Polyomavirus-associated Nephropathy Research Articles

#740 Very early BK polyomavirus viremia is associated with graft dysfunction in kidney transplant recipients

Abstract Background and Aims BK polyomavirus-associated nephropathy (BKN) is a leading cause of graft dysfunction and loss, mostly related to the lack of effective treatments. Increasing BK polyomavirus viremia (BKV) precedes BKN, and its monitoring is therefore the focus of preventive strategies; whether BKV itself and its timing of occurrence post-transplant are associated with graft dysfunction is not clearly understood. Method We designed a single-center retrospective study enrolling all patients who received a kidney transplant at our center from January 1st 2018 to June 30th 2022. The study received approval from local Ethical Committee (252/2023/OSS*/AOUMO SIRER ID 651-BKNEPHMO). We collected clinical and demographical data at baseline; donor and transplant characteristics, significant clinical parameter during follow-up. Patients were regularly screened for BK viremia (roughly, once every two weeks in the first 3 months, then monthly until 1 year, then at least every 3 months or on clinical indication). We defined sustained viremia as the presence of BK viremia confirmed in 3 different measurements; probable BKN as the presence of BKV with a viral load >3log10 copies/ml; presumptive BKN as BKV with a viral load >4log10 copies/ml. Results After exclusion of patients with less than 6 months of follow-up, we considered 138 subjects. We stratified patients in 3 groups: no viremia (group A), any BKV (after more than one month from transplant, group B) and very early BKV (within one month from transplant, group C). Clinical, demographic, transplant characteristics are reported in Fig. 1. Of note, group C had a significantly higher proportion of patients with diabetes mellitus (DM) and positive anti-HBc serology, and a lower proportion of Caucasians; there were no differences in transplant characteristics. During follow-up, patients in group C had a significantly stronger reduction in immunosuppressive treatment (especially with regards to MMF), had higher chance of receiving a graft biopsy and were more likely to develop donor-specific antibodies. While patients in group C had a higher probability of sustained BKV compared to those in group B, there were no differences in the proportion of probable, presumptive and histological BKN. With univariate logistic regression, pre-transplant DM and positive anti-HBc were significantly associated with development of very early BKV, while Caucasian ethnicity was a protective factor; only DM maintained statistical significance at multivariate analysis after adjustment for age and sex (OR 7959 with 95% CI 1593-39 771, p = 0.012). Time to eGFR <50% from baseline and time to graft failure were significantly shorter in group C (see Fig. 2). With univariate logistic regression, higher cold-ischemia time and positive BKV within one month from transplant were significantly associated with eGFR loss >50% at 12 months in our cohort; only BKV within one month maintained statistical significance at multivariate analysis after adjustment for age, sex and baseline eGFR (OR 22.17 with 95% CI 1.81-271.47, p = 0.015). Conclusion Patients who develop BKV very early after kidney transplantation are at increased risk of premature severe graft dysfunction (irrespective of the development of BKN); these patients should be offered close and intense surveillance with strategies aiming at identifying treatable causes of progressive kidney damage, and careful management of immunosuppressive treatment. Considering study design and low number of enrolled patients, further data are needed to confirm our findings, to elucidate risk factors for very early BKV and precise causes of rapid deterioration of graft function in these patients.

Concurrent JCPyV-DNAemia Is Correlated With Poor Graft Outcome in Kidney Transplant Recipients with Polyomavirus-associated Nephropathy.

