BKV-associated Nephropathy Research Articles

Characteristics, risk factors and outcome of BKV nephropathy in kidney transplant recipients: a case–control study

BackgroundFollowing kidney transplantation, BK virus associated nephropathy (BKVN) occurs in 1 to 10% of kidney transplant recipients (KTR) and represents a major cause of graft loss. We aim at identifying factors associated with biopsy proven BKVN among KTR.MethodsWe conducted a retrospective case–control study including all KTR with a biopsy-proven diagnosis of BKVN between 2005 and 2019. Clinical characteristics and outcome were described. For each case, one control KTR without BKV infection was identified and matched by age, transplant date, and donor status. Factors associated with BKVN diagnosis were identified using exact conditional logistic regression. Comparative survival was described using Kaplan–Meier estimator.ResultsSixty-four cases of BKVN were identified among 1737 new kidney transplantation (3.7% prevalence). Clinical characteristics did not differ between groups, except for a higher c-PRA among cases. BKVN occurred in a median time of 11 (5–14.5) months after KT, and was associated with a significantly impaired graft function at diagnosis. Following BKVN, 61 (95%) of the patients had immunosuppression reduction, which led to BKV DNAemia resolution in 49% of cases. In multivariate analysis, factors associated with BKVN diagnosis were lymphopenia < 500/mm3 and a prednisone dose > 7.5 mg/day. Median duration of follow-up was 40 months for both groups. BKVN was associated with a significantly increased risk of graft rejection (P = 0.02) and return to dialysis (P = 0.01).ConclusionsBKVN remains a severe complication in KTR and is associated with an increased risk for acute rejection and return to dialysis. Lymphopenia below 500/mm3 and corticosteroid maintenance therapy are significantly associated with biopsy-proven BKVN diagnosis.

Prevalence and Impact of BK Polyomavirus in the Ureters of Kidney Donors: Research Letter.

More than 75% of the population is seropositive for BK polyomavirus (BKV), which remains quiescent in the urothelium in immunocompetent hosts. However, it can reactivate in kidney transplant recipients (KTRs), and up to 30% of them will develop BKV viremia in the 2 years following transplant, with a risk of developing BKV-associated nephropathy (BKVAN). Viral reactivation is associated with the level of immunosuppression, but there is currently no way to predict which patients are at high risk for reactivation. As BKV originates from kidney donors, our primary objective was to determine the prevalence of detectable BKV in donor ureters. Our secondary objective was to see if there is a correlation between the presence of BKV in donor urothelium and the development of BKV viremia and BKVAN in KTR. Prospective cohort study. Single-center academic kidney transplant program. Prospective sequential KTRs that received a kidney transplant between March 2016 and March 2017. The presence of BKV in donor ureters was determined by TaqMan-based quantitative polymerase chain reaction (PCR; qPCR). We performed a prospective study which was done on 35 out of the 100 donors initially foreseen to take part in the study. During surgery, the distal part of donor ureter was kept and analyzed by qPCR (to establish the presence of BKV in the urothelium). The primary outcome was the development of BKV viremia in KTR over a period of 2 years after transplant. Secondary outcome was the development of BKVAN. Out of 35 ureters analyzed, only one had a positive qPCR for BKV (2.86%, 95% confidence interval [CI]: [0.07-14.92]). Considering the primary objective would not be met, the study was interrupted after 35 specimens. After surgery, 9 recipients had a slow graft function and 4 had delayed graft function, one of which never recovered graft function. Over the 2-year follow-up, 13 patients developed BKV viremia, while 5 patients developed BKVAN. The patient who received a graft from a positive qPCR donor eventually developed BKV viremia and nephropathy. The specimen analyzed was a distal rather than a proximal portion of the ureter. However, BKV replication is known to concentrate in the corticomedullary junction. BK polyomavirus prevalence in the distal part of donor ureters is lower than previously reported. It cannot be used as a predictor for the development of BKV reactivation and/or nephropathy.

