BK virus (BKV) infection is currently the most common infection seen in renal transplant recipients (1). This infection can be detected as asymptomatic BK viremia and BK viruria, BK viruria without viremia, or BKV nephritis (BKVN) with or without renal dysfunction. BKVN, if untreated, can progress to irreversible kidney graft failure (2). The clinical research on BKV infection has been focused on early identification, pathogenesis, and treatment strategies. Routine testing for BK viremia and viruria for all kidney transplant recipients is now the accepted screening at most centers. This approach toward testing all recipients is due to the lack of our ability to identify the subset of recipients, who are at highest risk for developing BKV infection. There is a paucity of studies that have addressed specific donor and recipient risk variables or specific immune tests for BKV infection that can predict posttransplantation BKV infection. We conducted a single-center prospective study and evaluated the impact of recipient and donor BKV-specific IgG antibody status before transplantation on the occurrence of posttransplantation BKV infection.
Of the 240 adult and 15 pediatric recipients, analysis was conducted on 192 adult and 11 pediatric transplant pairs with available donor and recipient IgG status.
Adult Study Groups
Of 192 adult recipients, 41 (21.5%) belonged to the D+R- group, 42 (22%) to the D+R+ group, 41 (21.5%) to the D-R+ group, and 68 (35%) belonged to the D-R- group. Details of the study subjects according to pretransplantation BKV-specific serostatus and subsequent posttransplantation BKV infection are shown in Figure 1.
Study Subject Variables
Baseline donor and recipient demographic variables, including BKV serostatus and transplantation and posttransplantation variables, are shown in Table 1. Approximately 68% of recipients in the D+R- group were females as opposed to 43% in the D+R+ group, 42% in the D-R+ group, and 32% in the D-R- group. The other risk variables such as donor demographics (age, sex, and race), recipient variables (age, race, and diabetes) and other transplantation variables (donor source and living vs. deceased donor), levels of pretransplantation panel reactive antibody (PRA), degree of human leukocyte antigen (HLA) matches or mismatches, cold ischemia time, induction antibody therapy, or occurrence of posttransplantation acute rejection were not statistically different between groups (P>0.05; Table 1).
Study Subjects With BK Virus Infection
A total of 89 of 192 (46%) adult study subjects developed detectable BKV infection, whereas 103 (54%) did not have BKV infection during the study period. Of the 89 study subjects who developed any BKV infection, 25 (61%) belonged to the D+R- group compared with lower prevalence in the D+R+ (n=22 [52%]), D-R+ (n=17 [41%]), and D-R (n=25 [36%]) groups (Table 2). A total of 15 (35%) cases of BKV infection occurred in patients with unknown donor–recipient immune status.
Study Subjects With Insignificant and Significant BK Viremia
During follow-up, 62 (32%) study subjects developed posttransplantation BK viremia. Of these, 33 (17% of 192) had insignificant viremia and 29 (15% of 192) had significant viremia (Table 2). Any level of BK viremia, both significant and nonsignificant, were seen in 20 of 41 (49%) study subjects in the D+R- group as opposed to lower prevalence of 14 of 42 (33%), 12 of 41 (29%), and 16 of 68 (23%) in the D+R+, D-R+, D-R- groups, respectively (P=0.014). On further subanalysis, significant BK viremia was noted in 10 of 41 (24%) study subjects in the D+R- group as opposed to lower prevalence of 5 (12%), 4 (9%), and 10 (14%) in the D+R+, D-R+, and D-R- groups, respectively (P=0.021). Of the total 29 study subjects with significant BK viremia, 10 (35%) were in the D+R- group. The prevalence of subclinical BKVN was numerically higher in the D+R- group (3 of 41 [7%]) compared with other groups (Fig. 1). Thus, D+R- study subjects had the highest likelihood of any BKV infection, any level of BK viremia, as well as significant BK viremia and a trend toward BKVN followed by the D-R- group as opposed to the other two groups (Table 2). To clarify the threshold of BKV-specific antibody level, additional analysis was performed using lower threshold for BKV seropositivity (1:4). However, this did not change the overall incidence of significant BK viremia and BKVN in the groups appreciably (data not shown).
