Urine BKV Research Articles

#2825 BK virus in transplant patients: an enemy to fear or a challenge to accept? The 17-year experience of our transplant center

Abstract Background and Aims Reactivation of the BK virus in immunosuppressed patients is a risk factor for the development of BKV nephropathy. Ten percent of transplant patients are affected by BKV nephropathy, and 50% of them experience graft loss. The purpose of this study is to assess the medium to long-term effects of an early immunosuppression reduction strategy in the presence of urinary and/or serum BKV replication in a large population of kidney transplant recipients from our center. Method A retrospective observational study was conducted on 681 patients from immediate post-transplant with at least 6 months of observation. 91.5% of them were on the same immunosuppressive therapy (MMF, Steroid, tacrolimus). A total of 16200 samples were collected from blood and urine simultaneously for PCR quantification of BKV over a post-transplant period of 54.4 ± 34.2 months. Immunosuppression reduction began after two repeated biweekly detections of viruria >105 copies/ml and/or viremia >103 copies/ml. Results 35% of cases tested positive for viruria, with 19% also positive for viremia. Onset and peak viruria and viremia were observed at 4.1 ± 3.3 months and 4.2 ± 3.5 months, and 5.3 ± 4.6 and 5.4 ± 4.1 months, respectively. Viruria negativization occurred in 79.4% of patients after 13.2 ± 9.7 months, while viremia disappeared in 94.6% after 9.2 ± 7.5 months. In 5 patients with persistent viruria and viremia, therapy with Cidofovir or Leflunomide was initiated following biopsy. 14.3% of patients with persistent viruria maintained low replication and stable renal function. No episodes of acute rejection occurred. Only one case of irreversible BKV nephropathy was observed. Conclusion Timely reduction of immunosuppression allows excellent control of BKV replication, stability of renal function in the medium to long term, and makes the occurrence of irreversible chronic BKV nephropathy rare.

319 Optimizing Urinary cell mRNA profiling of kidney allograft recipients: Development of a home processing protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy

OBJECTIVES/GOALS: Development of a user friendly home kit that enables kidney transplant recipients to process urine at home and post the lysate containing RNA to a Core Laboratory would simplify urinary cell mRNA profiling and facilitate longitudinal monitoring. We report our home processing protocol and investigation of its diagnostic performance characteristics. METHODS/STUDY POPULATION: We developed a home processing protocol (HPP) consisting of urine filtration and lysis of urinary cells, both performed at home by the kidney transplant recipients (KTR) themselves, followed by isolation of total RNA from the lysate and mRNA enrichment using a silica-membrane-based cartridge, both performed at the Core Laboratory. Using the HPP, total RNA was isolated from kidney allograft biopsy-matched urines and absolute copy numbers of CD3εmRNA, CXCL10 mRNA, and 18S rRNA, components of the Clinical Trials in Organ Transplantation 04 (CTOT-04) three-gene TCMR diagnostic signature, and urinary cell BKV VP1 mRNA copy number, were measured using customized RT-qPCR assays. RESULTS/ANTICIPATED RESULTS: CTOT-04 three-gene TCMR diagnostic signature scores in urine processed using HPP discriminated KTR with TCMR (12 TCMR biopsies from 11 KTR) from KTR with no TCMR/BKVN (29 No TCMR/No BKVN biopsies from 29 KTR) (P=0.0005, Mann-Whitney test), and AUROC was 0.84 (95% CI, 0.69 to 0.98). TCMR was diagnosed with sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of -1.213 (P=0.0016,Fisher exact test). BKV VP1 mRNA copy number in urine processed with HPP discriminated KTR with BKVN (n=7) from KTR with no TCMR/BKVN (n=29) and AUROC was 1.0 (95% CI, 1.00 to 1.00). BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5x10^8 BKV VP1 mRNA copies/μg of RNA (P DISCUSSION/SIGNIFICANCE: Urine processed using the HPP predicted TCMR and BKVN in KTR. The HPP represents not only a significant advance towards portability of urinary cell mRNA profiling but also should improve patient management by minimizing visits for urine collection.

Stepwise Reduction of Mycophenolate Mofetil with Conversion to Everolimus for the Treatment of Active BKV in Kidney Transplant Recipients: A Single-Center Experience in Vietnam.