Is Polyomavirus-Associated Nephropathy More Common in Kidney Transplant Recipients Exposed to Valganciclovir? A Retrospective Single Center Analysis

BK polyomavirus-associated nephropathy (PVAN) is a frequent complication in the early phase after kidney transplantation. The most important risk factor for PVAN is the intensity of immunosuppression. A recent study suggests that exposure to valganciclovir (VGC) could also be a risk factor. We performed a retrospective, single-center study to investigate the effect of valganciclovir exposure on the risk for PVAN during the first 100 days post transplant. Cytomegalovirus (CMV) seronegative recipients of a CMV seropositive donor kidney received VGC prophylaxis, whereas CMV seropositive recipients were managed by a pre-emptive CMV strategy. Cox regression analysis was used to identify risk factors for PVAN development with VGC treatment and strength of immunosuppressive therapy as time-dependent variables. A total of 211 adults who received a kidney transplant between 2014 and 2019 were included. Eighteen (9%) developed PVAN. Multivariate regression analysis showed that women have a lower risk of developing PVAN (hazard ratio [HR] 0.08 (confidence interval [CI] 0.01-0.58), P=.013), whereas age was associated with an increased risk for PVAN (HR 1.04 for every additional year [CI 1.00-1.08], P=.029). There was a trend toward a lower risk of PVAN for patients on reduced immunosuppressive therapy (HR 0.44 [CI 0.15-1.24], P=.12). VGC use was not associated with the risk for PVAN (HR 0.99 [CI 0.35-2.78], P=.98). In our study, VGC exposure was not associated with the risk for PVAN. Our study is the first to reassess in depth the hypothesis that VGC treatment increases the risk of PVAN. The unique strength of this study is the correction for the degree of immunosuppression and the statistical use of time-dependent covariates. This methodological approach can provide a foundation for further studies needed to confirm our findings.

Incidence, Risk Factors, and Outcomes of Kidney Transplant Recipients With BK Polyomavirus-Associated Nephropathy

BK polyomavirus-associated nephropathy (BKPyVAN) is associated with graft dysfunction and loss; however, knowledge of immunosuppression reduction strategies and long-term graft, and patient outcomes across the disease spectrum is lacking. This cohort study included 14,697 kidney transplant recipients in Australia and New Zealand (2005-2019), followed for 91,306 person years. BKPyVAN occurred in 460 recipients (3%) at a median posttransplant time of 4.8 months (interquartile range, 3.1-10.8). Graft loss (35% vs. 21%, P< 0.001), rejection (42% vs. 25%, P< 0.001), and death (18% vs. 13%, P= 0.002) were more common in the BKPyVAN group. The most frequent changes in immunosuppression after BKPyVAN were reduction (≤50%) in tacrolimus (172, 51%) and mycophenolate doses (134, 40%), followed by the conversion of mycophenolate to leflunomide (62, 19%) and tacrolimus to ciclosporin (20, 6%). Factors associated with the development of BKPyVAN included (adjusted hazard ratio [HR]; 95% confidence interval) male sex (1.66; 1.34-2.05), recipient age (≥70 vs.<20 [2.46; 1.30-4.65]), recipient blood group (A vs. B [2.00; 1.19-3.34]), donor age (≥70 vs.<20 [2.99; 1.71-5.22]), earlier era (1.74; 1.35-2.25), donor/recipient ethnic mismatch (1.52; 1.23-1.87), tacrolimus use (1.46; 1.11-1.91), and transplantation at a lower-volume transplant center (1.61; 1.24-2.09). The development of BKPyVAN was associated with an increased risk of all-cause (1.75; 1.46-2.09) and death-censored graft loss (2.49; 1.99-3.11), but not mortality (1.15; 0.91-1.45). BKPyVAN is associated with an increased risk of all-cause and death-censored graft loss, but not death. Interventional trials are urgently needed to evaluate the efficacy of immunosuppression reduction and novel strategies to minimize the adverse outcomes associated with BKPyVAN.

In silico design and evaluation of a novel mRNA vaccine against BK virus: a reverse vaccinology approach.

Human polyomavirus type 1, or BK virus (BKV), is a ubiquitous pathogen belonging to the polyomaviridae family mostly known for causing BKV-associated nephropathy (BKVN) and allograft rejection in kidney transplant recipients (KTRs) following the immunosuppression regimens recommended in these patients. Reduction of the immunosuppression level and anti-viral agents are the usual approaches for BKV clearance, which have not met a desired outcome yet. There are also debating matters such as the effect of this pathogen on emerging various comorbidities and the related malignancies in the human population. In this study, a reverse vaccinology approach was implemented to design a mRNA vaccine against BKV by identifying the most antigenic proteins of this pathogen. Potential immunogenic T and B lymphocyte epitopes were predicted through various immunoinformatic tools. The final epitopes were selected according to antigenicity, toxicity, allergenicity, and cytokine inducibility scores. According to the obtained results, the designed vaccine was antigenic, neutral at the physiological pH, non-toxic, and non-allergenic with a world population coverage of 93.77%. Since the mRNA codon optimization ensures the efficient expression of the vaccine in a host cell, evaluation of different parameters showed our designed mRNA vaccine has a stable structure. Moreover, it had strong interactions with toll-like receptor 4 (TLR4) according to the molecular dynamic simulation studies. The in silico immune simulation analyses revealed an overall increase in the immune responses following repeated exposure to the designed vaccine. Based on our findings, the vaccine candidate is ready to be tested as a promising novel mRNA therapeutic vaccine against BKV.