Thus, BKVN was predominantly seen in the D+R- and D-R- study subjects, illustrating BKV-specific immunodeficiency determining posttransplantation infection; the source of infection may be donor derived or exogenous.
Study Subjects With BK Virus Nephritis
Of the eight study subjects who developed subclinical BKVN, five received kidney from living donors, five were Caucasians (no African Americans developed BKVN), six were males, two received thymoglobulin as induction antibody, and only one had a preceding rejection. The mean class I and II PRA were 11% and 10%, respectively, the cold ischemia time was 345 min, and the mean of total mismatches was 5.
Onset of BK Virus Infection
Figure 2 shows the Kaplan-Meier curves from transplantation to the occurrence of posttransplantation BKV infection (n=192) for four different groups. The analysis revealed a significantly shorter time to occurrence of infection in the D+R- group compared with the other groups (P=0.011). The median time to the occurrence of BKV infection for D+R-, D+R+, D-R+, and D-R- groups was 11, 26, 21, and 25 months, respectively. Kaplan-Meier curves for 42 recipients without pretransplantation BKV-specific IgG values were distributed among all four groups (data not shown).
Donors With BK Virus–Specific Seropositivity
Higher prevalence of any BK viremia and significant viremia of 49% and 24%, respectively, were seen in study subjects who were in the D+R- group. When both recipient and donor were seropositive (D+R+), the prevalence of BK viremia and significant viremia was 33% and 12%, respectively, lower than the D+R- group. Thus, donor seropositivity increases the likelihood of infection in study subjects, more so, if recipients were seronegative at the time of transplantation.
Recipients With BK Virus–Specific Seropositivity
The prevalence of any BK viremia in the D+R+ and D-R+ was 33% and 29%, respectively. The corresponding values for significant viremia were 12% and 9%, respectively (Table 2). Thus, the overall prevalence of BK viremia in recipients who were seropositive was similar irrespective of the donor serologic status.
Pretransplantation BK Virus–Specific Titers and Posttransplantation BK Virus Infection
In our cohort, we did not see any relationship between the rising BKV titers in donors or recipients with posttransplantation BKV infection, including insignificant viremia, significant viremia, or BKVN (P>0.05, chi-square test; data not shown).
Biopsy-proven acute rejection was seen in 40 patients: 10 in the D+R- (ACR=9/AHR=1), 7 in the D+R+ (ACR=7/AHR=0), 7 in the D-R+ (ACR=6/AHR=1), and 17 were in the D-R- group (ACR=15/AHR=2). In our cohort, we did not see a definite relationship between intravenous pulse steroids for treatment of acute rejection and subsequent development of BKVN, nor did we see worsening of BK viremia with pulse steroids given for rejection that developed in response to reduction in immunosuppression for subclinical BKVN (3).
During the study period, there were 4 graft losses and 11 patient deaths. Three graft losses occurred in patients without BKV infection and one in a patient with BK viruria. Two graft losses were attributed to advance interstitial fibrosis tubular atrophy from prior humoral rejection and the third one lost the graft within 2 months of transplantation from primary nonfunction from donor-related kidney disease. The fourth graft loss was in patient with viruria alone and was attributed to chronic T-cell and humoral rejection. Two of the graft losses were in the D-R- group and one each in the D+R- and D-R+ groups. Eleven deaths occurred during the study period: (D+R-=1), (D+R+= 2), (D-R+=1), (D-R-=3), and (unknown group=4).