Background: No specific antiviral drug can effectively treat BKV reactivation after kidney transplantation. Thus, we evaluated stepwise-reduced immunosuppression to treat BKV reactivation. Methods: 341 kidney-transplant recipients were monitored for BKV infection (BKV-viremia, BKV-viruria). Positive samples with a significant virus load were nested PCR-genotyped in the VP1 region. In 97/211 patients presenting BKV viremia ≥104 copies/mL and/or BKV viruria ≥107 copies/mL, or BKV-nephropathy immunosuppression (i.e., mycophenolate mofetil [MMF]) was reduced by 50%. If viral load did not decrease within 28 days, MMF dose was further reduced by 25%, although calcineurin-inhibitor (CNI) therapy remained unchanged. If BKV viral load did not decrease within another 28 days, MMF was withdrawn and replaced by everolimus combined with reduced CNIs. Results: Only 41/97 BKV (+) cases completed the 6-month follow-up. Among these, 29 (71%) were in the BKV-I group and 12 (29%) were in BKV-IV. BKV viruria and BKV viremia were significantly decreased from 9.32 to 6.09 log10 copies/mL, and from 3.59 to 2.45 log10 copies/mL (p < 0.001 and p = 0.024, respectively). 11/32 (34.4%) patients were cleared of BKV viremia; 2/32 (6.3%) patients were cleared of BKV in both serum and urine, and 9/9 (100%) only had BKV viruria but did not develop BKV viremia. eGFR remained stable. No patient with BKV-related nephropathy had graft loss. There was a significant inverse relationship between changes in eGFR and serum BKV load (r = −0.314, p = 0.04). Conclusions: This stepwise immunosuppressive strategy proved effective at reducing BKV viral load in kidney transplant recipients that had high BKV loads in serum and/or urine. Renal function remained stable without rejection.

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy

BackgroundWe developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 800C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 800C, and shipment in cold containers for mRNA profiling. Furthermore, we trained kidney allograft recipients to perform the filtration of urine at home using the filter and post the urinary cell lysate containing the RNA at ambient temperature to our GEM Core for profiling. Here, we report our refinement of ZFBP and investigation of its diagnostic performance characteristics. MethodsTotal RNA was isolated from kidney allograft biopsy-matched urines using a filter-based protocol complemented by a silica-membrane-based cartridge for mRNA enrichment, the Weill Cornell Hybrid Protocol (WCHP). Absolute copy numbers of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA, components of the CTOT-04 three-gene TCMR diagnostic signature, and urinary cell BKV VP 1 mRNA copy number were measured using RT-qPCR assays. Mann-Whitney test, Fischer exact test, and receiver operating characteristic (ROC) curve analysis were used for data analyses. ResultsUrinary cell three-gene TCMR diagnostic signature scores in urines processed using the WCHP discriminated kidney allograft recipients with TCMR (12 TCMR biopsies from 11 patients) from those without TCMR or BKVN (29 No TCMR/No BKVN biopsies from 29 patients). The median (25th and 75th percentiles) score of the CTOT-04 three-gene TCMR diagnostic signature was −0.448 (−1.664, 0.204) in the TCMR group and − 2.542 (−3.267, −1.365) in the No TCMR/ No BKVN group (P = 0.0005, Mann-Whitney test). ROC curve analysis discriminated the TCMR group from the No TCMR/ No BKVN group; the area under the ROC curve (AUROC) was 0.84 (95% Confidence Intervals [CI], 0.69 to 0.98) (P < 0.001), and TCMR was diagnosed with a sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of −1.213 (P = 0.0016, Fisher exact test). BKV VP1 mRNA copy number in urines processed using the WCHP discriminated patients with BKVN (n = 7) from patients without TCMR or BKVN (n = 29) and the AUROC was 1.0 (95% CI, 1.00 to 1.00) (P < 0.0001) and BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at a specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5 × 108 BKV-VP1 mRNA copies per microgram of RNA (P < 0.0001, Fisher exact test). ConclusionUrine processed using the WCHP predicted TCMR and BKVN in kidney allograft recipients. WCHP represents not only a significant advance toward the portability of urinary cell mRNA profiling but also improved patient management by minimizing their visits for urine collection.

Outcomes of Living Kidney Donor Candidates and Living Kidney Recipient Candidates with JC Polyomavirus and BK Polyomavirus Viruria