Co-infection of JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) is uncommon in kidney transplant recipients, and the prognosis is unclear. This study aimed to investigate the effect of concurrent JCPyV-DNAemia on graft outcomes in BKPyV-infected kidney transplant recipients with polyomavirus-associated nephropathy (PyVAN). A total of 140 kidney transplant recipients with BKPyV replication and PyVAN, 122 without concurrent JCPyV-DNAemia and 18 with JCPyV-DNAemia were included in the analysis. Least absolute shrinkage and selection operator regression analysis and multivariate Cox regression analysis were used to identify prognostic factors for graft survival. A nomogram for predicting graft survival was created and evaluated. The median tubulitis score in the JCPyV-DNAemia-positive group was higher than in JCPyV-DNAemia-negative group (P = 0.048). At last follow-up, the graft loss rate in the JCPyV-DNAemia-positive group was higher than in the JCPyV-DNAemia-negative group (50% versus 25.4%; P = 0.031). Kaplan-Meier analysis showed that the graft survival rate in the JCPyV-DNAemia-positive group was lower than in the JCPyV-DNAemia-negative group (P = 0.003). Least absolute shrinkage and selection operator regression and multivariate Cox regression analysis demonstrated that concurrent JCPyV-DNAemia was an independent risk factor for graft survival (hazard ratio = 4.808; 95% confidence interval: 2.096-11.03; P < 0.001). The nomogram displayed favorable discrimination (C-index = 0.839), concordance, and clinical applicability in predicting graft survival. Concurrent JCPyV-DNAemia is associated with a worse graft outcome in BKPyV-infected kidney transplant recipients with PyVAN.

Cell-Free Mitochondrial DNA: An Upcoming Non-Invasive Tool for Diagnosis of BK Polyomavirus-Associated Nephropathy.

Mitochondria are essential organelles that possess their own DNA. Mitochondrial dysfunction has been revealed in many kidney diseases, including BK polyomavirus-associated nephropathy (BKPyVAN). In this study, we introduce an innovative approach for non-invasive monitoring of mitochondrial impairment through urinary donor-derived cell-free mitochondrial DNA (ddcfmtDNA), addressing the crucial challenge of BKPyVAN diagnosis. Urinary samples were collected at the time of biopsy from a total of 60 kidney transplant recipients, comprising 12 with stable function, 22 with T cell-mediated rejection, and 21 with biopsy-proven BKPyVAN. Our findings reveal that the ddcfmtDNA-to-ddcfDNA ratio exhibits superior capability in distinguishing BKPyVAN from other conditions, with a cutoff value of 4.96% (area under curve = 0.933; sensitivity: 71.4%; and specificity: 97.1%). Notably, an elevation of ddcfmtDNA levels is associated with mitochondrial damage, as visualized through electron microscopy. These results underscore the promise of non-invasive monitoring for detecting subtle mitochondrial damage and its potential utility in BKPyVAN diagnosis. Further investigations are required to advance this field of research.

BK polyomavirus infection: more than 50 years and still a threat to kidney transplant recipients

BK polyomavirus (BKPyV) is a ubiquitous human polyomavirus and a major infection after kidney transplantation, primarily due to immunosuppression. BKPyV reactivation can manifest as viruria in 30%–40%, viremia in 10%–20%, and BK polyomavirus-associated nephropathy (BKPyVAN) in 1%–10% of recipients. BKPyVAN is an important cause of kidney graft failure. Although the first case of BKPyV was identified in 1971, progress in its management has been limited. Specifically, there is no safe and effective antiviral agent or vaccine to treat or prevent the infection. Even in the current era, the mainstay approach to BKPyV is a reduction in immunosuppression, which is also limited by safety (risk of de novo donor specific antibody and rejection) and efficacy (graft failure). However, recently BKPyV has been getting more attention in the field, and some new treatment strategies including the utilization of viral-specific T-cell therapy are emerging. Given all these challenges, the primary focus of this article is complications associated with BKPyV, as well as strategies to mitigate negative outcomes.

Treatment of BK Polyomavirus-Associated Nephropathy in Paediatric Kidney Transplant Recipients: Leflunomide Versus Cidofovir.