The role of the DE and EF loop of BKPyV VP1 in the serological cross-reactivity between subtypes

BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV.We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA.We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.

Prevalence of JC and BK Polyomavirus Infection in Patients with Chronic Kidney Disease in the State of Pará, Brazil.

The polyomaviruses that infect humans, JC virus (JCV) and BK virus (BKV), can establish persistent infections in the cells that make up the renal system, causing nephritis and BKV-associated nephropathy in up to 10% of renal transplant patients, and of these, 90% lose the graft and return for hemodialysis. This study aimed to determine the prevalence of polyomaviruses (PyV) in the population with chronic kidney disease (CKD), classified into three groups (conservative, dialysis, and transplanted) and a control group. Urine samples were collected from 290 individuals, including 202 patients with CKD and 88 from the control group. PyV screening was performed by PCR amplification of a fragment of the VP1 region, and the JCV and BKV species were distinguished through enzymatic digestion with the restriction endonuclease BamHI from the amplification of a TAg region. All amplification products were visualized on a 3% agarose gel. The prevalence of PyV infection was correlated with clinical-epidemiological variables using the chi-squared and Fisher's exact tests. In the group with CKD, the prevalence of PyV was 30.2%, a higher rate being observed in conservative patients (36.66%; 22/60), followed by dialysis patients (30.48%; 25/82), and transplanted patients (20%; 12/60). In the control group, the prevalence was 46.59% (41/88). The differentiation between species revealed that JCV was present in 77.8% and BKV in 22.2% of the group with CKD. The prevalence of infection was higher in male patients (59.32%), whose most common pathology was systemic arterial hypertension (35.59%). In the group of transplanted patients, there was a statistically significant association between infection and the use of the immunosuppressant azathioprine (p = 0.015). The prevalence of PyV infection was higher in the control group than in the group with CKD, being predominant in males and in patients with systemic arterial hypertension.

1465. A Randomized Double-Blinded Placebo-Controlled Study to Determine Efficacy of Immunoglobulin Therapy to Treat BK Viremia in Renal Transplant Recipients

Abstract Background BK viremia in renal transplant recipients increases risk of BK virus-associated nephropathy (BKVAN), posing a threat to allograft function. Up to 30% of all renal transplant recipients develop BK viremia and 1-10% will develop BKVAN. With no effective antiviral, the mainstay of therapy to prevent BKVAN is immunosuppression reduction (IR). Case series showed potential efficacy of using intravenous immunoglobulins (IVIG) to treat BK viremia. However, a randomized placebo-controlled study is needed. Methods A multicenter prospective double-blinded randomized placebo-controlled proof-of-concept study was conducted at three transplant institutions. 14 adult renal transplant recipients diagnosed with BK viremia were randomized (1:1) to receive IVIG and IR versus placebo and IR and were followed for 12 months, with the primary endpoint defined as resolution of BK viremia by month 3. Samples were collected at 1, 2, 3, 6, and 12 months for viral load quantification and immunological assays. Results At enrollment, clinical characteristics of the IVIG group (n = 5) were similar to the control group (n = 9), except that a larger number of participants in the control group had delayed graft function (0% vs. 77.8%, p = 0.02) and a higher baseline BK viral load (11300 vs 99300 copies/mL, p = 0.04). At 3 months, 2 out of 5 patients in the treatment group and 4 out of 9 patients in the control group cleared their viremia (40% vs 44.4%, RR 0.89, 95% CI 0.21 – 3.35, p &amp;gt; 0.99). Patients who received IVIG and IR had reduction in viral load at 6 months only (11300 vs 141 copies/mL, p = 0.008). Those who received IR alone had continued reduction in viral load starting at 2 months (99300 vs 4700, 1542, 906, 278 copies/mL; p &amp;lt; 0.05). No participant progressed to BKVAN. Immunological profiles of each participant will be correlated to the viral load. Serum BK viral loads were higher in the placebo group than the IVIG group at enrollment, but not significantly different between the two groups at any of the follow-up time points. The dotted line represents threshold of viremia clearance at less than 1000 copies/mL. * p = 0.04. Bar – median (Mann-Whitney between group comparisons). Median serum BK viral loads of the IVIG group decreased at the 6 months follow-up time point. The dotted line represents threshold of viremia clearance at less than 1000 copies/mL. ** p = 0.008 (Mann-Whitney comparing enrollment to each follow-up time point). Median serum BK viral loads of the placebo group continued to decrease at each follow-up time point. The dotted line represents threshold of viremia clearance at less than 1000 copies/mL. * p = 0.04; ** p = 0.006; *** p = 0.0008, 0.0003 (Mann-Whitney comparing enrollment to each follow-up time point). Conclusion This proof-of-concept study illustrates that a clinical trial of IVIG versus placebo for the treatment of BK viremia in renal transplant recipients is feasible. IVIG may not be more effective than IR alone; its immunosuppressive effect may even limit BK clearance. A larger sample is needed to attenuate baseline differences between groups and to provide a higher level of evidence on IVIG therapy in BK viremia. Disclosures All Authors: No reported disclosures.

