Key words : BK polyomavirus nephropathy, Posttransplant complication, Renal transplantation

Introduction
Infections are among the most critical complications affecting graft survival in patients undergoing kidney transplant. Immunosuppressive therapy disrupts the balance between viral replication and cellular immune response, which can result in primary activation or reactivation of the virus and increase the incidence of viral infections.¹ BK polyomavirus (BKV) infection represents a serious complication that is common after kidney transplant and can cause graft dysfunction and loss. Approximately 80% of the adult population is seropositive for BKV. BK polyomavirus infection is typically observed as a nonspecific viral disease in early childhood and remains latent in the renal tubular cells throughout the uroepithelium and can be reactivated when the immune system is suppressed. Among kidney transplant patients, BKV can also contribute to graft dysfunction, nephropathy development, and graft loss.^2,3 BK polyomavirus-associated nephropathy (BKVN) is observed in 10% of cases, and 10% to 80% of these patients lose the graft.¹ The reported prevalence of BKVN in pediatric kidney transplant patients is 4.6%, and the rate of graft loss is 24%.⁴

Although the causes of BKV infection are not fully understood, risk factors include early transplant age, male sex, transplant from a deceased donor, immunosuppressive therapy (especially the use of tacrolimus), factors that damage the ureteral epithelium (eg, permanent ureteral stents), and acute rejection.^4-8

Our study aimed to determine the frequency of BKV infection in pediatric kidney transplant patients, the effects of BKV infection on graft function, and the risk factors of BKV infection.

Materials and Methods
Using medical records, we retrospectively reviewed the data of 144 patients (female/male: 67/77) who ranged in age from 0 to 18 years and who received kidney transplants in the past 10 years and were followed up for at least 1 year at our center. The demographic data, reasons for kidney failure, donor types, immunosuppressive treatments, and laboratory data were recorded.

The BKV DNA levels were measured by quantitative polymerase chain reaction (PCR) in the urine and serum, which were determined monthly during the first 6 months after transplant, periodically (every 3 months) during the next 6 months, and whenever elevated creatinine was observed.⁹ A urine BKV PCR viral load of greater than 107 copies/mL was defined as viruria, and a serum BKV PCR viral load greater than 10⁴ copies/mL was defined as viremia. Renal biopsies were performed in patients with graft dysfunction and viremia, and BKVN was diagnosed by detecting characteristic cytopathic changes in BKV and conducting immunohistochemical examinations.

The immunosuppressive doses for patients with BKV infection were reduced by 50%. Patients with viruria who received reduced doses were closely monitored. In patients with viremia and/or BKVN, together with immunosuppressive dose reduction, we used leflunomide (40 mg/day), cidofovir (0.25-1 mg/kg/dose), and/or intravenous immunoglobulin (IVIG) (2 g/kg).

Patients were grouped as those with BKV infection (BKV-positive group; n = 12 [7 male/5 female]) and those without BKV infection (BKV-negative group; n = 132 [60 male/72 female]). These groups were compared in terms of transplant age, sex, kidney failure etiology, donor type, immunosuppressive treatments, presence of ureteral stents, acute rejection episodes, and accompanying viral infections. We evaluated and compared the glomerular filtration rate and graft loss rate of the groups.

Statistical analyses
Statistical analysis was performed with SPSS for Windows version 22.0 (IBM). Categorical variables are expressed as frequencies (percentage); numerical data are presented as means ± SD for normally distributed data and medians (IQR) for nonnormally distributed data. Differences were analyzed using chi-square and Fisher exact tests. We used t tests for normally distributed data and Mann-Whitney U tests for nonnormally distributed data. Significance was defined as P < .05.

Results
We included 144 pediatric patients (77 females and 67 males, aged 0-18 years) who underwent kidney transplant in our center; the mean age at transplant was 12.79 ± 4.74 years. Congenital anomalies of the kidney and urinary tract (CAKUT; 33.3%), glomerulonephritis (27.1%), and tubular diseases (20.8%) were the top 3 causes of kidney failure. The rate of patients who underwent deceased donor kidney transplant was 19.4%. Tacrolimus plus mycophenolate mofetil and steroid treatment were administered in 70.1% of patients; cyclosporine plus mycophenolate mofetil and steroid treatment were administered in 28.5%; and mechanistic target of rapamycin inhibitors instead of calcineurin inhibitors were administered in 1.4% of patients. The mean period of follow-up posttransplant was 43.82 ± 29.68 months (Table 1).

Among patients included in the study, we detected BKV infection in 12 patients (8.3%). All of these patients had viruria (8.3%), 8 patients (5.5%) had viremia, and 4 patients (2.8%) had BKVN. The female-to-male ratio of patients with BKV infection was 5/7, and the mean age at transplant was 13.12 ± 6.9 years. We observed BKV infection at an average of 11.7 ± 3.2 months (IQR, 1-32 mo) posttransplant. Although 7 of the BKV-positive patients were asymptomatic, we performed kidney biopsies in the other 5 patients because of graft dysfunction accompanying viremia. The incidence of BKVN among all patients was 2.8% (n = 4 patients). Three of the patients with BKVN experienced acute rejection at the same time; the rate of accompanying acute humoral rejection was 75%. Graft loss occurred in 2 patients (1.4%) because of BKVN (Table 2).