At last follow-up, the D+R- group had a mean serum creatinine of 1.52 mg/dL, with a mean (range) estimated glomerular filtration rate (eGFR) of 51 (7 to >60) mL/min at a mean follow-up of 41 months. The D+R+ group had a mean serum creatinine of 1.35 mg/dL, with a mean (range) eGFR of 54 (32 to >60) mL/min at a mean follow-up of 42 months. The D-R+ group had a mean serum creatinine of 1.37 mg/dL, with a mean (range) eGFR of 52 (6 to >60) mL/min at a mean follow-up of 38 months. The D-R- group had a mean serum creatinine of 1.52 mg/dL, with a mean (range) eGFR of 51 mL/min (7 to >60 mL/min) at a mean follow-up of 41 months. Thus, at last follow-up, renal function measured by serum creatinine and eGFR was similar between four groups.
Pediatric Study Results
The donor and recipient demographics and transplantation variables of the 11 pediatric study subjects are shown in Table 3. These patients were followed for a mean (range) period of 22.7 (10–35) months after transplantation. All 11 recipients were BKV antibody seronegative before transplantation. There were 8 (73%) in the D+R- group and 3 (27%) in the D-R- group. During follow-up, BKV infection was seen in 5 of 11 (36%) recipients and all within the D+R- group. Posttransplantation BKV infection was detected 78, 116, 378, and 734 days after transplantation. Among the 8 study subjects in the D+R- group, 4 (50%) developed posttransplantation BKV infection and 3 (38%) developed BK viremia. Thus, lower prevalence of BKV-specific antibody was noted in pediatric recipients and higher prevalence of BKV infection was noted in the D+R- group.
BKV infection is currently the most common infectious complication after kidney transplantation. The prevalence of BKV infection at 1 year after transplantation varies between 40% and 80%, and 20% develop BK viremia. BKVN occurs in 1% to 10% of all renal transplants and may progress to irreversible kidney graft failure. BKVN is preceded by BK viremia. The current clinical approach has focused on detecting the virus before the occurrence of overt BKVN and institute appropriate intervention to prevent progressive renal failure. Previous research has not identified pretransplantation markers that can predict posttransplantation BKV infection. Predicting BKV infection before transplantation is critical to manage renal transplant recipients and optimize their short-term and long-term outcome. Recipients who have the highest risk of developing BKV infection should be monitored closely with appropriate intervention on detection of BKV infection. The current study was undertaken to evaluate differential pretransplantation BKV serostatus as a risk factor for posttransplantation BKV infection.
Donor, recipient, transplantation, and posttransplantation risk variables have been identified for the occurrence of BKVN. These risk variables predicted the occurrence of BKVN as opposed to BK viremia or viruria. The commonly accepted risk variables for BKVN include intensive triple immunosuppressive regimens (tacrolimus-mycophenolic acid), steroid pulses, prior acute rejection episode, age, placement of ureteral stents, new BK serotype, and total HLA mismatches (4–6). However, the abovementioned risk factors are common, have low positive and negative predictive values, and hence cannot be used as pretransplantation predictors of posttransplantation infection (7). Additionally, a multivariate logistic regression done in our cohort revealed only recipient race (African-American race) to be a predictor (negative) of subsequent development of any BKV infection or significant viremia (8).
BKV replication is known to occur in the early hours after transplantation before suppression of immune system by potent immunosuppressive agents (9). The BKV infection can occur either by reactivation of latent virus in the recipient or by transmission of the virus through the donor kidney (10). Bohl et al. suggested direct correlation between the levels of BKV-specific antibody titer in the donor to both transmissibility and activity of BKV infection. Transmissibility is decided by early onset of viruria and activity defined by higher peak and prolonged levels of viruria in the recipient (11, 12). High levels of BKV-specific IgG may suggest either active viral replication in the donor or higher virulence of BKV serotype. Two recipients receiving kidneys from the same seropositive donor developed infection with matched sequences of viral segments suggesting donor as the source of infection (11). Randhawa et al. (13) reported an inverse correlation between the pretransplantation recipient’s BKV-specific IgG titer and posttransplantation viremia. In a pediatric study, Ali et al. (14) reported that a combination of high donor BKV IgG titers and low recipient BKV-specific IgG titers is a risk factor for early BK viremia. Similarly, studies have also reported recipient BKV seronegativity as a risk factor for BKVN in pediatric population (15, 16). Thus, it is intuitive to consider low levels or absence of BKV-specific IgG to be an ideal environment for the transmission and activation of virus, from a donor with high BKV-specific IgG levels.