2105. Early-onset and Late-onset BK Polyomavirus-associated Nephropathy: A 10-year Retrospective Analysis

Abstract Background Polyomavirus-associated nephropathy (BKVAN) is one of the serious causes of allograft dysfunction and allograft loss in kidney transplant (KT) patients. Most of the patients were diagnosed with BKVAN within the first year after transplantation but some more cases of BKVAN occur later. A study focused on the differentiation between early and late-onset BKVAN is needed. Methods We conducted a retrospective cohort study among KT recipients at Ramathibodi Hospital between January 2010 – December 2020. The incidence of early-onset (diagnosed within 1 year) and late-onset (diagnosed after 1 year) BKVAN, as well as composite kidney allograft outcomes, were compared. Results From 1032 KT recipients, 645 (62.5%) were screened for BK viral infection. BKVAN was diagnosed in 83 (12.8%) patients. Of those, 46 (55.4%) and 37 (44.6%) were diagnosed with early-onset and late-onset BKVAN, respectively. Composite kidney allograft outcomes of GFR decline≥ 40% from baseline, graft loss or death were greater in early-onset BKVAN compared to late-onset BKVAN (7.5 vs 5.52 per 1000 person-months; hazard ratio 0.47; 95%CI, 0.23-0.95; P=0.037). In multivariate analysis, female gender and living-related kidney transplantation (LRKT) were independently associated with late-onset BKVAN compared to early-onset BKVAN. The more advanced the BKVAN diagnosis, the worse the allograft outcome occurs, even with optimizing immunosuppressive drugs and adjunctive therapy. Conclusion BKVAN could occur later after one-year post-transplantation, although relatively better composite kidney outcomes were observed compared to early-onset BKVAN. Female individuals who underwent living-related KT should be considered for BK virus preemptive monitoring beyond one year to detect this late-onset BK virus infection. Disclosures All Authors: No reported disclosures.

Complications of rabbit anti-thymocyte globulin induction immunosuppression in HIV-infected kidney transplant recipients.

Kidney transplantation in HIV-infected individuals with end-stage kidney disease is associated with improved survival compared to dialysis. Rabbit anti-thymocyte globulin (rATG) induction in HIV-infected kidney transplant recipients has been associated with a lower risk of acute rejection, but data on the rates of de novo malignancy and BK viremia in these patients is lacking. We performed a single-center retrospective cohort study of adult HIV-infected individuals who underwent kidney transplantation with rATG induction between January 2006 and December 2016. The primary outcome was the development of de novo malignancy. Secondary outcomes included the development of BK viremia, infections requiring hospitalization, HIV progression, biopsy-proven acute rejection, and patient and allograft survival. Twenty-seven HIV-infected individuals with end-stage kidney disease received deceased (n=23) or living (n=4) donor kidney transplants. The cumulative rate of malignancy at five years was 29%, of whom 29% died because of advanced malignancy. BK viremia was detected in six participants (22%), of whom one had biopsy-proven BK virus-associated nephropathy and all of whom cleared the BK viremia. Five-year acute rejection rates, patient survival and death-censored allograft survival were 17%, 85% and 80% respectively. rATG induction in HIV-infected kidney transplant recipients was associated with a low risk of acute rejection, but a potentially higher risk of de novo malignancies and BK viremia in this cohort. Screening strategies to closely monitor for BK virus infection and malignancy post-transplantation may improve outcomes in HIV-infected kidney transplant recipients receiving rATG induction.