BK polyomavirus-associated nephropathy is a clinicopathological entity that negatively affects graft function in kidney transplant recipients. We compared the efficacy of leflunomide and cidofovir to treat BK polyomavirus-associated nephropathy in pediatric kidney transplant recipients. Medical records of pediatric recipients with BK viremia for the period 2004 through 2019 were reviewed retrospectively, and patients diagnosed with BK polyomavirusassociated nephro-pathy were included in the study. A serum BK virus level above 104 copies/mL was accepted as BK viremia. We defined BK polyomavirusassociated nephropathy as detection of BK virus SV40 antigen on immunochemistry staining of renal graft tissue accompanied by signs of tubulointerstitial nephritis or elevated serum creatinine in addition to BK viremia. Of 304 kidney transplant recipients, 53 had persistent BK viremia; 36 of these patients (61.1% male) were included in the study with the diagnosis of BK polyomavirus-associated nephropathy. Twelve patients (33.3%) received cidofovir, and 14 (38.8%) received leflunomide. Results were similar between the cidofovir and leflunomide groups for serum creatinine level at last follow-up (0.91 ± 0.29 vs 0.94 ± 0.37 mg/dL, respectively; P = .843) and graft failure rate (8.3% vs 14.2%, respectively; P = .632). Graft failure was observed in 8.3% of patients with BK polyomavirus-associated nephropathy. Leflunomide and cidofovir showed similar efficacy for treatment of BK polyomavirus-associated nephropathy.

Is There Still a Role for Urine Cytology in Kidney Transplantation? Experience Report in a Teaching Hospital

Introduction: Urinary cytology has been used as a monitoring strategy for kidney transplant patients, live under chronic immunosuppression, as it is a non-invasive and low-cost method. The early detection of infections can prevent possible dysfunction and graft loss. However, its use has been decreased. Methods: We conducted a prospective study on voided urine samples from 29 consecutive patients undergoing kidney transplantation over a continuous period of 17 months, collected during their first-year post-transplantation. One technician prepared the samples by cytocentrifugation, Papanicolaou staining, and one pathologist analyzed them. Decoy cells (DCs) positive samples were prepared, by other two technicians, for contrast phase analysis, ultrastructural study, and immunofluorescence for SV40 T antigen. Results: We evaluated 252 urine samples from 26 patients. Two consecutive samples (0.8%) out of five, from the same patient (3.8%), referring to the fourth- and fifth-week post-transplant, showed low-grade squamous intraepithelial lesions; only rare epithelial cells with atypia of undetermined significance were detected (0.4%) in the sample corresponding to the sixth month post-transplant. The patient’s subsequent gynecological examination and Pap smear revealed no changes. Pseudohyphae and fungus yeasts were found in five samples (2.0%) from four patients (15.4%). Trichosporon sp. was identified in the urine of one of these patients. Decoy cells were detected in 25 samples (9.9 %) from six patients (23.1%). Two patients had sustained DCs shedding; one of them, presenting dirty background smears, developed Polyomavirus-associated nephropathy. Ultrastructural study of DCs showed icosahedral viral particles. Immunofluorescence (SV40 T antigen) was positive in DCs nuclei. Analysis by contrast phase was successful in samples with numerous DCs. Conclusion: Systematic urinary cytology after transplantation helps detect some infection signs. Patients with sustained DCs shedding and dirty background smears deserve special clinical attention. Electron microscopy and immunofluorescence (SV40 T antigen) are alternative techniques to detect polyomavirus reactivation. The findings suggest that urinary cytology still plays a role in kidney transplantation.

Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients

JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples a microfluidic droplet device with CRISPR-Cas13a and recombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.

A case of acute kidney injury due to native kidney BK polyomavirus-associated nephropathy in a human T-lymphotropic virus type 1 carrier

