肾移植术后BK病毒感染的检测及临床意义

目的 研究肾移植患者BK病毒感染的检测方法,并探讨其临床应用价值.方法 对132例两院同种异体肾移植随访患者,采集其血、尿标本进行BK病毒的检测,包括：尿沉渣decoy细胞、尿液BKV-DNA和血浆BKV-DNA,确定是否存在BK病毒复制.结果 132例肾移植患者中,37例（28.0%）尿decoy细胞阳性,出现decoy细胞阳性的中位时间为术后12个月;BK病毒尿症32例（24.2%）,中位时间为术后11个月;BK病毒血症16例（12.1%）,中位时间为术后15个月.5例BK病毒血症患者经病理学证实为BK病毒相关性肾病.结论 尿沉渣decoy细胞和体液BKV-DNA检测能早期发现BK病毒感染,为预防肾移植术后BK病毒相关性肾病提供重要的依据.     

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].     

BK virus: Current understanding of pathogenicity and clinical disease in transplantation

BK polyomavirus (BKV) is an important cause of graft loss in renal transplant recipients that continues to pose a significant challenge to clinicians due to its frequently unpredictable onset, persistence, and the lack of effective antiviral agents or prevention strategies. This review covers our current understanding of epidemiology, viral transmission and disease progression, and treatment and prevention strategies that have been used to manage this disease.     

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.     

KIR基因多态性影响肾移植受者微量巨细胞病毒和BK病毒DNA的研究

 目的:探讨肾移植受者杀伤细胞免疫球蛋白样受体（KIR）基因多态性对肾移植术后微量巨细胞病毒（CMV）和BK病毒（BKV）DNA的影响。方法:采用序列特异性引物聚合酶链反应（PCR-SSP）法检测48例肾移植受者KIR基因多态性。依照不同功能的KIR单倍体,将KIR基因组合型分为抑制型KIR基因组合型（AA型）和非抑制型KIR基因组合型（BX型,包括BB型和AA型）。采用实时荧光定量PCR法检测肾移植术的受者血清DNA中CMV和BKV的载量。分析AA和BX KIR基因型对肾移植术后1年内CMV和BKV DNA血症累积阳性率及血肌酐的影响。结果:不同KIR基因型间,免疫抑制剂浓度无明显差异（P>005）。相较KIR-BX基因型,KIR-AA基因型的BKV累积阳性率明显增加（P<0.05）;而KIR两型间,CMV病毒血症的发生率没有明显差异（P>005）。KIR-AA型术后1～12月平均血肌酐水平较BX型低,差别有统计学意义（P<0.05）;经3年随访,KIR-AA型受者血肌酐水平低于BX型（P<0.05）,而2组间血尿素氮和尿酸水平无统计学意义。结论:KIR-AA基因型肾移植受者术后1年内BKV DNA血症增加,而不影响CMV DNA血症。     

Glomerular changes in BK virus nephropathy

This study seeks to define the glomerular changes that are associated with human BK virus nephropathy (BKVN). It is based on histopathologic review of 124 biopsies showing light-microscopic changes of viral nephropathy. The diagnosis of BKVN was confirmed by immunohistochemistry or by in situ hybridization. Histological lesions were scored by the Banff 97 criteria for renal allograft pathology and were correlated with clinical parameters. Viral cytopathic effect in the parietal Bowman's capsular epithelium was seen in 21/124 (17%) biopsies. Immunohistochemistry showed infection of Bowman's capsular epithelium in an additional 15/124 (12%) biopsies. Crescents were found in 15/124 (12%) samples. Glomerulitis exceeding grade Banff g1 was only occasionally shown (4/124=3% biopsies). Other pathologic lesions documented include mild increase in mesangial matrix in 23% biopsies, aneurysmal dilatation of glomerular capillaries in 28%, ischemic glomerulopathy in 62%, and chronic transplant glomerulopathy graded as mild (cg1) in 62% of biopsies and as moderate (cg2) in 2/124 (1.9%) biopsies. These findings show that infection of the glomerular epithelium cells can occur in a subset of patients with BKVN, most often in biopsies with high viral load in the tubular epithelium. Isolated crescents can occur in BKVN biopsies, but rapidly progressive glomerulonephritis is not observed. Two biopsies showed electron-dense deposits on ultrastructural examination, but a cause and effect relationship to BK virus infection could not be established.     

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.     