Tumor necrosis factor-alpha blockade suppresses BK polyomavirus replication.

BK Polyomavirus (BKPyV) infection manifests as renal inflammation and can cause kidney damage. Tumor necrosis factor-α (TNF-α) is increased in renal inflammation and injury. The aim of this study was to investigate the effect of TNF-α blockade on BKPyV infection. Urine specimens from 22 patients with BKPyV-associated nephropathy (BKPyVN) and 35 non-BKPyVN kidney transplant recipients were analyzed. We demonstrated increased urinary levels of TNF-α and its receptors, TNFR1 and TNFR2, in BKPyVN patients. Treating BKPyV-infected human proximal tubular cells (HRPTECs) with TNF-α stimulated the expression of large T antigen and viral capsid protein-1 mRNA and proteins and BKPyV promoter activity. Knockdown of TNFR1 or TNFR2 expression caused a reduction in TNF-α-stimulated viral replication. NF-κB activation induced by overexpression of constitutively active IKK2 significantly increased viral replication and the activity of the BKPyV promoter containing an NF-κB binding site. The addition of a NF-κB inhibitor on BKPyV-infected cells suppressed viral replication. Blockade of TNF-α functionality by etanercept reduced BKPyV-stimulated expression of TNF-α, interleukin-1β (IL-1β), IL-6 and IL-8 and suppressed TNF-α-stimulated viral replication. In cultured HRPTECs and THP-1 cells, BKPyV infection led to increased expression of TNF-α, interleukin-1 β (IL-1β), IL-6 and TNFR1 and TNFR2 but the stimulated magnitude was far less than that induced by poly(I:C). This may suggest that BKPyV-mediated autocrine effect is not a major source of TNFα. TNF-α stimulates BKPyV replication and inhibition of its signal cascade or functionality attenuates its stimulatory effect. Our study provides a therapeutic anti-BKPyV target.

Conversion to mTOR-Inhibitors Plus IV Immunoglobulins in Kidney-Transplant Recipients with BKV Infection: A Retrospective Comparative Study.

BK virus-associated nephropathy (PvAN) increases the risk of graft failure justifying treatment. Conversion to mammalian target of rapamycin inhibitors (mTORi) and Human polyclonal immunoglobulins (IVIg) could prevent the risk of PvAN. Our retrospective study assessed the efficacy of mTORi associated with IVIg therapy (mTORi±IVIg group) versus standard immunosuppression reduction to clear BKV DNAemia. Among forty-three kidney-transplanted patients with positive BKV DNAemia, we included twenty-six patients in the mTORi±IVIg group and reduced immunosuppression therapy for seventeen patients. We focused on BKV DNAemia clearance on the first-year. Renal function, rejection rate, evolution to PvAN, and complications of immunosuppression were assessed. BKV DNAemia decreased faster and significantly in the control group as compared to the mTORi±IVIg group (p &lt; 0.001). Viral clearance was significantly higher in the control group compared to the mTORi±IVIg group (88% vs. 58%; p = 0.033). Death-censored graft loss, rejection rates and kidney-graft function at 12 months did not significantly differ. Multivariate analyses significantly associated BKV DNAemia clearance with reducing immunosuppression (OR = 0.11 (0.06-0.9), p = 0.045), female kidney donor (OR = 0.10 (0.01-0.59/)], p = 0.018) and time to first DNAemia, (OR = 0.88 (0.76-0.96), p = 0.019). In our study, the standard treatment for BKV DNAemia had better outcomes than an mTORi±IVIg conversion.

Host and viral RNA dysregulation during BK polyomavirus infection in kidney transplant recipients.

