EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Nephropathy due to BK virus infection is a major cause of graft dysfunction and loss. No specific treatment has been developed for the BK virus. Here, we compared the combination of intravenous immunoglobulin and leflunomide versus intravenous immunoglobulin to treat BK virus nephropathy after renal transplant.
Materials and Methods: This study was a randomized controlled clinical trial. Sixteen kidney transplant patients with BK virus infection were randomly divided into 2 groups; 1 group received intravenous immunog-lobulin, and another group received leflunomide and intravenous immunoglobulin. P < .05 was considered statistically significant.
Results: Results of a polymerase chain reaction test for BK virus after 2 months of treatment were negative in 3 patients in the intravenous immunoglobulin group and in 7 patients in the intravenous immunoglobulin + leflunomide group. The amount of BK virus decreased significantly in each group, and a significant difference was observed between the 2 groups after 3 months (P = .014). The average level of creatinine in the intravenous immunoglobulin group at 1, 2, and 3 months after treatment was 1.7 ± 0.23, 1.8 ± 0.5, and 1.5 ± 0.3, respectively, and in the intravenous immunoglobulin + leflunomide group was 2.1 ± 0.75, 1.76 ± 0.37, and 1.4 ± 0.18, respectively (P > .05).
Conclusions: Although BK viral load decreased significantly in both groups, there was a significant difference between patients who received intravenous immunoglobulin versus those who received the combination of intravenous immunoglobulin + leflu-nomide after 3 months. The addition of leflunomide to the intravenous immunoglobulin treatment seems to have a better effect in reducing BK viral load. However, further studies with a larger sample and longer duration are needed.

Key words : BK virus nephropathy, Immunocompromised patients, Kidney transplantation

The BK virus can be a major cause of nephropathy after renal transplant and can result in loss of the transplanted graft.¹ The primary BK infection mainly occurs in early childhood and is asymptomatic or minimally symptomatic. Almost 80% to 90% of healthy people are seropositive for this virus.^2,3 In immunocompromised patients, this virus, which is colonized in the urinary system’s epithelial cells, can be reactivated, leading to nephropathy or graft failure.⁴

So far, there is no specific treatment for this virus, so the common and effective standard of care is to reduce the level of maintenance immunosuppression for BK virus nephropathy or viremia. However, this approach is only successful in 50% to 80% of patients.^5,6 As an adjunctive therapy, in addition to reducing immunosuppression, use of leflunomide, cidofovir, intravenous immunoglobulin (IVIG), and fluoroquinolones has been evaluated in different studies in which each of these agents has shown both advantages and disadvantages.⁷ As a treatment strategy, IVIG seems to be safe and effective to treat or even prevent BK viremia and nephropathy, as well as graft loss in patients with an insufficient response to immunosuppression.^8-12

Leflunomide, another choice in the treatment of BK nephropathy, is an immunosuppressive drug. Reports of the use of leflunomide as a treatment option for BK nephropathy have increased recently. Leflunomide has been shown to reduce BK viral load levels by reducing immunosuppression.¹³ However, due to scarcity of robust evidence in studies on treating BK virus nephropathy with leflunomide, the role of leflunomide is still unclear.¹⁴

There is high prevalence of kidney transplant surgery in our country, and no comprehensive study has yet been conducted to compare the therapeutic effect of dual therapy with IVIG + leflunomide versus treatment with only IVIG in patients with BK virus infection after kidney transplant. Therefore, the pur-pose of our study was to compare these 2 methods.

Materials and Methods

Study design
The present study was a randomized controlled clinical trial performed among kidney transplant patients with BK virus infection who were referred to our center in 2022. Our main goal was to compare the therapeutic effect of double therapy with IVIG + leflunomide and treatment with only IVIG in patients with BK virus infection after kidney transplant.