When we examined the time of detection of BKV viremia, we observed that 5 of the BKV-positive patients developed viremia in the first 3 months after transplant, with 3 patients developing BKVN. We observed that BKV infection was shown in 3 patients at 24 months and in 1 patient at 32 months posttransplant. Viruria alone was observed in 3 of these patients (ie, no viremia or nephropathy was detected). We observed improvement in 66% of patients, but 2 patients (16.6%) with BKV infection had graft loss. The recovery rate was 25%, and the graft loss rate was 50% in patients diagnosed with BKVN with biopsy. Detailed data of the 12 patients with BKV infection are displayed in Table 2.

When BKV-positive and BKV-negative patients were compared for risk factors, no statistically significant differences were found in terms of sex, transplant age, donor type, or immunosuppressive treatment (P > .05). When both groups were compared in terms of kidney failure etiology, the rate of CAKUT was 30.3% in the BKV-negative group and 66.6% in the BKV-positive group, and the difference was statistically significant (P < .05). No significant dif-ferences between the BKV-positive and BKV-negative groups were observed in terms of the presence of a ureteral stent, acute rejection, or graft loss (P > .05). Although the incidence of cytomegalovirus (CMV) infection in the BKV-positive group was significantly higher than that in the BKV-negative group (P < .05), no differences were observed in the incidence of Epstein-Barr virus infection among the 2 groups (P > .05). The first- and third-year glomerular filtration rate values were similar among the patients in both groups (P = .10 and P = .23, respectively) (Table 3).

Discussion
We detected BKVN in 4 patients (2.8%), BKV viruria in 12 patients (8.3%), and BKV viremia in 8 patients (5.5%) in this study. Although the frequency of BKV infection in our study was 8.3%, which is lower than the 16% to 27% BKV infection rate reported in previous studies,^7,8,10-13 the rate of BKVN was 2.8%, which is similar to the 4.6% previously reported in a large-scale pediatric cohort study.4 Reported incidences in this regard are quite variable. Wide variation ranges are reported in the literature depending on the choice of induction and maintenance immunosuppressive therapy and the follow-up modality used for patients with BKV. Recently reported rates of BKV viruria, BKV viremia, and BKVN after kidney transplant were 60%, 13%, and 10%, respectively, and graft loss rate due to BKVN was reported to range from 5.7% to 7.5%.¹⁴ In our study, graft loss due to BKVN was 1.4% in all of our pediatric kidney transplant patients. From our 4 patients with BKVN, 2 patients (50%) had graft loss. Similarly, BKVN has been reported to cause graft loss in approximately 50% of cases,¹⁴ with a reported rate of graft loss of 24% in pediatric kidney transplant patients with BKVN.⁴

In our study, BKV positivity was detected at approximately 11 months posttransplant. In other studies conducted in pediatric patients, BKV infection has been observed in the early period, especially in the first 3 months.^10,15 Conversely, Hymes and colleagues reported a mean time until BKV viremia detection of 90 months in a pediatric prospective cohort study.¹⁶ In adults, BKV infection has been reported in the early period, especially in the first month after transplant and after an average of 75 to 90 days.^17,18 However, Sood and colleagues reported that BKV infection was also seen in the late period in their adult prospective cohort study.¹⁹ Favi and colleagues supported the idea that more than half of the patients with BKV viremia were diagnosed at an average of 180 days posttransplant; thus, in line with the Kidney Disease: Improving Global Outcomes (KDIGO) recommendation, monthly follow-ups were conducted during the first 6 months after transplant.²⁰ In our study, viremia was detected in 5 patients at approximately 3 months posttransplant, and BKVN developed in 3 of these patients. Three patients were diagnosed with BKV infection at month 24 and 1 patient at month 32. Viruria alone was detected in 3 of these patients. According to our study, as recommended by other studies and the KDIGO guidelines, we suggest that monthly monitoring (during the first 6 months posttransplant) of BKV infection in the early period is crucial.

Although viremia is a stronger predictor for the development of BKVN than viruria,¹³ we observed that viremia developed in patients with higher urinary BKV PCR viral load in our follow-up. Therefore, we suggest that closer monitoring of patients with viruria to detect viremia development is valuable for early treatment. Close monitoring of the serum viral load and creatinine value is imperative in patients who develop viremia.¹ In line with the study of Favi and colleagues,²⁰ we observed that the serum BKV PCR viral load was higher in patients with biopsy-diagnosed BKVN than in patients without BKVN. In addition, Viscount and colleagues reported that the sensitivity of BKV viremia for BKVN was 100%, and the positive predictive value was 50%.²¹ Although no definitive cutoff has been determined for the viral load associated with BKVN, some authors suggested that a viral load greater than 4 log copies/mL may be a precursor to BKVN.²² The KDIGO guidelines also recommend reducing the immunosuppressive dose if the BKV viral load is greater than 10 000 copies/mL.⁹ In the case of detecting graft dysfunction with BKV viremia, early kidney biopsy for BKVN diagnosis is crucial to prevent BKVN-related graft loss.¹ In addition, the sampling errors can reach 10% to 36.5% because of focal involvement in BKVN. Therefore, negative biopsy results do not exclude early BKVN. Although biopsy is considered the gold standard for the diagnosis of BKVN, when the biopsy is negative, patients with persistent serum BKV PCR levels greater than 10 000 copies/mL should be monitored closely under the diagnosis of “possible BKVN”.²³