Thus, there is ample evidence to support that differential donor–recipient BKV serostatus determines the risk of posttransplantation infection. Some of the studies mentioned above have evaluated various immunosuppressive regimens as a risk factor for the occurrence of posttransplantation BKV infection or evaluated a small pediatric study population. The current prospective single-center study was undertaken with a commonly used, Food and Drug Administration–approved, standardized immunosuppressive regimen and included both adult and pediatric subjects. The current study showed higher prevalence of posttransplantation BKV infection in the D+R- study subjects, confirming the relationship of pretransplantation donor and recipient BKV-specific antibody status to posttransplantation BKV infection.
Our study results showed that, in both adult and pediatric study subjects, D+R- had the highest incidence of BKV infection. For example, 68% of adult recipients with D+R- developed BKV infection, 48% developed BK viremia, and 24% developed significant viremia. These values were much higher than in the D+R+, D-R+, and D-R- groups (Table 2; Fig. 1). The prevalence of BKV infection, BK viremia, and significant viremia was lowest in the D-R- group. Similar results were obtained in a small cohort of pediatric study subjects (Table 3). In addition, the onset of BKV infection was much sooner in the D+R- study subjects (Fig. 2). In addition, donor seropositivity alone did not determine posttransplantation BKV infection, as also reported by Ginevri et al. albeit in a smaller study sample. As shown in our current study, recipient’s immune status is critical to determine the prevalence of BKV infection and nephritis. Recipient’s seropositivity resulted in lower prevalence of BKV infection and nephritis irrespective of the donor status, suggesting some degree of protection from prior exposure. Thus, our study shows that seronegative recipients had the highest incidence of viral activation when transplanted from a seropositive donor.
The limitations of our study include reliance on serology to determine previous exposure and immunity. The BKV-specific IgG antibody was developed against VP1 antigen. It is unclear if antibody against either large T or small T antigen would yield similar results. The prevalence of BK viremia in the D-R- study subjects, although lower than other groups, remains a bothersome finding of this study. Nevertheless, this may be related to BKV-specific IgG enzyme immunoassay (EIA) threshold lower than the proposed level of 1:8. Additional analysis done using lower threshold for BKV seropositivity (1:4) did not alter the incidence of significant BK viremia and BKVN in the groups appreciably. The significance and natural history of BKV-specific IgG level in normal subjects (donors) and recipients (end-stage renal disease) is not known and may not be similar. It is plausible that D-R- study subjects may be developing BKV infection other than donor as the source of infection. In addition, the current study outcomes described were any BKV infection, BK viremia, and significant BK viremia but not BKVN. However, numerically BKVN was higher in the D+R- group. Our single-center study needs to be validated by other prospective trials. In addition, longitudinal BKV-specific IgG levels with and without BKV infection were not available for all study subjects. In our cohort, we did not see any relationship between the rising BKV titers in donors or recipients to posttransplantation BKV infection. The strengths of the current study are its prospective nature, single laboratory estimation of BKV DNA, complete follow-up of all recipients, and a commonly used, Food and Drug Administration–approved, standard triple immunosuppressive therapy. In addition, the study results were also validated by a concurrent small pediatric study.
In conclusion, BKV seronegative renal transplant recipients of a seropositive donor have the highest likelihood of developing posttransplantation BKV infection. All donors and recipients should be tested for BKV-specific antibodies before transplantation to allow for risk stratification, screening, and potential immunosuppressive modification for posttransplantation BKV infection.