Introduction Recent data have emerged about a protective association between JCV viruria and chronic kidney disease (CKD). Material and Methods. Single-center retrospective cohort study; 230 living kidney donors (LKD) candidates and 59 potential living kidney receptors (LKR) were enrolled. Plasma and urinary JCV and BKV viral loads were measured in all LKD candidates and in nonanuric LKR candidates. Twenty-six living kidney transplant surgeries were performed. LKR were followed in order to evaluate BKV and JCV viremia and urinary viral shedding after KT. Results In LKD candidates, JCV viruria was negatively associated with proteinuria of >200 mg/24 hours (JC viruric LKD: 12.5% vs JCV nonviruric LKD: 26.7%, p=0.021, OR:0.393; 95% CI: 0.181–0.854). In a multivariate analysis, LKD candidates with JCV viruria had a lower risk of proteinuria of >200 mg/24 hours (p=0.009, OR: 0.342, 95% CI: 0.153–0.764), in a model adjusted for age, gender, presence of hypertension, and eGFR <80 mL/min. Prevalence of JCV viruria was higher in LKD candidates when compared with LKR candidates (40.0% vs 1.7%, p < 0.001). Among the 26 LKR, 14 (53.8%) KT patients evolved with JCV viruria; 71.4% received a graft from a JCV viruric donor. Conclusion Our data corroborate the recent findings of an eventual protective association between JCV viruria and kidney disease, and we extrapolated this concept to a South European population.

Analysis of the Risk Factors for Hemorrhagic Cystitis after Hematopoietic Stem Cell Transplantation

To analyze the risk factors affecting hemorrhagic cystitis(HC) after allogeneic hematopoietic stem cell transplantation(allo-HSCT). The clinical data of 153 patients underwent allogeneic hematopoietic stem cell transplantation in the First Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2018 were selected and retrospectively analyzed. The incidence, median time and treatment outcome of HC should be observed. Multivariate analysis was used to observe the risk factors of HC in patients, including sex, age, diagnosis, disease status before transplantation, transplantation type, ATG and CTX in the pretreatment scheme, stem cell source, neutrophil and platelet implantation time; CMV, EBV and BKV infection, and acute graft-versus-host disease(aGVHD). Among 153 patients underwent allogeneic hematopoietic stem cell transplantation, 25 (16.34%) patients had HC, the median occurance time was 31 days, all patients achieved complete remission after treatment, no bladder irritation and bladder contracture were left. The results of univariate and multivariate Logistic regression analysis showed that the type of transplantation, ATG, CMV viremia before treatment, aGVHD (r=1.036, 3.234, 3.298 and 2.817, respectively) were the independent risk factors of HC. The urinary BKV detections in the patients with HC are positive, mainly occured during the period from day +13 to days +56. HLA haplotype, pretreatment including ATG, and CMV viremia, and aGVHD are the independent risk factors for HC after allo-HSCT.

CD34+ Selected Hematopoietic Cell Boosts (HCB) for Treatment of Refractory Cytopenia Following T-Replete HLA-Haploidentical Transplantation

HLA-haploidentical donor transplants using T-replete grafts and posttransplant cyclophosphamide (HIDT) are increasingly utilized and have helped address the shortage of donors particularly among minority populations. While HIDT seem to produce similar survival outcomes to matched donor transplants, they may be subject to higher rates of early reactivation of some viruses. Myelosuppressive treatments for these viruses, other microbial infections and GVHD may produce prolonged periods of poor hematopoietic function and resulting cytopenia. Additional infusion of hematopoietic cells from the patient's donor that are ex-vivo depleted of T-cells, may help ameliorate these cytopenias and need for transfusions. Between June 2017 and June 2019 we performed 82 first HIDT at our center. Of these, seven patients (8.5%) underwent a T-cell depleted hematopoietic cell boost (HCB) following remobilization and collection of PBSC from the original donor in to correct refractory cytopenias. Patients were required to have &amp;gt;80% donor T-cell chimerism and have multilineage severe cytopenias for greater than three weeks after demonstrating prior engraftment. Active GVHD was not a contraindication to HCB. Patient characteristics were: Median age 69 (29-74); sex M4, F3; diagnoses - AML 3, NHL1, MPN 1, HL 1, SCD 1; prior autotransplant -2; regimen intensity of transplant - non-myeloablative -6, myeloablatve 1; median CD34 and CD3 doses of the original transplant were -5.01 (2.89-7.58) x 106/kg and 20.88 (3.7-41.6) x 107/kg respectively. All patients had 100% donor CD3 + cell and CD33+ cell chimerism in peripheral immediately prior to HCB. Median time between transplant and HCB was303 days (range 144-1048). Maximum grade acute GVHD and chronic GVHD pre-HCB were - gd 1(1), gd 2(3) gd 3(1) and severe chronic (1 patient). Four patients had active GVHD requiring systemic treatment at the time of HCB. Documented viral infections pre-HCB were: RTI- influenza (2) Parainfluenza(2), RSV(2) coronavirus(2) metapneumovirus(1) rhinovirus (1), adenovirus (1), HHV6 (1); Blood- CMV (3) adenovirus (3), HHV6 (1); Urine BKV (4). Original donors underwent repeat PBSC mobilization using G-CSF and leukapheresis targeting &amp;gt; 5 x 106 CD34 cells/kg. collection. The PBSC collection was CD34+ cell selected using a Miltenyi column (CliniMACS Plus CD34+ System) targeting &amp;lt; 1 x 105 CD3 cells/kg in the final product. No additional conditioning regimen was administered prior to HCB. Median doses infused for the HCB were CD34+ 4.89 x 106/kg (range 2.3-11.7) and CD3+ 0.21 x 105/kg (range 0.07-0.6). Median follow-up from day of HCB is 18.6m (13.2-37.2m). There were no infusional toxicities. One patient who had gd 1 aGVHD pre-HCB developed gd 3 aGVHD 4m post-HCB. However, this was felt to be related to withdrawal of immunosuppression. No other patient developed new GVHD or exacerbation of pre-existing GVHD. All patients demonstrated an increase in absolute neutrophil count (ANC) , platelet (plat) and hemoglobin (Hb) post HCB (Fig).Median values for maximal increase of ANC, plat and Hb from day of HCB to 60 days post HCB were 1.48 x 109/L, 73 x 109/L and 2.2 g/dL respectively (p&amp;lt;0.05 for all). All patients who were platelet and RBC transfusion dependent pre-HCB became transfusion independent within 60 days post-HCB. These data indicate that HCB using CD34+ cell selected re-mobilized PBSC collections from original haploidentical donor are safe and effective in correcting prolonged severe cytopenias following HIDT. Pre-existing GVHD and ongoing immunosuppressive therapy for GVHD are not a contraindication to their use Figure Disclosures No relevant conflicts of interest to declare.