Participants
The study population consisted of kidney transplant patients with BK virus infection. This was the first kidney transplant for all participants. We defined BK virus nephropathy as a persistent plasma BK viral load of more than 10 000 copies/mL for 4 weeks in the patients.¹⁵ A total of 16 patients were enrolled in the study. All patients underwent kidney transplant from deceased donors. Inclusion criteria were first kidney transplant experience, age of patients over 18 years, and chronic kidney disease (CKD) not due to an autoimmune disease in patients. Also, exclusion criteria were incidence of adverse side effects from drugs or a previous history of treatment due to BK virus and elevation of serum creatinine level, history of kidney sampling, and history of any transplant rejection. To measure the response to treatment, we monitored the patients in terms of viral load every month, and we used a significant decrease in the viral load and finally undetectable viral load as the criteria for treatment. It should be noted that in this project, to reduce costs and due to the noncoo-peration of patients, viremia was measured every month instead of every 2 weeks.¹⁶ Because of the COVID-19 pandemic at the beginning of the study and the decrease in the number of kidney transplants during this time period and to maintain the novelty of the study, the study population was small.

Randomization and masking
In this study, 16 kidney transplant patients with BK virus infection who met the inclusion criteria were randomly allocated into 2 groups. One group received IVIG (n = 8; note that 1 patient was excluded from the study due to rejection), and the other group received IVIG + leflunomide (n = 8). We used permutation block randomization for allocation sequence, with 4 blocks of 4. One patient from the IVIG group was excluded from the study due to graft rejection at the beginning of the study.

Procedures
All the patients in both groups received IVIG. Before the IVIG injection, 500 mL of normal saline was administered to all patients for hydration. The IVIG agent that we used was from Biotest Company of England, which is available in 100-mL vials containing 5 g of immunoglobulin. In the first group, IVIG was injected at 300 mg/kg once every 3 weeks (3 times). In the second group, in addition to IVIG, the participants received 100 mg oral leflunomide (20-mg tablet, Arena Life Science) in the first 5 days, then 40 mg/d for 2 months. Also, the serum level of the BK virus was checked monthly by polymerase chain reaction (PCR).

Before the trial, all the patients received 720 mg of mycophenolate mofetil twice daily. Mycophenolate mofetil was discontinued in the group that received the combination of leflunomide with IVIG. Also, the consumption of mycophenolate mofetil was reduced by half in those who received only IVIG. The prednisolone dose did not change during the trial compared with before the trial. The patients taking tacrolimus and cyclosporine continued to take these 2 drugs according to the previous routine. Moreover, in case of taking cyclosporine and tacrolimus during the study, the serum levels of the drugs were measured monthly, and the serum level of tacrolimus was maintained in the range of 4 to 6 ng/mL and cyclosporine in the range of 50 to 100 ng/mL. Before the study commenced, demographic data and history were recorded, including data for age, sex, disease symptoms, type and duration of dialysis, creatinine, and urea level. At the end of the study, the groups were followed up regarding serum levels of the BK virus and creatinine levels.

In case of an increase in creatinine of more than 25% of the baseline creatinine, kidney biopsy was performed on the patient, and if any type of rejection was reported, then the patient was excluded from the study.

Ethical considerations
The study was performed according to the Declaration of Helsinki, as approved by the National Committee for Ethics in Biomedical Research (ethical code No. IR.SUMS.REC.1399.607), and is registered in the Iran clinical trial system (https://www.irct.ir). At the beginning of the study, informed consent was obtained from all participants. All patient infor mation was kept confidential.

Statistical analyses
Analysis was performed with SPSS software (version 22). The quantitative data are shown as frequency and percentage, and we used the chi-square test for comparisons. The continuous variables are presented as mean values (±SD), which were analyzed by the Mann-Whitney test, as a nonparametric test of the t test, the Wilcoxon signed-ranks test as a non-parametric paired t test, and the Friedman test for the nonparametric repeated measurements. P < .05 was considered statistically significant. Due to hete-rogeneity of viral load in patients and abnormality of distribution, those data are not reported as mean values (±SD); instead, median values (with range) are reported.