MATERIALS AND METHODS
From July 2007 to July 2010, 240 adult study subjects were followed until December 2012. Concurrently, 15 pediatric recipients were enrolled for this study. Donor and recipient pretransplantation BKV-specific IgG antibody values were not available for 42 adult recipients and 4 pediatric recipients. In addition, 6 adult recipients were excluded due to early graft loss or inability to follow-up at the center. Thus, a total of 192 adult recipients and 11 pediatric recipients formed the study cohort. Study enrolment details have been published earlier by the same investigators (8). The adult and pediatric studies were approved by an institutional review board and an informed consent was obtained from each study subject as well as from all living donors.
BK Virus–Specific IgG Antibody Estimation
Blood samples from the study subjects and donors were collected for BKV-specific IgG antibody evaluation immediately before transplantation. Serum samples isolated from patients were diluted and frozen at −80°C until evaluation using BK virus-like particles (VLP) enzyme-linked immunosorbent assay. BK VLP-coated 96-well polystyrene flat-bottomed Polysorp plates (Nunc) are made by diluting BK VLPs to 0.3 μg/mL and adding 100 μL to each well. Antibody was measured using an enzyme-linked immunosorbent assay technique adopted from Viscidi et al. (17) using BK VLP. The EIA IgG levels were based on diluting the samples until they had an optical density in the assay equal to that of negative control (background). Based on the laboratory experiments, BKV IgG EIA control sample remained negative until 1:640 titers or below 8 EIA units. Thus, a positive BKV-specific humoral antibody status was defined as those with BKV-specific IgG EIA ≥8 units. The detailed methodology of BKV-specific IgG antibody has been published by our group (18). The current study population was divided into four groups according to donor and recipient serostatus: D+R+, D+R-, D-R+, and D-R-.
Immunosuppression consisted of induction antibody with three to five doses of thymoglobulin or two doses of interleukin-2 receptor blocker based on our center’s immunologic risk stratification. Maintenance therapy consisted of mycophenolate mofetil, tacrolimus and prednisone.
BK Virus DNA Testing Methods and Monitoring
Monitoring of viral load by real-time polymerase chain reaction was performed using combined VP2 and VP3 segments of BKV as targets. Primers employed were synthesized according to Hoffman et al. (19). Details of BKV DNA polymerase chain reaction testing have been published earlier (17). Quantitative plasma BKV DNA alone was tested at the time of transplantation and quantitative plasma and urine BKV DNA was monitored at 1, 3, 6, 12, and 24 months after transplantation (9).
Standard of Care Follow-up
All adult subjects were followed for a mean (range) period of 37 (18–55) months after transplantation. Study subjects received standard-of-care follow-up with periodic blood testing for renal function, tacrolimus levels, and infection prophylaxis (20). For adult recipients, kidney biopsy was performed on detection of significant BK viremia. In pediatric patients, a kidney biopsy was performed if any level of BK viremia was found in the context of decreased graft function.
The groups based on the donor–recipient immune status were analyzed for occurrence of BKV infection (any degree of viruria or viremia), BK viremia (any level of BK viremia), and significant viremia (≥10,000 copies/mL plasma). The threshold for BKV DNA level used to define significant viremia was based on a positive predictive value of more than 80% for BKVN (2, 21). The diagnosis of BKVN was based on typical renal histologic findings and labeled as subclinical BKVN in the absence of changes in renal function.
SPSS version 16 was used for statistical analysis. Paired t test and repeated-measures analysis of variance were used for continuous variables. Kaplan-Meier curves were constructed to study the difference between the various groups. P<0.05 was considered significant.
The authors thank the Medical College of Wisconsin clinical research staff (Charlotte Klis and Patricia Lyman), Children’s Hospital Research staff (Theresa Kump and Shelley Chapman), Blood Center of Wisconsin (Amoa Childs), and Wisconsin Donor Network (Jay Campbell) for their support and effort in this study; Brian DuChateau, Ph.D., for establishing BKV-specific antibody testing (Blood Center of Wisconsin); Ray Viscidi, Ph.D. (Johns Hopkins School of Public Health) for providing VLPs; and Rimas Orentas, Ph.D. (currently at the National Institutes of Health, Bethesda, MD) for establishing BKV DNA testing at the Medical College of Wisconsin.
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