Clinical Analysis of Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation

Objective: To analyze the risk factors and survival of the patients with hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT).Methods: Propensity score Matching was conducted for HC patients among 523 patients who received HSCT in Hematopoietic Stem Cell Transplantation Center, Institute of Hematology &amp; Blood Disease Hospital, Chinese Academy of Medical Sciences from August 2016 to August 2019. Non-HC patients were selected with a ratio of 1:2 (HC: non-HC) and then statistically analyzed with SPSS 22.0. Results:130 HC patients and 233 non-HC patients were included in the study. There were HC of grade I-II in 114/130 cases(87.69%) and of grade III-IV in 16/130 (12.31%), The median time of onset was 33 days after HSCT(range 3-339 days).Univariate analysis indicated that HC occurrence were associated with the source of stem cells, infusion CD34+ cells count, morphologic remission before transplantation, HLA matching, urinary BK virus load and the drug of Bu in conditioning regimens. Multiple regression analysis showed that morphologic remission before transplantation, HLA matching and infusion CD34+ cells count were associated with HC. K-M survival analysis showed that the 1-year survival rate of HC and non-HC was 91.8% vs 86.3%, and the 2-year survival rate was 52.5% vs 50.6%, respectively, showing no statistical difference. ROC curve showed that when the level of BKV is higher than 5×108 copies /ml, the risk of HC would increase. Conclusion: HC was associated with morphologic remission before transplantation, HLA matching and infusion CD34+ cells count. There was no significant difference in survival between HC and Non-HC groups. the level of urinary BKV load higher than 5×108 copies /ml may cause hemorrhagic cystitis symptoms. Key words: hemorrhagic cystitis; Allogeneic hematopoietic stem cell transplantation; Risk factors; Prognosis Disclosures No relevant conflicts of interest to declare.

Developing adoptive T cell therapy for Polyomavirus-associated diseases using HLA-defined peptide platform

Background & Aim Polyomavirus infection is a growing threat among immunocompromised patients. Reconstitution of T cell immunity has been shown to control the infection. Hence, the adoptive transfer of polyomavirus-specific T cells is a promising approach to revive T cell immunity and tackle viral reactivation and disease progression. We aimed at mapping T cell epitopes across multiple viral antigens of BKV and JCV with broad HLA coverage. Methods, Results & Conclusion Methods Proteome wide analysis of T cell responses against BKV and JCV antigens was carried out in a cohort of healthy volunteers and transplant recipients to identify immunodominant T cell epitopes. This epitope screening was conducted using overlapping peptides from VP1, VP2, VP3, LTA and STA antigens of BKV and JCV. HLA restriction and fine mapping of these epitopes was confirmed using peptide loaded HLA-matched and mismatched targets. Finally a novel pepmix based on these epitopes was used for optimization of GMP-compliant ex vivo expansion of BKV and JCV-specific T cell therapy. Results We have mapped 57 novel epitopes from BKV and JCV restricted to HLA class I and class II alleles. Many of these epitopes show cross protective potential where BKV-specific T cells could detect JCV peptide and vice versa. Molecular analysis of T cell directed against polyomaviruses showed a unique transcriptional signature when compared to T cell specific for other persistent viruses. Using this knowledge we have developed a rapid in vitro T cell expansion process for manufacturing of adoptive T cell therapy which can be used for the treatment of BKV and JCV-associated diseases. We have successfully used this optimized protocol to generate BKV-specific T cells for treating two patients who were unresponsive to standard clinical management. Patient 1 who had hemorrhagic cystitis was injected with 4 doses of autologous BKV-specific T cells and Patient 2 who had BK virus associated nephropathy was administered with 10 doses of HLA-matched third party BKV-specific T cells. Both patients completely cleared BKV in both urine and serum with no further reactivation. Conclusion Development of a novel HLA-defined peptide platform for BKV and JCV provides an unique opportunity to offer rapid autologous or “off-the-shelf” allogenic T cell therapy. More importantly, preliminary clinical studies indicates that the adoptive T cell therapy manufactured using this pepmix offers safe and effective clinical responses in untreatable disease settings.