Early detection of BK polyomavirus (BKPyV) infection in kidney transplant recipients (KTRs) would enhance their quality of life and save the allograft. Still, many patients lose their grafted kidneys because of this infection. BKPyV microRNAs (miRNAs) have been detected in KTRs during viral infection. BKPyV produces two mature miRNAs that are named BKV-miR-B1-5p and BKV-miR-B1-3p. Additionally, BKPyV associated nephropathy (BKVAN) in kidney transplanted patients cause changes in the expression level of host genes and miRNAs such as IFN-ɣ, BCLA2A1, has-miR-10, and has-miR-30a. BKVAN can alter viral genes and miRNAs expression level, too, like viral miRNAs and T-Ag. However, their potential value as viral infection markers and the regulatory network produced by their expression during viral-host interactions needs more consideration since there are no approved medications for treating BKPyV-related diseases in KTRs. Hence, it is vital to recognize complicated facts regarding the impact of BKPyV infection on the distribution of miRNAs and mRNAs within the host cell and the virus. This article is categorized under: Translation > Regulation RNA Processing > Processing of Small RNAs RNA in Disease and Development > RNA in Disease.

Thiết lập quy trình PCR-RFLP xác định kiểu gen vi rút BK phân lập từ bệnh nhân ghép thận

The monitoring and treatment of patients after kidney transplant is important in maintaining transplant kidney function, including BK polyomavirus surveillance. BKV is a common opportunistic pathogen in kidney transplant patients and can cause BKV-associated nephropathy, which represents a major problem for these patients, causing graft dysfunction and graft loss. BKV is classified into four genotypes, I, II, III and IV. Here, thirty urine samples from kidney transplant patients were collected and quantified BKV-DNA loads by real-time PCR, viral loads were 105-1010 copies/ml. We successfully amplified and purified the specific VP1-BKV region with 580 bp in length using primers BK_(S+AS). Additionally, we also identified BKV genotypes by PCR-RFLP using the restriction enzymes RsaI and AluI. In total 20 out of 30 cases (66.7%) were genotype I and 10 cases (33.3%) were genotype IV in collected urine samples. Comparing the obtained PCR-RFLP results with direct sequencing analysis achieved complete concordance (30/30, 100%). We successfully developed a cost-effective and accurate BKV genotyping assay using PCR-RFLP, that contributes to the monitoring of patients after kidney transplantation.

Tối ưu quy trình phân lập hệ gen virus BK từ mẫu máu, nước tiểu của bệnh nhân sau ghép thận

BK polyomavirus (BKV) is a common opportunistic pathogen in the community, infecting humans in their early life stages, often remaining in a latent state. BK virus-associated nephropathy occurs in 2 to 10% of kidney recipients, and up to 40 to 90% of these patients have permanent loss of graft function due to delay in diagnosis. In the present study, we have successfully optimized the component and thermal cycling for the long-PCR assay to amplify the BKV genome, including middle-concentration samples such as plasma (concentration about 105 copy/ml). We have successfully amplified the BKV genome in 41/50 samples, including 04 plasma and 39 urine samples. The optimized amplification protocol of full-length BKV genome includes the following steps: 1) Extraction of DNA-BKV from plasma and urine samples and quantification of BKV-DNA concentrations to ensure that about 105 copies/ml (plasma) and 107 copies/ml (urine); 2) Each full-length genome reaction in the volume of 50 μl containing 1X Phusion HF master mix (Thermo Scientific, USA); 0.5 μM of wBK_a(F+R) primers, 3-5 μl of genomic DNA. Cycling conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s; 58°C for 20 s; 72°C for 2 min, and 72ºC for 6 min and a 4oC hold; 3) All 5.1 kb purified PCR were verified and confirmed by restriction enzymes XbaI, EcoRI, and direct sequencing.

Longitudinal viral kinetic study of BK virus in renal transplant patients - A single-center study in Sri Lanka

BackgroundThere is a strong association between high-level BK viraemia and the development of BK virus-associated nephropathy. Therefore, understanding the kinetics of BK virus replication following transplantation would be helpful in predicting high-level viraemia and the development of BKVAN. ObjectivesThis study was aimed to assess the incidence of BKV infection and the BK viral kinetics among renal transplant recipients in their first year following the transplant. MethodsThis study was a retrospective descriptive study involving renal transplant recipients treated at National Hospital, Kandy, Sri Lanka. The patients were recruited from January 2018 to December 2019 and their demographic details and quantitative BKV PCR assay results were analysed during the first year following transplantation. Results297 patients were transplanted during the study period wherein, males predominated with 78.1% and 43.67 years old being the mean age (IQR: 35.50–53.0). The cumulative incidence of BK viraemia was 24.9% (n = 74). The mean age of the cohort with BK viraemia was 44.4 years (IQR: 36–54). Twenty percent (n = 13) of the female population had BKV viraemia, while 26.29% (n = 61) of the male population. Among patients with BK viraemia 12 had significant viraemia of 104IU/ml giving a cumulative incidence of significant viraemia was 4.04% and 10 of them (83.33%) had low levels of viraemia before showing significant viraemia. ConclusionThe presence of low-level viraemia in 83% of patients with significant viraemia proves the value of early routine screening to predict the development of significant BK viraemia and to take preventive measures.