BackgroundBK polyomavirus-associated nephropathy (BKPyVAN) has become a major cause of kidney dysfunction and graft loss in kidney transplant recipients. On rare occasion, polyomavirus has also been known to affect native kidneys of immunocompromised individuals. Only a small number of opportunistic infections have been reported in the carrier phase of human T-lymphotropic virus type 1 (HTLV-1). This is the first reported case of BKPyVAN in native kidneys of an HTLV-1 carrier.Case presentationA 61-year-old man was referred to our hospital from a primary care physician for work-up and treatment of pneumonia. He was diagnosed with Pneumocystis pneumonia and identified as a HTLV-1 carrier who had not yet developed adult T-cell leukemia (ATL). The pneumonia was successfully treated with sulfamethoxazole-trimethoprim. He had never been diagnosed with any kind of kidney dysfunction. Laboratory investigations showed a serum creatinine of 5.3 mg/dL, and urinary sediment showed cells with nuclear enlargement and inclusion bodies suggesting viral infection. The urinary Papanicolaou stain showed inclusions in swollen, ground-glass nuclei, typical of “decoy cells”. Renal biopsy showed degeneration of tubules with epithelial enlargement, vacuolar degeneration, nuclear inclusion bodies, and detachment from the tubular basement membrane. Tubular nuclei showed positive staining positive for simian virus 40 large-T antigen. Polymerase chain reaction tests for BK polyomavirus DNA of both urine and plasma were positive. These findings confirmed a diagnosis of BKPyVAN. Intravenous immunoglobulin therapy did not improve renal function, necessitating maintenance hemodialysis therapy.ConclusionsBKPyVAN should be considered when acute kidney injury occurs with opportunistic infection. HTLV-1 carriers can develop opportunistic infections even before the onset of ATL.

Tissue miRNA Profile Is Associated with Acute Tubular Necrosis, Rejection Phenotypes and BK Polyomavirus-Associated Nephropathy in Human Kidney Allografts

Introduction: MicroRNAs (miRNAs), short noncoding RNAs, are involved in the modulation of gene expression, mainly by inhibiting the translation of mRNAs. Under physiological conditions, miRNAs are involved in viral infections and immune responses, among others; aberrant miRNA expression has been associated with kidney transplant pathologies, but a comprehensive comparison of later, particularly in tissue sections, is still pending. Methods: We used the genome-wide screening of miRNAs to identify those potentially involved in the disease processes after kidney transplantation. RNA was isolated from formalin-fixed paraffin-embedded kidney biopsy samples. Study included 8 patients with acute tubular necrosis (ATN), 8 patients with antibody-mediated rejection (ABMR), 10 patients with T-cell-mediated rejection (TCMR), 10 patients with BK polyomavirus-associated nephropathy (BKPyVAN), and 12 surveillance biopsies from patients with stable allograft function and no major abnormalities (normal allografts, CTRL). Results: We found 136 miRNAs differentially expressed in diseased kidney transplant tissue compared with normal allografts; of these, 74 miRNAs were differentially expressed in ABMR, 65 in ATN, 62 in BKPyVAN, 69 in TCMR, and 16 miRNAs were not associated with a specific disease phenotype. In addition, 29 miRNAs were differently expressed between ABMR and ATN, 39 between BKPyVAN and TCMR, and 20 between BKPyVAN and ABMR, and 38 between ABMR and TCMR. Conclusion: Our findings show that miRNA derived from kidney allograft biopsy samples represent an additional diagnostic tool to distinguish different disease phenotypes. This finding has the potential to assist clinicians in therapeutic decision-making and to translate to noninvasive monitoring of patients, e.g., blood samples.

Predictive Factors of BK Virus Development in Kidney Transplant Recipients and the Effect of Low-Dose Tacrolimus Plus Everolimus on Clinical Outcomes.

This study aimed to determine the predictive factors of BK virus viremia/nephropathy in kidney transplant recipients and to evaluate the effects of low-dose tacrolimus plus everolimus. This study included 3654 kidney transplant recipients. The patients were divided into 2 groups: group 1 were BK virus negative (n = 3525, 96.5%) and group 2 were BK virus positive (n = 129, viremia 3.5%, nephropathy 1%). Predictive factors were determined by receiver operating characteristic curve analysis and logistic regression models.We also divided and analyzed patients with BK virus viremia/nephropathy into 2 groups according to immunosuppressive changes. Group 2a had been switched to low-dose tacrolimus plus everolimus (n = 54, 41.9%), and group 2b had been switched to other immunosuppressive protocols (n = 75, 58.1%). We found that use of anti-T-cell lymphocyte globulin and tacrolimus, deceased donor transplant, and rejection were predictive factors for BK virus viremia/nephropathy. In addition, patients who had low-dose calcineurin inhibitor plus mammalian target of rapamycin inhibitor regimens showed a low rate of BK virus development(only 6.2% of all cases). In Group 2a, both the BK polyomavirus-associated nephropathy rate (n = 23 [42.6%] vs n = 12 [16%] in group 2b; P = .001) and viral load (DNA > 104 copies/mL) (n = 49 [90.7%] vs n = 27 [36%] in group 2b; P = .001) were increased versus group 2b. Graft function, graft survival, viral clearance, and rejection rate were similar between the groups after protocol change. BK virus viremia/nephropathy rate was lower in patients who received low-dose calcineurin inhibitor plus mammalian target of rapamycin inhibitor protocols; the low-dose tacrolimus plus everolimus switch protocol after BK virus was more effective and safe than other protocols.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