Effects and long-term follow-up of using umbilical cord blood-derived mesenchymal stromal cells in pediatric patients with severe BK virus-associated late-onset hemorrhagic cystitis after unrelated cord blood transplantation.

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood-derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus-associated late-onset hemorrhagic cystitis (BKV-HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV-HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1×106 /kg once a week until the symptoms improved. The median follow-up time was 1432 (89-2080) days. The median frequency of MSC infusion was 2 (1-3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36-56.9) ×108 /mL before MSC infusion and 2.67 (0-56.3) ×108 /mL after MSC infusion; the difference was not significant (P=.219). There were no significant differences in the overall survival, disease-free survival, and the incidence of relapse and acute and chronic graft-versus-host disease between the MSC infusion group and non-MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein-Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood-derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV-HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long-term infection, or survival of patients with leukemia.

Urinary polyomavirus: novel biomarker of congenital ureteropelvic junction obstruction

Pregnancy is associated with reactivation and transmission of latent polyomavirus to fetus. Polyomavirus is also known to cause ureteral stenosis and hydronephrosis. The aim of this study was to investigate whether the urinary polyomavirus could be used as a potential biomarker in newborns with ureteropelvic junction obstruction (UPJO). Urinary polyomavirus virus was measured by PCR in 42 newborn infants with fetal hydronephrosis history. Random urine samples were obtained from newborns immediately after birth and from their mothers at the time of delivery. Results were compared with 25 healthy infants matched for gestational and postnatal ages. The diagnosis of UPJO was established by diuretic renal scintigraphy. UPJO was graded according to the Society for Fetal Urology (SFU) classification. The urine samples of healthy infants showed no detectable polyomavirus. No statistically significant difference was found in the median urinary polyomavirus level between grade 1 (1000 copies/mL) and grade 2 (1500 copies/mL) UPJO infants. When the median urinary BKV values were compared for each grade of UPJO, patients with grade 3 and 4 had significantly higher urinary polyomavirus levels than those with grades 1 or 2 (P<0.001). There was a strong correlation between the median polyomavirus in the urine of pregnant women and the urine of newborns with UPJO (P<0.001). Data suggest that routine screening of urinary polyomavirus may help to identify infants with severe obstruction in whom early surgical intervention could reduce the risk of developing progressive kidney disease. To the best of our knowledge this is the first prospective study to present the role of urinary polyomavirus in newborn infants with UPJO to distinguish between patients who would benefit from early surgical intervention. Urinary polyomavirus is a potential biomarker of UPJO in newborns with fetal hydronephrosis.

Cytomegalovirus prevention strategies and the risk of BK polyomavirus viremia and nephropathy.

Polyomavirus BK (BKV) is the cause of polyomavirus-associated nephropathy resulting in premature graft loss. There are limited data regarding the role of cytomegalovirus (CMV) infection and its prevention in developing BKV viremia and PVAN. In a prospective study, we analyzed 207 consecutive renal transplant recipients previously enrolled in 2 randomized trials evaluating different CMV prevention regimens with routine screening for BKV and CMV. Of these, 59 received valganciclovir and 100 valacyclovir prophylaxis; 48 patients were managed by preemptive therapy. At 3years, the incidence of BKV viremia and PVAN was 28% and 5%, respectively. CMV DNAemia developed in 55% and CMV disease in 6%. Both BKV viremia (42% vs 23% vs 21%, P=.006) and PVAN (12% vs 2% vs 2%, P=.011) were increased in patients treated with valganciclovir prophylaxis compared to valacyclovir and preemptive therapy. Using multivariate Cox proportional hazard regression, valganciclovir prophylaxis was independent predictor of BKV viremia (hazard ratio [HR]=2.38, P=.002) and PVAN (HR=4.73, P=.026). In contrast, the risk of subsequent BKV viremia was lower in patients with antecedent CMV DNAemia (HR=0.50, P=.018). These data suggest valganciclovir prophylaxis may be associated with increased risk of BKV viremia and PVAN. CMV DNAemia did not represent a risk for BKV.

