-BKVAN patients, 45 were negative for Q-BKDNA; 4 were positive in plasma with a median viral load of 4.8 log10 copies/ mL, and 10 were positive in urine with a median viral load of 4.8 log10 copies/mL. Receiver operating characteristic curve analysis showed that a cutoff of 4.5 log10 copies/mL in plasma and a cutoff of 5.9 log10 copies/mL in urine had a sensitivity of 100% and a specificity of 96.4%, respectively. A combined cutoffs of 4 log10 copies/ mL in plasma and 6 log10 copies/mL in urine had better performance with a sensitivity of 100% and a specificity of 98.2% than each cutoff of urine or plasma. QBKDNA with the combined cutoffs could reliably diagnose BKVAN in renal transplant recipients.     

Real-time quantitative analysis of polyoma BK viremia and viruria in renal allograft recipients

Polyoma BK virus (BKV) remains dormant in the urinary tract and circulating leucocytes and becomes reactivated during immunosuppression. BK viruria is prevalent in renal allograft recipients and BK viremia may be related to nephropathy and allograft rejection. How BK viruria and viremia are related in renal allograft patients is undefined. In this study, BKV copies in paired urine and serum samples of renal allograft recipients were measured by a real time quantitative polymerase chain reaction (Q-PCR) to test the hypothesis that their quantitative relationship might help to delineate viral reactivation patterns in these tissues. Urine and plasma samples from 44 renal allograft recipients with stable graft function were collected during outpatient follow-up and the genome copies of BKV were determined by Q-PCR. All patients showed quantifiable viremia and two groups of patients were identified: one group of patients (n=35) showed low viral load (median: 270/ml, range: 108-1000/ml) and the other group (n=9) with high viral load (median: 5x10(4)/ml, range: 2x10(4)-6x10(4)/ml). The corresponding median levels of viruria were 2000 and 900 ml. BK viremia and viruria were not related quantitatively. BK viremia/viruria were also not related to age, immunosuppression, time and source of renal grafts and serum creatinine levels. The absence of a quantitative relationship between BK viremia and viruria may reflect independent BKV reactivation in different tissues during immunosuppression.     

Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.     

Mechanisms of BK virus infection of renal cells and therapeutic implications

BK virus (BKV) causes BKV nephritis in renal transplant patients and contributes significantly to the increase of probability of graft loss. BKV, being latent in the urogenital tract, is likely to be transported with the donor kidney to recipients and following reactivation replicates in the nucleus of renal epithelial tubular cells. BKV daughter viruses are released and enter other renal epithelial cells to spread infection. There are still a lot of unknown factors about the mechanism and kinetics of BKV infection. The treatment of BKV infection, with exception of reduction in immunosuppression which increases the risk of allograft rejection, is almost exclusively limited to application of anti-viral drugs with rather inconsistent results. The shortcomings of anti-viral therapies demand the understanding of early steps of infection of permissive cells by BK virus in hope that adequate interventional therapies preventing infection of cells with BK virus could be developed. This review describes the BKV entry in target human cells, intracellular trafficking pathways of BKV particles and potential therapeutic implications based on understanding of mechanisms of BKV infection of renal cells.     

Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction.

DNAs of the human polyomaviruses BK virus (BKV) and JC virus (JCV) were amplified by the polymerase chain reaction (PCR) by using a single pair of 20-base oligonucleotide primers that were complementary to the same regions of both viruses. The sequences flanked by the primers were unique for each virus and could be differentiated by hybridization with 40-base, 32P-labeled oligonucleotide probes or by cleavage with BamHI. The DNA fragments resulting from amplification of BKV and JCV were 176 and 173 nucleotide pairs, respectively. The sensitivities of PCR for amplification of cloned BKV and JCV DNAs were 10 and 100 copies, respectively. Hybridization with the oligonucleotide probes was specific for each virus. A total of 57 urine samples from three groups of subjects were processed by DNA extraction or boiling and were tested by PCR. Urine samples collected from immunosuppressed patients (n = 11) and previously documented to be positive for BKV, JCV, or both were positive by PCR. Ten percent of urine samples from healthy adults (n = 30) that were previously negative for BKV and JCV were positive for one or both viruses by PCR. Urine samples (n = 16) from four seronegative bone marrow transplant recipients were uniformly negative for BKV. JCV was detected in deparaffinized brain tissue from a patient with progressive multifocal leukoencephalopathy. Specific diagnosis of virus in clinical specimens could be made within 1 day of receipt of the specimens. The PCR method is attractive for use in diagnosing polyomavirus infections because of its sensitivity, specificity, and rapid turnaround time.     

