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.     

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。     

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.     

Molecular characterization of BK polyomavirus subtypes in renal transplant recipients in Brazil

BK polyomavirus (BKV) is highly prevalent in the world population. Different reports indicate that BKV subtypes and subgroups present an uneven geographical distribution which might be correlated with human migration. However, there is a lack of data on the BKV subtype distribution in the South American population. The occurrence of BKV subtypes and subgroups detected in 51 kidney transplant recipients in Rio de Janeiro, Brazil is described. According to genetic studies, the population in this region descends mainly from European or African immigrants, with a relatively low genetic background from the Amerindians. By sequencing the VP1 region of BKV, subgroups Ib1 and Ia of subtype I were found in 34 (67%) and 15 (29%), respectively, of samples, while subtype II was present in 2 (4%) of the samples. Subtypes III and IV were not detected. Phylogenetic analysis indicated similarities between Brazilian BKV subgroup Ia and East African lineages; and subgroup Ib-1 with Asian and North American lineages, while subtype II samples were similar to sequences from Japan and the UK. This is the first report that describes distribution of BKV subtypes in South America. The high prevalence of BKV subgroup Ia probably reflects the high proportion of African descendants in this population. On the other hand, the predominance of subgroup Ib-1 and the absence of Ib-2 in an area with a high proportion of European ancestry was unexpected. Further studies in South American populations are needed to provide a better understanding of the epidemiology of BKV in this region.     

BK virus-associated nephropathy in kidney transplant recipients

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.     

Systematic screening of BK virus by real-time PCR prevents BK virus associated nephropathy in renal transplant recipients

BK virus associated nephropathy occurs in approximately 5% of renal transplant recipients. Quantitation of BKV DNA in serum/plasma early in the course of disease has been suggested to be an important diagnostic tool for polymavirus-associated nephropathy (PVAN). The aim of this study was to develop a BKV real-time PCR (qPCR), which could be included in a diagnostic qPCR platform. Additionally, the significance of the assay as a surrogate marker for PVAN was investigated. Quantitation of BKV DNA by qPCR was carried out on 234 serum samples from a retrospective study including 31 renal transplant recipients monitored for at least 6 months post-transplantation. BKV viremia was detected in 9 out of 31 patients. Four patients had a viral load of >10,000 copies/ml at least on one occasion. In two of these patients, PVAN was diagnosed clinically during the study period. In retrospect, these patients were shown to be BKV positive before the clinical diagnosis of PVAN was made. Another two patients had a permanent graft dysfunction, but were never clinically diagnosed with PVAN. None of the remaining five patients with BKV DNA (<10,000 copies/ml) had renal impairment. Based on these results, an algorithm was introduced at the study center in 2006 and to date, August 2011, no cases of PVAN with loss of graft have been observed. The concept of including different PCR protocols in a common qPCR platform allows laboratories with small sample numbers to perform regularly a variety of assays at a reasonable cost.     

Measurements of global cell-mediated immunity in renal transplant recipients with BK virus reactivation

The Cylex ImmuKnow Test (Cylex, Columbia, MD) measures immune cell function (ICF) and is based on the amount of adenosine triphosphate (ATP) released when T cells are stimulated by phytohemagglutinin. This preliminary study sought to determine if ICF measurements can be used to stratify kidney transplant recipients according to the risk for developing BK virus infection. ICF measurements were done in 15 samples from 8 patients with BK viremia, 38 samples from 25 patients with BK viruria, and 243 samples from 148 patients with no BK viruria or viremia. The mean+/-SD amounts of ATP released in these 3 groups were 102.9+/-58.6, 227.2+/-146.4, and 231.8+/-150.8 ng/mL, respectively (P= .002, viremia vs all other samples). Within the viruria group, lower ICF values were associated with higher urinary viral load (P= .037). These results show that a decreased ICF test result correlates with active viral replication in kidney transplant recipients.     

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.     

Testing for polyomavirus type BK DNA in plasma to identify renal-allograft recipients with viral nephropathy

BACKGROUND: Reactivation of polyomavirus type BK (BK virus) is increasingly recognized as a cause of severe renal-allograft dysfunction. Currently, patients at risk for nephropathy due to infection with the BK virus are identified by the presence of cells containing viral inclusion bodies ("decoy cells") in the urine or by biopsy of allograft tissue. METHODS: In a retrospective analysis, we performed polymerase-chain-reaction assays for BK virus DNA in plasma samples from 9 renal-allograft recipients with BK virus nephropathy; 41 renal-allograft recipients who did not have signs of nephropathy, 16 of whom had decoy cells in the urine; and as immunocompromised controls, 17 patients who had human immunodeficiency virus type 1 (HIV-1) infection (stage C3 according to the classification of the Centers for Disease Control and Prevention) and who had not undergone transplantation. RESULTS: In all nine patients with BK virus nephropathy, BK virus DNA was detected in the plasma at the time of the initial histologic diagnosis (a mean [+/-SD] of 46+/-28 weeks after transplantation) and during the course of histologically diagnosed, persistent BK virus disease. In three of the six patients with nephropathy who were studied serially after transplantation, BK virus DNA was initially undetectable but was detected 16 to 33 weeks before nephropathy became clinically evident and was confirmed by biopsy. Tests for BK virus DNA in plasma became negative and the nephropathy resolved after the doses of immunosuppressive drugs were decreased in two patients and after removal of the renal allograft in three patients. BK virus DNA was found in the plasma of only 2 of the 41 renal-allograft recipients who had no signs of nephropathy and in none of the patients with HIV-1 infection. CONCLUSIONS: Testing for BK virus DNA in plasma from renal-allograft recipients with use of the polymerase chain reaction is a sensitive and specific method for identifying viral nephropathy.     