We reduced the immunosuppressive dose in all of our patients with BKV infection, and we used leflunomide and cidofovir treatment in patients with viremia and BKVN. Three of these patients did not improve with cidofovir. We administered IVIG to these patients who had acute humoral rejection accompanying BKVN. Because of the suppression of the immune system with immunosuppressive treatment after transplant, BKV can be reactivated, and BKV infection is observed with replication of the virus. Therefore, the basic and first-line treatment to reduce and prevent virus replication is immunosuppressive dose reduction.^1,10,24 In addition to immunosuppressive dose reduction for the treatment of posttransplant BKV infection, leflunomide, cidofovir, and IVIG treatments are used as second-line treatments.²⁵ A 66% improvement was observed in the first 6 months after our patients started treatment. In previous pediatric and adult studies, the remission rates were 68% to 100% with 2 to 6 months of treatment.^{4,10,16,18,20} Also, the recovery rate was 25% in those with BKVN.

Risk factors for BKV infection were evaluated in many studies, but the results have been inconsistent. For this reason, the risk factors for BKV infection in kidney transplant patients are controversial. A systematic review found that many studies have reported risk factors of immunosuppressive treatment (especially tacrolimus treatment), acute rejection, male sex, presence of a ureteral stent, and transplant from a deceased donor; studies have also reported risk factors of previous transplant history, delayed graft function, and age.⁶ We found no differences between the BKV-positive and BKV-negative groups in our study in terms of tacrolimus use, acute rejection, sex, the presence of a ureteral stent, donor type, and age at transplant. Transplant from a deceased donor under 8 years of age has been reported as a risk factor, and the incidence of BKV infection has been found to be lower for transplants from living donors.^4,7,19,24,26 The presence of a ureteral stent has been identified as a risk factor for BKV infection because of uroepithelial damage and increased risk of BKV infection in the damaged area.^5,18,27 The risk of BKV infection was specifically increased for long-term stents (>3 weeks), whereas short-term stents were not associated with BKV infection.²⁸ Acute rejection and the use of tacrolimus have been identified as the most important risk factors for BKV infection in many studies.^6,10,29-33

Although previous studies have not demonstrated a relationship with the etiology of kidney failure, our study found that the rate of CAKUT in BKV-positive patients was significantly higher than that in patients who were BKV-negative. This may be related to the low number of BKV-positive patients in our study, the fact that our group consisted of pediatric patients, and the fact that CAKUT is the most common cause of kidney failure in pediatric patients in our country. However, it has been shown that damage of the ureteral epithelium is a risk factor for BKV infection.⁶ Therefore, it is thought that this result may be due to uroepithelial damage associated with CAKUT.

We found that the incidence of CMV in BKV-positive patients was significantly higher than that in BKV-negative patients. Cytomegalovirus, one of the viral infections accompanying BKV infection, has been identified as a risk factor for BKV infection.^6,34,35 This situation may be associated with the increased risk of other opportunistic infections due to the use of immunosuppressive therapy, as well as the increased risk of BKV infections. However, although no prophylactic treatment is available for BKV infection, all BKV-negative and BKV-positive patients received valganciclovir treatment during the first 3 months posttransplant because of the risk of developing CMV infection. Under valganciclovir prophylactic treatment, BKV infection was accompanied by CMV infection. However, Favi and colleagues also mentioned a lower risk of BKV infection in patients who received CMV prophylaxis because of the lower incidence of CMV reactivation in these patients.²⁰ No difference was found between our BKV-positive and BKV-negative patients in terms of another opportunistic infection (Epstein-Barr virus).

The risk factors and treatment methods for BKV infection are still controversial. Therefore, comprehensive multicenter and prospective studies are warranted to gather more knowledge about BKVN risk factors and alternative anti-BKV treatment modalities to reduce BKVN-related graft loss.

In conclusion, although the frequency of BKV infection in pediatric patients with kidney transplants in our center was lower than other previously reported studies, the rate of BKVN was consistent with data reported on pediatric patients. Infection with BKV is a serious complication with a risk of graft loss when BKVN develops in pediatric kidney transplant recipients. Although no specific treatment is available, development of nephropathy as well as graft loss can be prevented by early detection and treatment of viruria and viremia through close periodic control and adequate evaluation of risk factors, especially in the first 6 months after transplant.

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