C Blackwell Munksgaard 2005 Copyright
American Journal of Transplantation 2005; 5: 582–594 Blackwell Munksgaard
doi: 10.1111/j.1600-6143.2005.00742.x
Incidence of BK with Tacrolimus Versus Cyclosporine and Impact of Preemptive Immunosuppression Reduction Daniel C. Brennana, ∗ , Irfan Aghab , Daniel L. Bohla , Mark A. Schnitzlerb,c , Karen L. Hardingerd , Mark Lockwooda , Stephanie Torrenced , Rebecca Schuesslerd , Tiffany Robye , Monique Gaudreault-Keenerd and Gregory A. Storche
Received 6 August 2004, revised 15 October 2004 and accepted for publication 18 October 2004
Introduction a
Washington University School of Medicine, Department of Internal Medicine b The St. Louis University Department of Internal Medicine c St. Louis University Center for Outcomes Research d Barnes-Jewish Hospital and e Washington University School of Medicine, Department of Pediatrics, St. Louis, Missouri, USA ∗ Corresponding author: Daniel C. Brennan,
[email protected] Our purposes were to determine the incidence of BK viruria, viremia or nephropathy with tacrolimus (FK506) versus cyclosporine (CyA) and whether intensive monitoring and discontinuation of mycophenolate (MMF) or azathioprine (AZA), upon detection of BK viremia, could prevent BK nephropathy. We randomized 200 adult renal transplant recipients to FK506 (n = 134) or CyA (n = 66). Urine and blood were collected weekly for 16 weeks and at months 5, 6, 9 and 12 and analyzed for BK by polymerase chain reaction (PCR). By 1 year, 70 patients (35%) developed viruria and 23 (11.5%) viremia; neither were affected independently by FK506, CyA, MMF or AZA. Viruria was highest with FK506-MMF (46%) and lowest with CyA-MMF (13%), p = 0.005. Viruria ≥ 9.5 log 10 copies/mL was associated with a 3-fold increased risk of viremia and a 13fold increased risk of sustained viremia. After reduction of immunosuppression, viremia resolved in 95%, without increased acute rejection, allograft dysfunction or graft loss. No BK nephropathy was observed. Choice of calcineurin inhibitor or adjuvant immunosuppression, independently, did not affect BK viruria or viremia. Viruria was highest with FK506-MMF and lowest with CyA-MMF. Monitoring and preemptive withdrawal of immunosuppression were associated with resolution of viremia and absence of BK nephropathy without acute rejection or graft loss. Key words: BK, kidney transplant, immunosuppression, polyomavirus, preemptive 582
BK virus infection occurs early in life, appears to be asymptomatic and results in a seroprevalence of 65–90% in the general adult population (1). After transplantation, 30–60% of renal transplant recipients develop BK viruria, 10–20% develop BK viremia and 5–10% develop BK virus nephropathy (2–9). Up to 70% with BK nephropathy lose the transplant from the infection, and those that do not lose the transplant often remain with renal dysfunction (10,11). Thus BK virus has emerged as one of the most important problems in renal transplantation. The BK virus was originally discovered and deemed to be the cause of obstructive uropathy in a patient treated with azathioprine (AZA) and prednisone (12). BK infection in transplant recipients occurred rarely throughout the 1970s and 1980s when only AZA and prednisone were available as maintenance immunosuppressive therapy. However, a single-center analysis of archived renal biopsy specimens showed an increase in the incidence of the disease contemporaneous with the introduction and use of more intense immunosuppression with tacrolimus (FK506), mycophenolate mofetil (MMF) (CellCept, Hoffman-La Roche, Nutley, NJ) or the combination compared to previous eras (7–9,11–14). BK viruria is the first sign of active virus replication and progression to BK viremia appears to be a prerequisite for the development of BK nephropathy (5,6). There is no known safe and effective antiviral agent, and reduction of immunosuppression, in an attempt to clear the virus, risks precipitation of acute rejection and graft loss. The primary purpose of this study was to determine the effect of the calcineurin inhibitors, tacrolimus (FK506) or modified cyclosporine A (CyA), on the incidence of BK virus infection in de novo renal transplant patients. The secondary purpose was to test the safety and efficacy of a management strategy consisting of intensive monitoring for BK viruria and viremia with preemptive reduction of adjuvant immunosuppression, MMF or AZA, upon detection of BK viremia to prevent progression to BK nephropathy.
