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

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, approximately 5.9%). 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 > or =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 > or =7.0 log(10) copies/ml.     

Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants

Fifty-three recipients of bone marrow transplants were monitored prospectively for urinary excretion of human polyomaviruses by enzyme-linked immunosorbent assays of urinary supernatants and DNA hybridization assays of urinary cells. Excretion of BK virus was demonstrated in 47 percent of the transplant recipients and was the result of the reactivation of latent virus. Hemorrhagic cystitis of long duration (greater than or equal to 7 days) was associated with BK viruria. The disease occurred four times more frequently in patients who excreted BK virus than in those who did not, and the virus was identified in 55 percent of the urine specimens during episodes of cystitis as compared with 8 to 11 percent of the specimens during cystitis-free periods. BK viruria often preceded or coincided with the onset of the disease. Among 19 patients with BK viruria lasting seven days or longer, hemorrhagic cystitis occurred in 15. Occurrence of the disease was related to the source of marrow. The disease occurred in 50 percent of 38 recipients of allogeneic marrow and in 7 percent of 15 recipients of syngeneic or autologous marrow. Among recipients of allogeneic marrow, the disease was observed in 71 percent of the 21 patients excreting BK virus and in 24 percent of the 17 not excreting the virus. An association of BK virus with hemorrhagic cystitis was demonstrated in 16 of the 18 cases of the disease that were adequately characterized. We conclude that reactivation of BK virus may account for a substantial proportion of late-onset, long-lasting hemorrhagic cystitis in recipients of bone marrow transplants.     

Human polyomavirus (BK) infection and ureteric stenosis in renal allograft recipients.

Human polyomavirus (BK) was detected in two renal allograft recipients as a result of routine examination of Papanicolaou-stained smears of urinary sediment in the light microscope. Infection with this recently identified virus was confirmed by virus isolation and electron microscopy. The cytological, histological, and ultrastructural changes due to the virus are described, and virus excretion is correlated with the clinical progress of the patients and the pathological findings. The transplant ureters in both patients were found to be ulcerated and stenosed, and virus-infected cells were observed in the ureteric epithelium. We suggest that the administration of high-dose steroids in transplantation may permit active infection with human polyomavirus to occur in ureteric epithelium which has been damaged by ischaemia or inflammation.     

Quantification of BK polyoma viruria in Japanese children and adults with hemorrhagic cystitis complicating stem cell transplantation

Polyoma BK virus (BKV) is frequently found in the urine of stem cell transplantation (SCT) patients with hemorrhagic cystitis (HC), but also occurs in SCT patients without HC. How BK viruria relates to the development of HC in SCT patients, especially in children, has not yet been fully evaluated. In the present study, we analyzed the relationship of several factors including urinary BKV load to HC development in children and adults undergoing SCT. We employed a quantitative PCR assay and evaluated 37 patients (aged 9 months–62 years) of whom 12 developed HC and 25 did not. Older age was a risk factor for the development of HC; however, other factors such as sex, primary disease, type of SCT, conditioning regimen and aGVHD were not. Peak urinary BKV values in HC patients were not higher than those in non‐HC patients. Severity of HC also did not correlate with urinary BKV loads. However, in some patients who secreted higher urinary BKV loads, the peak loads were closely related with the onset of HC. Higher BKV loads may be a risk factor for the development of HC in conjunction with other coexisting factors. J. Med. Virol. 80:2108–2112, 2008. © 2008 Wiley‐Liss, Inc.     

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.     

Incidence of JC viruria is higher than that of BK viruria in Taiwan

To investigate the prevalence of human polyomaviruses in Taiwan, urine samples from immunocompetent (healthy), transient immunocompromised (pregnant), and prolonged immunosuppressed (autoimmune disease) individuals were collected throughout the island. The viral DNA in the urine was detected by the polymerase chain reaction (PCR) and Southern blot. The viral genotypes were determined by DNA sequencing within the regulatory region. The overall results, including cases reported previously, show that 13.3% (10/75) of immunocompetent individuals, 26.0% (20/77) of pregnant women, and 37.5% (18/48) of autoimmune disease patients are JCV positive. All of the immunocompetent individuals are BKV negative, but 3.9% (3/77) of the pregnant women and 6.2% (3/48) of autoimmune disease patients are BKV positive. Twenty-four percent (48/200) of the examined urine samples were JCV positive, but only 3% (6/200) were BKV positive. JCV positive individuals were mainly infected with CY (42%) and TW-1 (52%) subtypes. These results suggest that the incidence of urinary excretion of human polyomaviruses in immunosuppressed individuals is higher than that of immunocompetent individuals. The prevalence of JCV appears to be higher than that of BKV in Taiwan. In addition, CY and TW-1 are the predominant subtypes of JCV prevalent in the Taiwanese population. J. Med. Virol. 52:253–257, 1997. © 1997 Wiley-Liss, Inc.     