Post Transplantation Cyclophosphamide (PTCY) Is Associated with Increased Risk of BK Virus-Associated Hemorrhagic Cystitis (BKHC) in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant (HSCT).

Introduction BK virus hemorrhagic cystitis is a well-recognized complication in patients undergoing allogeneic HSCT and is associated with significant morbidity. PTCY has been used for GVHD prophylaxis in patients undergoing haploidentical or mismatched unrelated donor HSCT. We hypothesized that PTCY is associated with increased incidence of HC associated with BK virus. Objectives • To identify the risk factors for developing BKHC in allogeneic HSCT • To assess the effect of PTCY in development of BKHC in patients with myeloablative (MA) regimen containing total body irradiation (TBI) • To evaluate the risk of concurrent CMV infection and BKHC in allogeneic HSCT with MA conditioning regimen Methods We identified 116 consecutive patients who underwent allogeneic HSCT at our institution between January 2015 and July 2018. The MA regimen (n=42) consisted of fludarabine 50 mg/m2/day IV for 5 days and busulfan 3.2 mg/kg/day IV for 4 days with or without TBI of 200 cGy/day for 2 days (FBT or FB4). The reduced intensity conditioning (RIC) regimen (n=74) consisted of fludarabine 30 mg/m2/day IV for 5 days and busulfan 3.2 mg/kg/day IV for 2 days (FB2). In terms of GVHD prophylaxis all recipients except those undergoing haploidentical and mismatched unrelated transplant received Thymoglobulin 2 mg/kg/daily IV days -2, -1 and 0 (n=80). Recipients of haplo- related and mismatched unrelated donor transplantation received cyclophosphamide 50 mg/kg IV on day +3 and +4 along with Mesna (n=36). All patients received post-transplant immunosuppression with Tacrolimus and Mycophenolate Mofetil. Patients with urinary symptoms were tested for BKV in urine by PCR. Results The median age of patients was 56 years (range 20–76). A total of 31% of patients (n=13) received PTCY in FBT group which was similar to 31% of patients (n=23) in FB4-FB2 (p=0.98). Overall 21.5% patients were diagnosed with BKHC. The patients who received ATG, the incidence of BKHC was 27.5% in FBT group and 11.7 % in FB4-FB2 (p=0.073). The patients who received PTCY, the incidence of BKHC was significantly increased to 69.2% in FBT vs 9% in FB4-FB2 (p=0.0001). The risk of developing concurrent CMV viremia and BKHC was increased to 10.3% in FBT compared to 5.8% in FB2-FB4 (p=0.662). The patients who received PTCY following FBT, the incidence of concurrent CMV viremia and BKHC was increased to 23.1%. as compared to 0% with FB2-FB4. Conclusion Our result shows the addition of PTCY to TBI containing regimen significantly increased the incidence of BKHC. We did not observe increase in the incidence of BKHC in patients receiving PTCY with FB2 or FB4 regimens. Our study revealed an increased incidence of concurrent BKHC and CMV viremia in patients receiving PTCY with FBT which could be due to increased immunosuppression. Our findings suggest that using PTCY as GVHD prophylaxis in patients undergoing MA conditioning regimen including TBI is a risk factor for BKHC.

Polyomavirus BK, BKV microRNA, and urinary neutrophil gelatinase-associated lipocalin can be used as potential biomarkers of lupus nephritis.