Everolimus reduces BK polyomavirus infection by suppressing its replication and spread of infection

BK polyomavirus-associated nephropathy is one of serious complications in transplant recipients. Everolimus–a mammalian target of rapamycin inhibitor–has been shown to reduce the incidence of BK polyomavirus infection in transplant recipients. In this study, the effects of everolimus were examined on viral replication and the spread of infection in BK polyomavirus-infected cultures. BK polyomavirus replicated in renal and pulmonary cells, contrary to that in hepatocytes, and spread as diffusely scattered patterns of infected cells, unlike plaque formation through the cell-to-cell mode. BK polyomavirus is stable to heat up to 65 °C with a particle per infectivity ratio of 5000, and the replication cycle was for approximately 34 h. Everolimus administration remarkably reduced the viral replication to 20% in cells treated with 0.1–10 ng/mL, the concentration at which everolimus reached the serum of transplant recipients. In addition, it reduced the amount of viral capsid protein 1 at 5 ng/mL without reducing the ratio of viral capsid protein 1 versus β-actin, and it also retained the pattern of viral capsid protein 1 localization in the nuclei. Everolimus suppressed the number of infected cells to 32.8% during a 14-day treatment, indicating the reduction of BK polyomavirus-infected cell mass to 18.8% of untreated cultures by modifying cellular functions. The reduction in the total number of BK polyomavirus infected cells by everolimus indicates that everolimus alleviates BK polyomavirus infection, including nephropathy in transplant recipients.

Detection of BK polyomavirus-associated nephropathy using plasma graft-derived cell-free DNA: Development of a novel algorithm from programmed monitoring.

Graft-derived cell-free DNA (GcfDNA) is a promising non-invasive biomarker for detecting allograft injury. In this study, we aimed to evaluate the efficacy of programmed monitoring of GcfDNA for identifying BK polyomavirus-associated nephropathy (BKPyVAN) in kidney transplant recipients. We recruited 158 kidney transplant recipients between November 2020 and December 2021. Plasma GcfDNA was collected on the tenth day, first month, third month, and sixth month for programmed monitoring and one day before biopsy. ΔGcfDNA (cp/mL) was obtained by subtracting the baseline GcfDNA (cp/mL) from GcfDNA (cp/mL) of the latest programmed monitoring before biopsy. The receiver operating characteristic curve showed the diagnostic performance of GcfDNA (cp/mL) at biopsy time and an optimal area under the curve (AUC) of 0.68 in distinguishing pathologically proven BKPyVAN from pathologically unconfirmed BKPyVAN. In contrast, ΔGcfDNA (cp/mL) had a sensitivity and specificity of 80% and 84.6%, respectively, and an AUC of 0.83. When distinguishing clinically diagnosed BKPyVAN from clinical excluded BKPyVAN, the AUC of GcfDNA (cp/mL) was 0.59 at biopsy time, and ΔGcfDNA (cp/mL) had a sensitivity and specificity of 81.0% and 76.5%, respectively, and an AUC of 0.81. Plasma ΔGcfDNA (cp/mL) was not significantly different between TCMR [0.15 (0.08, 0.24) cp/mL] and pathologically proven BKPyVAN[0.34 (0.20, 0.49) cp/mL]. In conclusion, we recommend programmed monitoring of plasma GcfDNA levels after a kidney transplant. Based on our findings from the programmed monitoring, we have developed a novel algorithm that shows promising results in identifying and predicting BKPyVAN.