Prospective study of the human polyomaviruses BK and JC and cytomegalovirus in renal transplant recipients.

Forty eight renal transplant recipients were investigated prospectively for evidence of infection with the polyomaviruses BK and JC and cytomegalovirus. An active polyomavirus infection was shown in 31 patients (65%) and cytomegalovirus in 30 (62.5%). Half of the BK and JC virus infections occurred within the first three months after transplantation compared with 93% of the cytomegalovirus infections. Very late polyomavirus infections two or more years after the transplant were also shown. Cytology was useful in identifying polyomavirus but not cytomegalovirus infections, and 21 (68%) of the 31 polyomavirus infected patients excreted inclusion-bearing cells. Only three patients had symptoms possibly associated with the polyomavirus infection. One patient with BK virus infection developed ureteric stenosis and a second patient had malaise and vomiting. One patient with JC virus infection developed pericarditis and effusion. Renal function became impaired at the time of the polyomavius infection in eight patients (26%) and ureteric obstruction and pericarditis developed in two patients treated with methyl prednisolone for possible rejection. At the end of the study 25 of the 31 polyomavirus infected patients (81%) had functioning renal grafts. The detection of polyomavirus infection is important as increased immunosuppression needs to be avoided to prevent possible complications such as ureteric stenosis in transplant recipients.     

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.     

Association between viral infections and post-transplant malignancies in renal transplant recipients

Immunosuppressive treatment is associated with an increased incidence of different malignant diseases. The etiology of posttransplant malignancies is multifactorial and includes decreased immune response to different viral infections, inappropriate removal of damaged cells, and impaired ability to repair DNA. EBV, HHV-8, Merkel cell virus, hepatitis B virus, hepatitis C virus and BK virus are all considered to be involved in the etiology of post-transplant malignancies. CMV has been considered as a potential causative factor in the development of colon cancer. However, current knowledge is mainly based on case reports. Further studies are needed to establish the causative role of different viruses in the etiology and pathogenesis of different malignant diseases in renal transplant population.     

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 histopathologic disease severity does not predict allograft outcome in renal transplant recipients

AimsBK polyomavirus nephropathy (BKPyVN) is an important cause of allograft failure after renal transplantation. Despite early screening for the virus, allograft loss from BKPyVN is still experienced in up to 14% of all renal transplant recipients. The aim of this study was to investigate the association between BKPyVN histopathologic disease severity and allograft outcome at our center.MethodsKidney transplant recipients who had undergone transplantation between 2002 and 2014 with biopsy proven BKPyVN were eligible for this retrospective study. Each biopsy was re-evaluated by a single pathologist blinded to the clinical data and scored according to the Banff criteria for rejection and BKPyVN. Serum creatinine and BK viral load at the time of biopsy diagnosis as well as allograft outcomes to include allograft survival and serum BK viremia resolution were collected for each recipient to determine if BK virus histopathologic disease severity could predict allograft outcome.ResultsTwenty cases of BKPyVN were identified from 1031 total renal transplants performed. There was no statistical association between allograft loss and BKPyVN histopathology (p = 0.49). There was also no statistical association between BKPyVN histopathology and BK viral load at the time of biopsy diagnosis (p = 0.38) or serum BK viremia resolution (p = 0.16).ConclusionsBKPyVN histopathology does not appear to be useful in predicting renal allograft outcome in those recipients diagnosed with BKPyVN which is in contrast to some previously published data.     

Neutrophil functions in renal transplant recipients.

Neutrophils obtained from peripheral blood of renal allograft recipients were studied for their ability to kill Gram-positive and Gram-negative bacteria as well as to enhance intracellular metabolism measured by the reduction of NBT salts. In addition, the influence of sera these patients on normal cells was investigated. At the same time, these cells were also tested for candidacidal activity. The data derived from these studies indicate that phagocytic cells from these patients are impaired with respect to their capacity to fight the pathogenic microorganisms as well as their sera do not promote normal killing of microorganisms, while the NBT reaction is not changed significantly. Large doses of steroids and rejection crises do not appear to affect dramatically these functions, while an ATG therapy abolishes neutrophil killing ability.     

Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay.

BACKGROUND: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients. It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. OBJECTIVE: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. STUDY DESIGN: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. RESULTS: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r=0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r=0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200,000 polymorphonuclear leukocytes (PMNLs) is log4.095 copies per ml of blood. CONCLUSIONS: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.     

Viral infections in renal transplant recipients.

Cytomegalovirus (CMV) infections are common in renal transplant recipients. We studied 23 recipients prospectively to determine whether infections by other herpes-group and non-herpes-group viruses were also present. Sera, obtained at the time of surgery and periodically thereafter, were tested for antibody to CMV, herpes simplex virus (HSV), Epstein-Barr virus (EBV), parainfluenza viruses types 1, 2, and 3, and the viruses of measles and rubella. We found no evidence of an unusual incidence of primary or secondary infection by the non-herpesviruses tested. Rises to CMV, HSV, and EBV antibody titers occurred in 43, 38, and 32% of patients, respectively. All serological rises to herpes-group viruses occurred in patients seropositive at the time of transplantation, with the exception of three patients who experienced primary CMV infections. We conclude that reactivation of all herpes-group viruses tested may occur in transplant recipients. Morbidity was associated only with primarly CMV infection.     

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients

BackgroundSensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice.Objectives/study designUsing 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS^® AmpliPrep/COBAS^® TaqMan^® CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS^® AMPLICOR MONITOR CMV tests.ResultsThe median time between transplantation and testing samples was 57 days (range, 0–207 days). The median CMV load (log₁₀) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (?0.15 [95% CI, ?0.18 to ?0.13] copies/mL), but slope differences indicated only limited co-linearity.ConclusionsThe CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.