BK polyomavirus in renal transplants: role of electron microscopy and immunostaining in detecting early infection

Reactivation of BK polyomavirus (BKV) is increasingly recognized as a cause of failure of renal allografts. Since no specific treatment is available for this infection, early diagnosis is important, as it allows for early intervention and possible recovery of renal function. Forty-four consecutive renal transplant biopsies performed over a 2-year period were included in the study. In addition to evaluation of renal biopsy tissue sections using routine histochemical stains, CD3, CD20, BK virus immunostains using the specific BK virus and the SV40 antibodies and electron microscopy studies were performed. None of the transplant cases but one exhibited classical histologic viral changes. Viral particles were seen by EM in 19%, and BK-virus positivity was identified in only 43% of these cases. CD20-rich inflammatory infiltrates predominated in cases in which either positive BK stain and/or viral particles were identified ultrastructurally. A combined approach using electron microscopic and immunohistochemical evaluation can be utilized effectively to identify BK virus-associated nephropathy at an early phase facilitating early clinical intervention.     

Low frequency of BK virus in prostatic adenocarcinomas

BK virus (BKV) exhibits many oncogenic properties and has been associated with a variety of tumors in humans. BKV has not been well studied in the context of prostate neoplasia; however, an association of BKV with prostatic adenocarcinoma has been suggested based on the detection of viral DNA sequences and expression of viral proteins in clinical samples. To further investigate the reported association of BKV with prostatic adenocarcinoma and the potential role of the virus in prostate tumorigenesis, 30 cases of adenocarcinoma of the prostate were analyzed for evidence of BKV infection by in situ hybridization and immunohistochemistry. In situ hybridization analysis detected BKV DNA in 2 of 30 (7%) prostatic adenocarcinomas, with positive signals focally identified in less than 1% of the neoplastic cells in both cases. However, none of the tumors evaluated demonstrated evidence of BKV large tumor antigen expression by immunohistochemistry. Among prostatic adenocarcinomas that showed no evidence of BKV infection, BKV DNA was focally observed in the adjacent non-neoplastic prostate tissue in four cases by in situ hybridization in the absence of BKV large tumor antigen immunoreactivity. The findings of the present study indicate rare cases of prostatic adenocarcinoma may be associated with BKV infection. However, lack of localization of BKV to a large population of the neoplastic cells and absence of BKV large tumor antigen expression suggest that the virus does not play a role in the pathogenesis of prostate cancer.     

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.     

Stability of BK polyomavirus IgG seroreactivity and its correlation with preceding viremia

BackgroundRecently we showed that the level of BK polyomavirus (BKPyV) IgG seroreactivity in kidney donors predicted viremia and BKPyV-associated nephropathy in kidney transplant recipients (KTRs). This observation could be explained by assuming a direct association between BKPyV seroreactivity and the amount of persistent infectious virus in the renal allograft.ObjectivesSince the renal BKPyV reservoir is probably sowed by viremia during primary BKPyV infection, we systematically analysed the dynamics of BKPyV IgG seroreactivity in relation to preceding BKPyV viremia in KTRs and healthy individuals.Study designA cohort of 85 KTRs consisting of BKPyV viremic and nonviremic subjects was analysed for BKPyV IgG seroreactivity at five fixed time points until one year after transplantation. A cohort of 87 healthy blood donors (HBDs) was used as controls.ResultsBaseline BKPyV seropositivity was high in both KTRs and HBDs, and the baseline mean BKPyV IgG level comparable. BKPyV IgG levels in nonviremic KTRs and HBDs remained stable during follow-up, while a considerable increase was observed in viremic KTRs (p = 0.015). The increase of BKPyV seroreactivity in viremic KTRs was associated with the duration and peak level of BKPyV viremia.ConclusionsBKPyV IgG seroreactivity was stable over time in immunocompetent subjects, which enables the use of this potential pretransplantation biomarker in kidney donors. The observed dose-dependent relationship of BKPyV IgG seroreactivity with preceding BKPyV replication is in agreement with the assumption that BKPyV seroreactivity reflects past BKPyV activity and correlates with the amount of latent BKPyV residing within a kidney allograft.     

Detection of specific IgA antibodies against BK virus by ELISA.

The development and evaluation of an ELISA for analysis of anti-BKV specific IgA antibodies in human sera are described. All children with cancer with a primary BKV infection developed specific IgA antibodies, without any specific symptoms during the infection. Specific IgA was found in 61% of sera from healthy persons containing BKV IgG antibodies, using the chosen cut-off value, and BKV IgM in 4%. These results indicate that IgA production is more persistent than IgM. The high frequency of specific IgA antibodies could either be explained by frequent reactivations or long-lasting persistence of antibodies.     