Genomic mutations of viral protein 1 and BK virus nephropathy in kidney transplant recipients

Genomic variability in the viral protein 1 region of BK polyomavirus (BKV) may change the ability of the virus to replicate. The significance of such changes was studied in clinical samples taken from kidney transplant patients with and without BKV nephropathy. A 94 base‐pair fragment of viral protein 1 was amplified from 68 urine, 28 blood, and 12 renal biopsy samples from eight patients with BKV nephropathy, and from 100 urine samples, 17 blood and three renal biopsy samples from 41 of 218 controls. The DNA was sequenced and the amino acid changes were predicted by the Expert Protein Analysis System program (ExPASy, Swiss Institute of Bioinformatics, Geneva, Switzerland). Single base‐pair mutations were detected more frequently in the samples from the BKV nephropathy patients than in the controls, and this was the only statistically significant finding of the study (P < 0.05), thus suggesting a greater genetic instability in BKV nephropathy associated strains. The amino acid changes were distributed at random in both BKV nephropathy patients and controls. However, one aspartic acid‐to‐asparagine substitution at residue 75 was detected in all samples of the one patient with BKV‐associated nephropathy, who developed disease progression confirmed by histology, and not in any of the other patient or control samples. Whether this specific amino acid change plays a role in disease deserves further study. J. Med. Virol. 81:1385–1393, 2009. © 2009 Wiley‐Liss, Inc.     

Decoy cells and malignant cells coexisting in the urine from a transplant recipient with BK virus nephropathy and bladder adenocarcinoma

The search for decoy cells (DC) in urine is widely used as screening for BK virus (BKV) reactivation in transplant recipients. BKV cytopathic effect of DC must not be confused with high-grade urothelial carcinoma. This report presents a case of coexistence of DC and malignant cells in the urine from a transplant recipient with BKV-associated nephropathy (BKVN) and bladder adenocarcinoma. A 38-year-old female with type 1 diabetes mellitus and end-stage renal disease underwent a simultaneous pancreas and kidney transplant. Four years post-transplantation, BK virus studies were performed for renal dysfunction. Isolated DC and DC in casts were identified in urine. Also, the tests for BKV DNA were positive in serum and renal allograft biopsy. BKVN was treatment-resistant and the patient returned to hemodialysis. A kidney transplant nephrectomy was performed 2 years later. The next urine cytology showed, in addition to DC, other distinct cells with nuclear atypia highly suggestive of malignancy. Some cells showed both, malignant and DC features. A bladder adenocarcinoma was diagnosed on biopsy and BKV proteins were demonstrated on tumor cells, supporting a possible role for BKV in the oncogenic pathway in this clinical setting. The presence of DC in the urine from a transplant recipient is the hallmark of BKV activation, but it does not exclude the existence of carcinoma. Furthermore, the presence of highly atypical cells should raise, not eliminate, the possibility of neoplastic transformation of the bladder.     

BK virus infection in human immunodeficiency virus-infected patients

The aim of this study is to evaluate the prevalence of BK virus (BKV) infection in HIV-positive patients receiving highly active antiretroviral therapy (HAART) in our hospital. The presence of BKV was analysed in urine and plasma samples from 78 non-selected HIV-infected patients. Clinical data were recorded using a pre-established protocol. We used a nested PCR to amplify a specific region of the BKV T-large antigen. Positive samples were quantified using real-time PCR. Mean CD4 count in HIV-infected patients was 472 cells/mm3 and median HIV viral load was <50 copies/mL. BKV viraemia was detected in only 1 HIV-positive patient, but 57.7% (45 out of 78) had BKV viruria, which was more common in patients with CD4 counts>500 cells/mm3 (74.3% vs 25.7%; p=0.007). Viruria was present in 21.7% of healthy controls (5 out of 23 samples, p=0.02). All viral loads were low (<100 copies/mL), and we could not find any association between BKV infection and renal or neurological manifestations. We provide an update on the prevalence of BKV in HIV-infected patients treated with HAART. BKV viruria was more common in HIV-infected patients; however, no role for BKV has been demonstrated in this population.     

肾移植后BK病毒感染者实时荧光定量PCR检测

背景：对于BK病毒感染、BK病毒相关性肾病的认识缺乏规范的实验室诊断程序和标准化的无创性检验方法。 目的：建立肾移植后患者尿液和外周血BK病毒感染负荷实时荧光定量PCR检测方法。 方法：针对BK病毒基因组,自主设计特异性引物BKV-F和BKV-R,Taqman荧光探针BKV-P,Taqman荧光探针BKV-P的5’端标记有荧光基团,在除5’端外的任意一个位置上标记有淬灭基团;然后处理待测样本,进行PCR反应。 结果与结论：将检测阳性的扩增产物进行基因测序,测序结果经BLAST比对后证实为BK病毒基因序列;利用上述方法对56份样本进行检测,其中,20份BK病毒血清样本及20份BK病毒尿液样本检测均为阳性,有S型扩增曲线。动态范围测定显示在103~1010 copies/mL之间标准曲线具有良好的相关性。5份健康人尿液样本,5份血液样本及6份临床常见的其他病原体血液样本检测均为阴性,无S型扩增曲线。结果表明该方法可进行定性、定量检测,特异性好,反应快速,一般30 min即可得到反应结果,并且成本低、假阳性少。