The study was performed at Abu Ali Sina transplant hospital in Shiraz, Iran. The study population consists of 16 patients who had their first kidney transplant experience, but 1 patient from the IVIG group was excluded due to graft rejection. Finally, a total of 10 men and 5 women who had received kidney transplants from deceased donors were enrolled in the study (Table 1). The mean age of the participants was 48.2 ± 2.5 years.

Of 15 participants, the most reported cause of CKD was hypertension, which was observed in 5 patients (33.3%). Also, the frequency distribution of patients according to the cause of CKD before the trial is mentioned in Table 1.

The average serum creatinine levels in the IVIG group before treatment and at 1 month and 2 months after treatment were 1.7 ± 0.23, 1.8 ± 0.53, and 1.52 ± 0.26, respectively, and in the IVIG + lefluno-mide group were 2.1 ± 0.75, 1.76 ± 0.37, and 1.4 ± 0.18, respectively (P > .05). Blood urea levels before treatment and after 1 month and 2 months of treatment are shown in Table 1.

Four patients (57.1%) in the IVIG group and 5 patients (62.5%) in the IVIG + leflunomide group had living donor kidney transplants, and the remaining patients in both groups had deceased donor transplants. In both groups, before treatment and after 1 month and 2 months of treatment, the results of all urine analyses and cultures were negative.

The PCR results for BK virus were positive in all participants before starting the trial and 1 month after starting the trial; however, after 2 months, 3 patients (42.9%) in the IVIG group and 7 patients (87.5%) in the IVIG + leflunomide group showed negative results for PCR.

Quantitative comparison of median BK virus serum level (minimum, maximum) in patients treated with IVIG at 1, 2, and 3 months after treatment showed 470 000 (2000, 280 000), 12 000 (2800, 120 000), and 850 (350, 2000), respectively, and in the IVIG + leflunomide group showed 76 500 (1830, 998 000), 13 754.5 (2800, 93 000), and 80 (8, 3400), respectively (Table 2). The serum level of BK virus in each group decreased significantly (P < .05), and a statistically significant difference was observed between the 2 groups after 3 months (P = .014).

As previously mentioned, BK virus nephropathy is a main cause of transplanted kidney dysfunction and failure. The activity of the BK virus eventually leads to transplanted organ rejection. The incidence of BK virus nephropathy is 1% to 15% for kidney transplant patients.^17,18

Therefore, because of the importance of the BK virus and the lack of a comprehensive study on the comparison of the 2 methods of IVIG and IVIG + leflunomide, in the present study, we investigated and compared the effect of IVIG and leflunomide in patients with a kidney transplant who had BK virus infection as confirmed by the PCR method. We observed that the levels of BK virus in both groups were reduced during these 3 months, and significant differences were observed in the median levels of serum BK in these 3 time periods. Also, the comparison of BK serum levels between the IVIG group versus the IVIG + leflunomide group showed a significant difference between the 2 groups after 3 months. The BK serum level in the IVIG + leflunomide group was lower compared with the IVIG group, which can decrease the graft loss rate in patients receiving IVIG + leflunomide compared with patients receiving IVIG after 3 months.

Benotmane and colleagues investigated whether early administration of IVIG prevents replication of the BK virus. The study group of 174 kidney transplant patients was divided into 3 subgroups based on the titer of BK virus-neutralizing antibodies on the day of transplant: (1) high-risk patients who received IVIG for the first 3 months after transplant; (2) patients who were not treated with IVIG and were at low risk; and (3) patients who were not treated with IVIG and were at high risk. Twelve months after transplant, the incidence of BK virus viremia was 6.8% in the high-risk group under treatment, 36.6% in the high-risk group without treatment, and 10.1% in the low-risk group. Therefore, according to the results of this study, IVIG may be a valuable strategy to prevent the BK virus. Although IVIG treatment is expensive, the cost is justified by the fact that BK virus nephropathy is a serious cause of kidney allograft dysfunction and kidney graft loss in transplant recipients.¹⁰