ObjectiveLupus nephritis (LN) frequently progresses to end-stage renal disease. Finding a biomarker for LN and a predictor for the development of chronic kidney disease (CKD) is important for patients with systemic lupus erythematosus (SLE).MethodsNinety patients with SLE were divided into biopsy-proven LN (n = 54) and no kidney involvement (non-LN) (n = 36) groups and followed up for 54 months.ResultsOf 36 patients with LN, 3 (5.6%) had class II disease, 3 (5.6%) had class III, 35 (64.8%) had class IV, 10 (18.5%) had class V, and 3 (5.6%) had class VI (advanced sclerosis). Compared to the non-LN group, patients in the LN group had higher autoimmunity evidenced by a higher proportion of low C3 and C4 levels, positive anti-double-stranded DNA antibody levels, and lower estimated glomerular filtration rates (eGFR). Urinary neutrophil gelatinase-associated lipocalin (uNGAL) levels were significantly higher in the LN group (LN vs non-LN, 670 vs 33 ng/mL, respectively). The patients with LN had a higher urinary polyomavirus BK (BKV) load (3.6 vs 3.0 log copies/mL) and a lower urinary BKV miRNA (miR-B1) 5p level (0.29 vs 0.55 log copies/mL, p = 0.025), while there was no significant difference in the level of miR-B1-3p. Urinary miR-B1-5p level but not urinary BKV load was negatively correlated with uNGAL level (r = -0.22, p = 0.004). At the cutoff value of 80 ng/mL, the receiver operating characteristic curve analysis showed that uNGAL level as a predictor of the presence of LN had a high sensitivity (98%) and specificity (100%) (area under the curve [AUC], 0.997; p < 0.001). During the 54-month follow-up period, 14 (7%) patients with LN and none of the non-LN patients developed CKD. Multivariate Cox regression analysis revealed that baseline uNGAL level was the only predictive factor for CKD development, while baseline serum creatinine level and eGFR were not.ConclusionAn elevated urinary BKV viral load with a decreased level of miR-B1 implies the presence of LN. In addition, an increased uNGAL level is a good biomarker not only in predicting the presence of LN but also for prediction of CKD development in patients with SLE.

Viral Infections After Kidney Transplantation in a Cohort of Children: A Retrospective Single-Center Study

BackgroundViral infections are known to be common complications after kidney transplant (KTx), causing significant numbers of mortality and morbidity. ObjectivesWe aimed to highlight the pattern of viral infections after KTx in children and its impact on allograft function. MethodsWe included children who underwent KTx between 2012 and 2017. Baseline demographics, immunosuppressive agents, episodes of viral infections with cytomegalovirus (CMV), BK virus, and Epstein-Barr virus (EBV), and serum creatinine were collected. All children received induction agent followed by maintenance immunosuppression. Oral valganciclovir was given to all high-risk patients for CMV for 180 days as prophylaxis. CMV and EBV polymerase chain reactions were monitored every 2 weeks initially until the ninth month, then monthly until the end of the second year, and then every 3 months. Urine BKV polymerase chain reactions were monitored monthly in the first year and then every 3 months. ResultsA total of 18 children received transplants. There was 1 episode of CMV infection (5.6%), 2 episodes (11.1%) of isolated BK viruria (1 of the 2 with an episode of BK viremia [5.6%] with no associated BK nephropathy [0%]), and no episodes of EBV or lymphoproliferative disease (0%). Allograft functions continued to be stable with mean serum creatinine of 52.2 μmol/L during the study period with 2 episodes (11.1%) of acute cellular rejection and 1 episode (5.6%) of early antibody-mediated rejection. ConclusionProlonged prophylaxis and strict viral monitoring protocol can be effective ways of controlling viral infections after KTx.

Etiological Study of Late Onset Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation in Children

To prospectively study the correlation BKV with the occurrence and development of late onset hemorrhagic cystitis (LOHC) after allogeneic hematopoietic stem cell transplantation(allo-HSCT). The clinical data of a total of 276 patients with allo-HSCT in our department between January 1998 and March 2016 were analyzed ratrospectvely. Quantitative Real-time PCR assay was used to prospectively monitor the BKV DNA load of the urine and plasma for 23 patients accepting allo-HSCT from August 2015 to March 2016. LOHC(24.28%) occurred in 67 of 276 cases with allo-HSCT. Univariate analysis showed that age older than 6 years, different diseases, unrelated donor, pretreatment with BU, Ⅲ-Ⅳ aGVHD significantly correlated with LOHC. Multivariate analysis demonstrated that age older than 6 years (P<0.01), pretreatment with BU(P<0.05), and aGVHD of grade Ⅲ-Ⅳ （P= 0.011） were the independent risk factors for LOHC. Among 23 patients after allo-HSTC, 10 of which were positive of urine BKV, and LOHC occurred in 6 cases. The positive rate of urine BKV (85.7%)in group LOHC+ was significantly higher than that in the group LOHC-(25.0%）(χ2=5.043, P<0.01). The incidence of LOHC positively correlated with the positive rate of BKV (r=0.564, P<0.01), and the severity of LOHC positively correlated with urinary BKV load (r = 0.502, P<0.01). And 5 of 6 petriatic patients with LOHC had aGVHD. All of them were subject to the strengthened antiviral treatment, and 4 of them accepted intensive immunosuppression therapy. Age ≥6 years old, precenditioning regieme with BU and aGVHD of grade Ⅲ-Ⅳ are independent risk factors for LOHC after allo-HSCT, the positive rate of urine BKV load positively correlates with the severity of LOHC after allo-HSCT.