CC Genotype of GNAS c.393C>T (rs7121) Polymorphism Has a Protective Effect against Development of BK Viremia and BKV-Associated Nephropathy after Renal Transplant

The GNAS gene encodes the alpha-subunit of the stimulatory G-protein (Gαs) in humans and mice. The single-nucleotide polymorphism of GNAS, c.393C>T, is associated with an elevated production of Gαs and an increased formation of cyclic adenosine monophosphate (cAMP). In the present study, we analyzed the effect of this GNAS polymorphism on a renal allograft outcome. We screened a cohort of 436 renal allograft recipients, who were retrospectively followed up for up to 5 years after transplant. GNAS genotypes were determined with polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays. The 393T allele was detected in 319 (73%) recipients (113 recipients with TT and 206 with CT genotype) and the CC genotype in 117 (27%). The CC genotype was associated with a significantly lower frequency of BK viremia (CC, 17 recipients (15%); T 84 (26%)); p = 0.01; TT, 27 vs. CC, 17, p = 0.07; TT, 27 vs. CT, 57, p = 0. 46; CT, 57 vs. CC, 17, p = 0.01) and BKV-associated nephropathy (CC, 3 recipients (3%); T, 27 (8%); p = 0.03; TT,10 vs. CC, 3, p = 0.04; TT, 10 vs. CT,17, p = 0.85; CT, 17 vs. CC,3, p = 0.04) after transplant. BKV-associated nephropathy-free survival was significantly better among CC genotype carriers than among T allele carriers (p = 0.043; TT vs. CC, p = 0.03; CT vs. CC, p = 0.04; TT vs. CT, p = 0.83). Multivariate analysis indicated an independent protective effect of the CC genotype against the development of both BK viremia (relative risk. 0.54; p = 0.04) and BKV-associated nephropathy after renal transplant (relative risk. 0.27; p = 0.036). The GNAS 393 CC genotype seems to protect renal allograft recipients against the development of BK viremia and BKV-associated nephropathy.

310.5: Torque teno virus Titre Changes Under Immunosuppression Reduction Protocol for BK Virus-Associated Nephropathy: A Single Centre Pilot Study

Introduction: In BK virus-associated nephropathy (BKVaN), balancing the risk of immunosuppression (IS) reduction with the potential benefits can be challenging. A common approach is to empirically stop antiproliferative agents in the first instance and reduce the dose of calcineurin inhibitors (CNIs) later. Torque Teno virus (TTV), a novel ubiquitous DNA virus, has emerged as a better predictor of IS burden in some studies. We examined the changes in BKV and TTV titres in response to IS reduction as part of BKVaN treatment. Method: 20 renal transplant patients with stored blood samples were included in the study cohort from a population transplanted between 2017-19, in remission from an episode of clinically significant BK viraemia. They received standard IS; tacrolimus, antiproliferative (mycophenolate mofetil/azathioprine) and weaning dose prednisolone. BKV and TTV titres, 30-day average tacrolimus concentration, the dose of antiproliferative and prednisolone were collected at 4-time points. These included the last negative BK PCR; the onset of viraemia; the peak BK titre and BK titre <500 copies/ml. TTV DNA was extracted from stored frozen plasma and PCR was performed with Thermofisher 7500 Fast platform and TTV R-GENE® kit. The relationships between the variables were assessed using a multivariable linear regression model. Data were collected from electronic patient records and analysed with R 4.0.3. Result: The log-transformed mean TTV titre at the 4 time points were 5.02 (2.07), 5.52 (1.74), 6.23 (2.14) and 4.56 (1.27) respectively. In the multivariable model, log-transformed BK virus titre increased by 0.25 for every 1 ng/mL increase in tacrolimus concentration (p 0.0008). However, every 1 log rise in TTV titre led to 0.57 (0.17) log copies increase in BKV titre (p 0.0009), once adjusted for tacrolimus concentration. The relationship between tacrolimus and BKV became statistically insignificant when TTV titre was added as a predictor variable. This implies that TTV titre is a more sensitive predictor of BK viral load than tacrolimus concentration. Although statistically significant, all changes in antiproliferative or prednisolone dose were unable to predict linear changes in BKV titres. 5 patients who lost their graft in the mean follow up of 2.8 years had significantly low TTV titres (p< 0.05) at baseline, indicating suboptimum IS burden. In this small cohort, there was no relationship between TTV titres and rejections (n=5) or de novo DSA (n=3). An ongoing adequately powered prospective study will address these questions more decisively.Conclusion: Our study demonstrates that TTV titres more accurately indicate the effect of IS/CNI changes on BK titre and demonstrates its potential as a sensitive assay to objectively guide IS modification compared to current empirical practice in the management of BKVaN. A randomised control trial is warranted comparing empirical practices to TTV titre guided adjustment of immunosuppressive medications.