In another study, patients diagnosed with BK virus nephropathy received 100 mg/kg/d IVIG for 5 days. Treatment with IVIG showed a promising result by showing a drastic decrease in plasma BK virus load and the negativity of decoy cells in the urine. Notably, IVIG therapy in all patients diminished simian virus 40 large T antigens.⁹ Sener and colleagues investigated the effect of IVIG in kidney transplant patients and treated the patients with IVIG at 2 g/kg divided across 2 to 5 days. The result showed that, after a mean follow-up of 15 months after treatment with IVIG, 88% of the patients had retained a functional transplanted kidney, and although kidney function in the patients was still impaired, they had ceased dialysis.¹⁹ In a study conducted on 19 patients with BK virus infection, Dheir and colleagues reported that IVIG treatment effectively prevented graft rejection and prolonged graft survival, which is in line with our study.²⁰

Yamazaki and colleagues investigated the effect of leflunomide and everolimus in 4 patients with the BK virus. Patients were treated with leflunomide when immunosuppressive drugs were tapered. Participants were treated with leflunomide (20 mg/d) for 3 months. They reported that leflunomide has immunosuppressive and antiviral effects and can be used as an antiviral agent while reducing the chance of transplant rejection.¹³ A leflunomide dose of at least 60 mg/d was needed to achieve therapeutic levels and viral clearance in most patients in the study by Nesselhauf and colleagues. Also, the virus clearance days in patients differed from 17 to 476, and delay in treatment might result in progressive BK virus infection.²¹

According to the studies conducted by Santeusanio and colleagues, based on institutional immunosup-pressive protocols and posttransplant outcomes, patients with BK virus nephropathy or viremia can benefit from leflunomide or cidofovir or IVIG treatment, but treatment should be indi-vidualized to maximize clinical efficacy while limiting side effects.²² So far, no comprehensive study has been conducted to compare IVIG + leflunomide with IVIG.

According to a study by Vu and colleagues, after 1 month of treatment with leflunomide and reduction of immunosuppression, BK viral load in 30 patients decreased. After 12 weeks, 27 of 30 patients had positive responses. After IVIG treatment, the formation of immunoglobulin complexes affects inflammation and autoimmune diseases, shows potential antiviral properties against BK virus, and is safe and effective to treat BK virus infection and BK virus nephropathy.⁸

In the present study, although the decrease in creatinine level was higher in the IVIG + leflunomide group than in the IVIG group, there was no significant difference between the creatinine level before and after the intervention in the 2 groups. Also, similar to the present study, Kable and colleagues investigated the effect of IVIG combined with antiviral therapy to treat patients but did not observe a significant difference in creatinine levels.¹² However, in a case report by Zavos and colleagues of a 46-year-old patient treated with 100 mg of leflunomide, his serum creatinine level improved significantly after 1 year. Although we used leflu-nomide in combination with IVIG in the present study, the duration of drug administration was longer in the study by Zavos and colleagues compared with our present study.²³

However, IVIG can be associated with adverse side effects, ranging from mild and transient flushing, headache, malaise, or fever to more serious complications like renal impairment, thrombosis events, hemolytic anemia, arrhythmia, aseptic meningitis, and transfusion-related acute lung injury. In general, these adverse effects are rarely debili-tating or fatal.²⁴

Also, the small number of samples can be considered the main limitation of this study, which reduces the study’s accuracy.

According to the results of the present study and the quantitative analysis of the mean serum level of BK virus, we observed a statistically significant difference between patients who received IVIG versus patients who received the combination of IVIG + leflunomide after 3 months, and addition of leflunomide to the IVIG regimen had a better effect to reduce BK viral load; therefore, this regimen leflunomide can have a better effect on graft loss in these patients. However, because of the small study population, further studies with a larger study population and a longer study period are needed. It is worth noting that another study with the same goal has been planned at our center with a larger sample size and a longer study period.

Volume: 21 Issue: 10 October 2023

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