An observational cohort study of histological screening for BK polyomavirus nephropathy following viral replication in plasma

Systematic screening for BKPyV-DNAemia has been advocated to aid prevention and treatment of polyomavirus associated nephropathy (PyVAN), an important cause of kidney graft failure. The added value of performing a biopsy at time of BKPyV-DNAemia, to distinguish presumptive PyVAN (negative SV40 immunohistochemistry) and proven PyVAN (positive SV40) has not been established. Therefore, we studied an unselected cohort of 950 transplantations, performed between 2008-2017. BKPyV-DNAemia was detected in 250 (26.3%) transplant recipients, and positive SV40 in 91 cases (9.6%). Among 209 patients with a concurrent biopsy at time of first BKPyV-DNAemia, 60 (28.7%) biopsies were SV40 positive. Plasma viral load showed high diagnostic value for concurrent SV40 positivity (ROC-AUC 0.950, 95% confidence interval 0.916-0.978) and the semiquantitatively scored percentage of tubules with evidence of polyomavirus replication (pvl score) (0.979, 0.968-0.988). SV40 positivity was highly unlikely when plasma viral load is below 4 log10 copies/ml (negative predictive value 0.989, 0.979-0.994). In SV40 positive patients, higher plasma BKPyV-DNA load and higher pvl scores were associated with slower viral clearance from the blood (hazard ratio 0.712, 95% confidence interval 0.604-0.839, and 0.327, 0.161-0.668, respectively), whereas the dichotomy positivity/negativity of SV40 immunohistochemistry did not predict viral clearance. Although the pvl score offers some prognostic value for viral clearance on top of plasma viral load, the latter provided good guidance for when a biopsy was unnecessary to exclude PyVAN. Thus, the distinction between presumptive and proven PyVAN, based on SV40 immunohistochemistry, has limited clinical value. Hence, management of BKPyV-DNAemia and immunosuppression reduction should be weighed against the risk of occurrence of rejection, or exacerbation of rejection observed concomitantly.

Time-dependent variations in BK polyomavirus genome from kidney transplant recipients with persistent viremia

BK polyomavirus (BKPyV) is a human DNA virus that resides latent in the host’s renal tissue. Reactivation occurs occasionally and in case of kidney transplantation, it can lead to polyomavirus-associated nephropathy (PVAN). Due to the lack of specific antivirals for BKPyV and despite the risk of allograft rejection, reduction of immunosuppression remains the main approach for treating PVAN. Current data suggests that mutations can accumulate over time in the major capsid protein VP1 and can lead to neutralization escape in kidney transplant recipients. Herein, we show that mutations occur throughout the entire BKPyV genome, including in VP1. Changes were identified by per-patient comparison of viral genome sequences obtained in samples from 32 kidney recipients with persistent viremia collected at different post-transplant time-points. Amino acid changes were observed in both earlier and later post-transplant samples, although some of them were only found in later samples. Changes in VP1 mainly consisted in the introduction of a new amino acid. A switch back to the conservative amino acid was also observed. This should be considered in future approaches for treating BKPyV infection in kidney transplant recipients.

Clinical and immunological characteristics for BK polyomavirus-associated nephropathy after kidney transplantation.

BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) can cause a significant risk of allograft impairment after kidney transplantation (KT). Intact BKPyV-specific immunity is associated with viral containment. This study investigated BKPyV-specific immunological factors among KT recipients. This prospective study in a single transplant center from January 2019 to August 2019 assessed associations between clinical and immunological characteristics, with a focus on BKPyV-cell-specific immunity and BKPyVAN, among KT recipients aged ≥15 years. The numbers of interferon-gamma (IFN-γ)-producing CD4+ T, CD8+ T, natural killer (NK), and natural killerT (NKT) cells were measured after stimulation with large T antigen and viral capsid protein 1 (VP1). In total, 100 KT recipients were included (mean age ± SD, 42 ± 11 years); 35% of the recipients were female patients, and 70% had received induction immunosuppressive therapy. The 1-year cumulative incidence of high-level BKPyV DNAuria (possible BKPyVAN) and (presumptive BKPyVAN) was 18%. Among 40 patients with immunological factor data, pre-KT %NK cells (hazard ratio [HR], 1.258; 95% confidence interval [CI], 1.077-1.469; p = .004) and %VP1-specific NK cells (HR, 1.209; 95% CI, 1.055-1.386; p = .006) were factors independently associated with possible and presumptive BKPyVAN. KT recipients with possible and presumptive BKPyVAN were more likely to exhibit significant mean coefficients of %NK, %VP1-specific NK, and %NKT cells at 1 month after KT than before KT (all p < .05). Individuals with nonspecific and VP1-specific NK cells before KT and increasing numbers of these cells after KT may be at risk for high-level BKPyV DNAuria and presumptive BKPyVAN. Further studies are needed to determine the utility of BKPyV-specific innate immune surveillance in predicting the occurrence of BKPyVAN.

Importance of Follow-Up Biopsies in the Prediction of Renal Allograft Survival Following Polyomavirus-Associated Nephropathy.

Allograft biopsy is the gold standard for diagnosing polyomavirus-associated nephropathy. We aimed to establish the effects of histopathologic findings proposed by the Banff Polyomavirus Working Group on graft outcome. We also aimed to understand the clinical importance of follow-up biopsies for patients with polyomavirus-associated nephropathy. Our study included 22 patients with polyomavirus-associated nephropathy. All biopsies were classified according to the latest Banff Polyomavirus Working Group classification. Follow-up biopsies of all patients were evaluated in detail. The mean interval between polyomavirus-associated nephropathy and transplant was 10 ± 1.6 months. Of 22 patients, biopsy revealed stage 1 in 3 (13.6%), stage 2 in 17 (77.3%), and stage 3 in 2 patients (9.1%). Fourteen patients (63.6%) had polyomavirus viral load 3, 5 (22.7%) had polyomavirus viral load 2, and 3 had polyomavirus viral load 1. Among patients included in analyses, 18.2% had antibody-mediated rejection and 27.2% had T-cell-mediated rejection simultaneously with polyomavirus-associated nephropathy. Graft loss increased with increasing polyomavirus-associated nephropathy class and polyomavirus viral load (P = .015 and P = .002, respectively). The mean time of graft survival decreased with increasing degree of tubulitis, interstitial inflammation, plasma infiltration, and neutrophil infiltration. Patients with interstitial fibrosis, glomerular polyoma, and cortical plus medullar involvement showed earlier graft loss. Follow-up biopsies showed that diffuse interstitial fibrosis or persistent inflam-mation negatively influenced graft loss. The Banff Polyomavirus Working Group's schema significantly correlated with graft outcome. Early detection of polyomavirus-associated nephro-pathy and subsequent detection of persistent inflammation and interstitial fibrosis and tubular atrophy in follow-up biopsies and modification of immunosuppressive therapy can successfully prevent graft loss.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

Exploring the mechanism of BK polyomavirus-associated nephropathy through consensus gene network approach.