High incidence of BK virus-associated hemorrhagic cystitis in children after second or third allogeneic hematopoietic stem cell transplantation.

BKV-HC is a serious complication of allogeneic HSCT. To characterize the incidence, risk factors, and clinical outcomes of post-HSCT BKV-HC, we retrospectively analyzed 112 patients who underwent one or more allogeneic HSCTs at our hospital between 2001 and 2017. Twenty underwent second or third HSCT thereafter. Ten patients developed BKV-HC at a median of 30days after HSCT. The 100-day cumulative incidences of grade 0-4 and grade 2-4 BKV-HC were 7.8% and 6.2%, respectively. HSCTs performed in 2011-2017 associated with significantly higher 100-day cumulative incidence of grade 2-4 BKV-HC (14.0%) than HSCTs performed in 2001-2010 (1.3%, P=0.004). On multivariate analysis, second or third HSCT was the only independent significant risk factor for development of grade 2-4 BKV-HC (P=0.015). Serial PCR monitoring of urine and blood BKV load did not predict BKV-HC. The recent increase in the incidence of BKV-HC may reflect recent innovations in transplant technologies that facilitate second or third HSCT, which are known to cause prolonged immune deficiency. If safe and effective treatment or prophylaxis becomes available, it could be used to target the high-risk patients for BKV-HC.

DNA detection of JC and BK virus in archival urine cytospin slides.

To identify decoy cells, cytological examination was performed in urine cytospin slides. Decoy cells are related to Polyomaviruses (JC virus [JCV] and BK virus [BKV]), which are recognized worldwide due to potential infection and morbidity in kidney transplant recipients. Cytologically, it is difficult to evaluate the cytopathic effect of JCV and BKV in urine of patients with urothelial neoplasia. For this reason, there is a need for molecular approaches. To evaluate the incidence of BKV and JCV DNA in archival slides of urine cytospin material with benign and malignant characteristics. A total of 176 urine specimens were used for cytological examination of neoplastic or decoy cells. The samples were analyzed for the presence of JCV and BKV, by polymerase chain reaction (PCR) in DNA Isolated from archival slides of urine cytospin material. A typical samples (n = 48) were compared with the remaining 128 samples without atypia/neoplasia for the presence of JCV or BKV DNA. A statistically nonsignificant result was observed correlating the presence of JCV or BKV. The results show that DNA Isolated from archival slides of urine cytospin material can be used for detection of BKV and JCV.

The association between polyomavirus BK strains and BKV viruria in liver transplant recipients.

BK virus (BKV) is a polyomavirus that cause of allograft dysfunction among kidney transplant recipients. The role of BKV infection in non-renal solid organ transplant recipients is not well understood neither for the relationship between various BKV strains with occurrence of BKV viral viruria. This study aimed to understand the prevalence of BKV infection and identified of BKV various strains in the urine of liver transplant recipients. There was not significant difference of renal outcome between high BKV viruria and low BKV viruria in the liver transplant recipients. The WW-non-coding control region (NCCR) BKV detected in urine was associated with higher urinary BKV load, whereas the Dunlop-NCCR BKV was detected in the urine of low urinary BKV load. An in vitro cultivation system demonstrated that WW-BKV strain exhibiting the higher viral DNA replication efficiency and higher BKV load. Altogether, this is the first study to demonstrate the impact of BKV strains on the occurrence of BK viruria in the liver transplant recipients.

Pre-transplant shedding of BK virus in urine is unrelated to post-transplant viruria and viremia in kidney transplant recipients.

BK virus-(BKV) associated nephropathy (BKVN) is a major cause of allograft injury in kidney transplant recipients. In such patients, subclinical reactivation of latent BKV infection can occur in the pre-transplant period. The purpose of this study was to determine whether urinary BKV shedding in the immediate pre-transplant period is associated with a higher incidence of viruria and viremia during the first year after kidney transplantation. We examined urine samples from 34 kidney transplant recipients, using real-time quantitative polymerase chain reaction to detect BKV. Urine samples were obtained in the immediate pre-transplant period and during the first year after transplant on a monthly basis. If BKV viruria was detected, blood samples were collected and screened for BKV viremia. In the immediate pre-transplant period, we detected BKV viruria in 11 (32.3%) of the 34 recipients. During the first year after transplantation, we detected BKV viruria in all 34 patients and viremia in eight (23.5%). We found no correlation between pre-transplant viruria and post-transplant viruria or viremia (p = 0.2). Although reactivation of latent BKV infection in the pre-transplant period is fairly common among kidney transplant recipients, it is not a risk factor for post-transplant BKV viruria or viremia.