Clinicopathologic analysis of patients with BK viruria and rejection-like graft dysfunction

BK virus infection can be associated with interstitial inflammation, tubulitis without viral cytopathic effect, and negative in situ hybridization for viral DNA. We evaluated the consequences of increased immunosuppression in 32 viruric patients, with such acute cellular rejection-like changes in allograft biopsies (n = 50). When follow-up information was available, complete creatinine response, decrease in urine viral load (VL), and improvement in overall Banff grade for acute rejection were only seen in 13 (27%) of 49, 7 (21%) of 33, and 10 (39%) of 26 episodes of graft dysfunction, respectively. Histologic response was not always accompanied by clinical response. This low rate of response to antirejection therapy suggests that interstitial nephritis in a subset of these patients was secondary to viral infection. The presence of high VL (>1.0E+05 copies/mL) was associated with low immune cell function values (129 ± 99 ng of adenosine triphosphate per milliliter, P = .08) and with significant development of viremia after antirejection treatment (5/9 [56%] versus 0/24 [0%] in patients with low VL, P < .001).     

BK polyomavirus interstitial nephritis in a renal allograft recipient

Human polyomavirus (PV) interstitial nephritis has recently been recognized as a cause of severe renal allograft dysfunction. It occurs in immunosuppressed patients after reactivation of the latent virus PV type BK (BK virus) in the renal epithelium. BK disease is defined as a morphologically manifest renal infection with cytopathic signs accompanied by varying degrees of interstitial inflammatory cell infiltrates and functional impairment. It is also identified by the presence of cells containing viral inclusion bodies (decoy cells) in the urine. The authors report a case of BK PV interstitial nephritis in a 36-year-old renal allograft recipient. Under light microscopy the chief diagnostic indicator was detection of intranuclear viral inclusions, which were found exclusively in tubular epithelial cells. Cells with viral changes were often enlarged with nuclear atypia and chromatin basophilia. Widespread interstitial plasma cell infiltrates associated with tubulitis were present. Intranuclear paracrystalline arrays of virus particles 35-38 nm in diameter were present as characteristic ultrastructural indicators. Urine samples revealed decoy cells with ground-glass-type intranuclear inclusions positive for BK virus by electron microcopy.     

Neopterin levels in long-term renal allograft recipients

In this paper we present results dealing with neopterin levels in the urine of 21 kidney grafted patients during long-term allograft survival. Data dealing with results of urinary neopterin excretion to predict acute cellular allograft rejection during the immediate post-transplant period have been reported previously. The evaluation of patients included a physical investigation and numerous biochemical parameters as described below. We found that neopterin excretion is normal or elevated, but if elevated levels remain stable the prognosis for the allograft seems to be good.     

Invasive fungal infections in renal allograft recipients

Invasive fungal infections contribute significantly to morbidity and mortality in renal allograft recipients. We identified 29 cases of invasive mycoses on histological and/or cytological examination, out of the total 1231 renal transplants performed at our centre over a period of last 15 yrs (1989-2003). A detailed clinical analysis was performed. The time interval between transplant and the occurrence of invasive fungal infection ranged from 15 days to 10.5 yrs. Candida and Aspergillus were the most frequent offenders (66%); Candida alone accounting for 45% of the cases. The most common risk factors were post transplant Cytomegalovirus infection, diabetes mellitus and episodes of acute rejection. Fine needle aspiration cytology, bronchoalveolar lavage and esophageal brush smears aided in prompt diagnosis. Disseminated infection was associated with a high mortality (80%). Management of renal transplant recipients requires identification of risk factors and early clinical suspicion of infection. The role of prophylaxis needs further evaluation.     

Ultrastructural analysis of peripheral blood mononuclear cells in renal allograft recipients.

Renal transplanted patients receiving immunosuppressive therapy with glucocorticoids (GC) and azathioprine show in the peripheral blood numerous large mononuclear cells. The origin and the lineage of these cells was not clearly established. In the present study we investigated the surface phenotype and the ultrastructural characteristics of this subset constituted by large mononuclear cells. From peripheral blood mononuclear cells (PBL) of allograft recipients, a cell preparation exhaustively depleted of the relatively numerous monocytes was obtained. At ultrastructural examination two main cellular populations were distinguished: the predominant one (congruent to 85%) was constituted by cells with lymphoid morphology while the other (congruent to 15%) showed myeloid appearance. The large lymphoid cells, T4 or T8 positive, did not express Ia molecules on the surface and were morphologically suggestive of cells in an intermediate stage of the cell cycle between resting and activated lymphocytes. The myeloid population was constituted by promyelocytes, myelocytes and metamyelocytes. Promyelocytes and myelocytes capable of cell division are responsible for the increase of 3H-thymidine incorporation observed in transplanted patient PBL. In conclusion our data suggest that in allograft recipients the immunosuppressive therapy with GC and azathioprine may inhibit the lymphocyte blast transformation and can influence the release of immature myeloid cells responsible for the PBL increased 3H-thymidine incorporation.     