Cell-mediated immune responses to BK virus in normal individuals

A lymphoproliferative assay was developed to study cell-mediated immunity (CMI) to BK virus (BKV), a human papovavirus, in healthy volunteer subjects. Responses to ultraviolet-inactivated antigen prepared from BKV-infected fibroblasts were compared to those elicited against a mock antigen preparation and an unrelated control antigen (tetanus toxoid, TET). CMI to BKV and TET were contrasted with humoral immunity as measured by enzyme-linked immunosorbent assay (ELISA). Specificity of the assay was confirmed by absence of response to mock antigen in all subjects studied. Positive response to BKV antigen was observed in all of 15 seropositive individuals but not in 5 neonates or 1 seronegative child. Similarly, all TET seropositive (n = 13) but no seronegative subjects (n = 2) responded to TET. The magnitude of lymphoproliferation to either antigen did not correlate with antibody titer. Additionally, the frequency of peripheral blood BKV-specific proliferating lymphocytes was determined by limiting dilution analysis (LDA). The frequency was approximately tenfold less than that observed for TET in the same group of subjects (1/30,300 vs 1/2,700). This may be due to differences in route and frequency of antigen exposure, both of which are unknown, at present, for BKV.     

尿液细胞学检测方法在肾移植患者多瘤病毒BK感染中的临床应用

目的 探讨尿液细胞学检测方法在肾移植患者多瘤病毒(BKV)感染筛查和监测中的价值.方法 收集2006年1月至2008年12月期间129名肾移植患者的尿液标本,镜检查找诱饵细胞(decoy细胞).再利用聚合酶链反应(PCR)检测Decoy细胞阳性患者(病毒尿症)预留的血液标本中相应的核酸并随访.血肌酐升高及持续性病毒血症、病毒尿症者进行移植肾穿刺活检术,对检测结果进行分析评价.结果 25名患者(19.4％,25/129)的439份尿液标本检查到decoy细胞,首次检测到decoy细胞的中位时间为移植后69(14～540)天;8名患者(6.2％)存在病毒血症,最早出现在移植后119(56 ～540)天.3名患者(2.3％)病理证实存在BK病毒肾病(BKVAN),全部来自持续性decoy细胞(+)组且存在病毒血症.最早检测到decoy细胞时间比病毒血症和病理诊断分别提前52和81天.Decoy细胞检测预警BKVAN的敏感度为100％,特异性82.5％,阳性预测值12.0％,阴性预测值100％.结论 尿液细胞学中的Decoy细胞镜检方法是一项简单而且敏感的筛查BK感染的方法,和病毒血症相比能更早的提示BK病毒感染.     

A 3-month course of ciprofloxacin does not prevent BK virus replication in heavily immunosuppressed kidney-transplant patients

BackgroundIn vitro and retrospective studies of kidney-transplant patients have shown that quinolones can efficiently prevent BK virus (BKV) replication. However, in a prospective study, a 3 month-course of levofloxacin did not decrease the rate of BK viruria in kidney-transplant patients treated with standard immunosuppression.ObjectivesThe aim of this study was to assess the effect of a 3-month course of ciprofloxacin prophylaxis on BKV replication in kidney-transplant patients that had received heavy immunosuppression (plasma exchange or immunoadsorption and rituximab) to achieve desensitization before undergoing HLA- and/or ABO-incompatible (ABOi) transplantation.Study designTwenty-nine patients were given ciprofloxacin (500 mg/d) for 3 months, starting immediately after transplantation. The results were compared with results from a previous study where patients had received a similar immunosuppression regimen without ciprofloxacin prophylaxis (n = 43). Around 60% of patients had undergone a retransplantation. After transplantation, all patients were given induction therapy, tacrolimus, mycophenolic acid and steroids. BK viruria and viremia were monitored at months 1, 3, 6 and 12 post-transplantation.ResultsThe rates of BK viruria, BK viremia, and BKV-associated nephropathy did not differ between patients who were given or not given ciprofloxacin prophylaxis. These rates were also identical when patients received quinolones at any time within the first year after transplantation compared to those that had not. The rate of bacterial infection was also similar in patients who had or had not received ciprofloxacin.ConclusionThe use of quinolones seemed to not have any beneficial effect in preventing BKV replication in kidney-transplant patients receiving heavy immunosuppression.