BK polyomavirus-associated nephropathy occurs in kidney transplant recipients under immunosuppressive treatment. BK polyomavirus is implicated in cancer development and invasion, and case reports of renal cell carcinoma and urothelial carcinoma possibly associated with BK polyomavirus has been reported. Further, it has been suggested that the immune responses of KT-related diseases could play a role in the pathogenesis and progression of renal cell carcinoma. Thus, we thought to examine the relationship between BK polyomavirus-associated nephropathy and renal cell carcinoma in terms of gene expression. To identify the common and specific immune responses involved in kidney transplantation-related diseases with a specific focus on BK polyomavirus-associated nephropathy, we performed consensus weighted gene co-expression network analysis on gene profile datasets of renal biopsy samples from different institutions. After the identification of gene modules and validation of the obtained network by immunohistochemistry of the marker across kidney transplantation-related diseases, the relationship between prognosis of renal cell carcinoma and modules was assessed. We included the data from 248 patients and identified the 14 gene clusters across the datasets. We revealed that one cluster related to the translation regulating process and DNA damage response was specifically upregulated in BK polyomavirus-associated nephropathy. There was a significant association between the expression value of hub genes of the identified cluster including those related to cGAS-STING pathway and DNA damage response, and the prognosis of renal cell carcinoma. The study suggested the potential link between kidney transplantation-related diseases, especially specific transcriptomic signature of BK polyomavirus associated nephropathy and renal cell carcinoma.

The relation of NCCR variations and host transcription factors gene regulation in BK polyomavirus infected kidney transplant patients

BackgroundBK polyomavirus (BKPyV) infection in immunocompromised patients can led to polyomavirus-associated nephropathy (BKPyVAN) especially after kidney transplantation. The polyomavirus genome contains enhancer elements that are important transcription activators. In this study, the association between viral and host gene expression and NCCR variations was evaluated in kidney transplant recipients (KTRs) with BKPyV active, and BKPyV in-active infection. Methods and ResultsBlood samples were collected from selected KTRs who divided to patients with active and in-active BKPyV infection. Transcriptional control region (TCR) anatomy was compared to the genomic sequence of archetype BKPyV strain WW using nested PCR method and sequencing. The expression level of some transcription factor genes was evaluated using in-house Real-time PCR (SYBR Green) technique. Most changes were observed after TCR anatomy detection in the Q and P blocks. The expression level of VP1 and LT-Ag viral genes were significantly higher in patients with active infection compared with non-infected ones. Transcription factor genes SP1, NF1, SMAD, NFκB, P53, PEA3, ETS1, AP2, NFAT and AP1 were significantly higher in BKPyV active group in comparison in-active and control groups. The analyses revealed that viral load level and mutations frequency has significant correlation. ConclusionsBased on the results, increasing of NCCR variations were associated with higher viral load of BKPyV especially in Q block. Host transcriptional factors and viral genes all had higher express level in active BKPyV patients versus no in-active ones. Detection of the relation between NCCR variation and BKPyV severity in KTRs need to be confirmed in further complicated studies.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1 × 107 copies/ml in urine (viruria) or 1 × 104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (&amp;gt;18 years) patients who underwent renal transplantation (RT) at the State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Healthcare institutions “Brest Regional Clinical Hospital”, “Vitebsk Regional Clinical Hospital Belarus”, “Mogilev Regional Clinical Hospital”. These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2 × 1012 copies/ml in urine and 5.9 × 107 copies/ml in serum. JCV loads were up to 3 × 109 copies/ml in urine and 1.2 × 108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the Fig. 1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1 × 107 copies/ml in urine or 1 × 104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1×107 copies/ml in urine (viruria) or 1×104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (&amp;gt;18 years) patients who underwent renal transplantation (RT) at the State Institution "Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology", Healthcare institutions "Brest Regional Clinical Hospital", "Vitebsk Regional Clinical Hospital Belarus", "Mogilev Regional Clinical Hospital". These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2×1012 copies/ml in urine and 5.9×107 copies/ml in serum. JCV loads were up to 3×109 copies/ml in urine and 1.2×108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the fig.1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1×107 copies/ml in urine or 1×104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.