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.     

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.     

Problems associated with pregnancy in renal allograft recipients

Of 18 pregnancies in 11 renal transplant recipients, three were terminated and in the remaining 15 (in 8 women) there were 10 live births (including one set of twins), five intrauterine deaths, and one spontaneous abortion. Graft function deteriorated in six women, from obstruction of the transplanted ureter in two, recurrent glomerulonephritis in two, rejection in one, and pelvi-ureteric junction obstruction in one. Hypertension worsened or developed in all but one of the pregnancies and proteinuria appeared in eight. Of the 10 live births only one reached 38 weeks gestation (mean 35 weeks) and four neonates were small for gestational age. One infant died early from intraventricular hemorrhage and hyaline membrane disease, one fetus had hydrocephalus, and the others were normal. Factors associated with a poor fetal outcome were deterioration in graft function during pregnancy, pre-existing hypertension, or the development of hypertension before the third trimester.     

Natural killer-cell activity in cyclosporine-treated renal allograft recipients

We prospectively studied natural killer (NK)-cell activity in 16 cyclosporine-treated renal transplant recipients. NK function remained intact in the group as a whole in the initial 6 months following transplantation. The percentage of CD16-positive cells within the peripheral blood mononuclear-cell population was highly correlated with NK activity both prior to and following transplantation in the absence of rejection. During rejection, the correlation was poor. A marked increase in NK activity occurred during 9 of 12 rejection episodes; similar increases in NK activity were rarely observed in the absence of rejection. Significant infiltrates of NK cells, as determined by expression of CD16, were not demonstrated in stained biopsy specimens obtained from rejecting allografts. Pretransplant NK activity did not predict clinical outcome of the allograft. Our results indicate that NK cells are activated during allograft rejection in cyclosporinetreated patients, but their exact role in the rejection process is unknown.     

Variable sources of Bk virus in renal allograft recipients

BK virus is the causative agent of polyomavirus-associated nephropathy, a major cause of kidney transplant failure affecting 1%-10% of recipients. Previous studies that investigated the viral source on the kidney recipient pointed that the donor is implicated in the origin of human polyomavirus BK (BKPyV) infection in recipients, but giving the low genetic variability of BKPyV this subject is still controversial. The aim of this study was to determine if BKPyV replicating in kidney recipients after transplantation is always originated from the donor. Urine and blood samples from 68 pairs of living donors and kidney recipients who underwent renal transplantation from August 2010-September 2011 were screened for BKPyV by real time polymerase chain reaction. Only three recipients presented viremia. When both donors and recipients were BKPyV positive, a larger fragment of VP1 region was obtained and sequenced to determine the level of similarity between them. A phylogenetic tree was built for the 12 pairs of sequences obtained from urine and high level of similarity among all sequences was observed, indicating that homology inferences for donor and recipient viruses must be cautiously interpreted. However, a close inspection on the donor-recipient pairs sequences revealed that 3 of 12 pairs presented considerably different viruses and 4 of 12 presented mixed infection, indicating that the source of BKPyV infection is not exclusively derived from the donor. We report that about 60% of the renal recipients shed BKPyV genetically distinct from the donor, confronting the accepted concept that the donor is the main source of recipients’ infection.     

Diagnosis of cytomegalovirus infection in cyclosporin-treated renal allograft recipients

The relative merits of antibody response and virus shedding as markers of cytomegalovirus (CMV) infection among cyclosporin-treated renal allograft recipients were analysed. CMV-specific antibody was assayed by IgG-specific radioimmunosorbent test (RIST) and by complement fixation test (CFT). CMV shedding was assayed by virus isolation and by the rapid test immediate early nuclear antigen detection (IENAD). RIST and CFT detected seroconversion in similar numbers of patients, but the former test was the more sensitive when CMV antibody was sought in pretransplant sera to differentiate primary from recurrent infection. IENAD detected or excluded CMV shedding for more urine specimens than virus isolation (462/515 [90%] vs. 366/515 [71%]), but the reverse applied to saliva specimens (33/57 [58%] vs. 54/57 [95%]). The high specificity of IENAD allowed positive results by IENAD to be accepted when virus isolation failed to provide a result. IENAD was, however, less sensitive than virus isolation even when specimens yielding CMV by IENAD, but no result by virus isolation, were included in the analysis (27/44 [61%] vs. 38/44 [86%]). Assays of both antibody response and virus shedding were required to maximise the diagnosis of recurrent CMV infections, but the former assay detected all primary CMV infections. The diagnostic implications of these results are discussed.