BK, Calcineurin Inhibitor and Preemptive Therapy
Materials and Methods Study design This was an open-label prospective trial of de novo renal transplant recipients randomized, prior to transplantation, in a 2:1 block-design fashion stratified by race and gender to receive FK506 or CyA at a single center. The Washington University Human Studies Committee approved the study.
Inclusion and exclusion criteria All adult patients (age >18 years) were considered for enrollment. Exclusion criteria were previous treatment with rabbit anti-T-cell polyclonal agents or allergy to rabbit proteins, malignancy, except skin cancers in the previous 2 years, serologic evidence of infection with HIV-1, HTLV-1 or hepatitis B, use of an investigational agent in the prior 4 weeks or inability to comply with the protocol in the opinion of the investigators. Pregnant or nursing females were not included.
Immunosuppression All patients except 6 antigen-matched living-related allograft recipients were induced with rabbit anti-thymocyte globulin [Thymoglobulin (TMG), SangStat Medical Corp., Fremont, CA]. Four doses of TMG (1.5 mg/kg each) were administered; the first intra-operatively followed by doses on postoperative days 1–3. Dose adjustments were made for leukopenia or thrombocytopenia. Calcineurin inhibitors were instituted upon a brisk diuresis but no later than 4 days post-operatively. Cyclosporine was started at 8 mg/kg per day in two divided doses targeting 12-h whole blood trough levels of 250–300 ng/mL, using monoclonal fluorescent polarization immunoassay (FPIA) (Abbott Laboratories, Abbott Park, IL) during the first 3 months and 100–250 ng/mL, subsequently. FK506 was initiated at 0.1 mg/kg per day divided in two doses aiming at trough levels of 5–10 ng/mL by microparticle enzyme immunoassay (MEIA) (IMx, Abbott Laboratories, Abbott Park, IL). Patients were assigned to receive AZA at a dose of 5 mg/kg per day for 3 days then 2.5 mg/kg per day orally as the primary adjuvant agent. The dosage was adjusted per WBC count. Under certain circumstances (glomerulonephritis or other immunologic basis for end-stage renal disease (ESRD); second transplant; high sensitization with panel reactive antibody (PRA) >20%; or history of gout) MMF was substituted for AZA at 1000 mg orally twice a day. Methylprednisolone (7 mg/kg IV) was infused intra-operatively. Prednisone was started at 1 mg/kg per day, orally, postoperatively and tapered by month 3 to 5–7.5 mg daily.
Cytomegalovirus (CMV) prophylaxis Recipients at risk for CMV received oral ganciclovir 1000-mg t.i.d. for 3 months if D−/R+, 6 months if D+/R+ and 12 months if D+/R−.
Monitoring Undiluted whole blood, plasma and urine samples for BK virus polymerase chain reaction (PCR) were collected pre-transplant, weekly for 16 weeks, and at months 5, 6, 9 and 12. BK viruria was defined by presence of BK virus DNA in the urine. BK viremia was defined by detection of BK virus DNA in plasma. Sustained viruria and viremia were defined as two or more consecutive positive urine or plasma samples, respectively, spanning 3 or more weeks, and ‘transient’ otherwise. An early internal analysis compared the results of PCR performed on DNA extracted from 419 blood samples: 26 were positive in both plasma and whole blood, 11 positive in plasma but negative in whole blood while 2 were positive in whole blood but negative in plasma and 380 negative in both
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plasma and whole blood (p = 0.03, McNemar test). Based on these results, all subsequent testing of blood samples was performed only on plasma. Plasma samples were tested on all dates that the urine sample from the same date was positive for BK virus. This was based on another internal analysis in which plasma PCR was performed and found to be uniformly negative on 150 samples for which the corresponding urine was negative for BK virus. A smaller internal analysis of six urine samples, positive for BK virus by PCR, showed that decoy cells could be detected in less than half and was deemed an unreliable method to follow patients at our institution. Patient contact, clinic and laboratory follow-up were maintained throughout the study, with at least bi-weekly review of all patient data.
Polymerase chain reaction methods For purposes of this study, qualitative PCR for detection of BKV DNA was used for patient management. DNA was extracted and purified from urine and ethylene-diamine-tetra-acetic acid (EDTA) anti-coagulated plasma using QIAamp spin columns (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. Extracted DNA was tested for BK virus DNA by real-time PCR using the LightCycler System (Roche Diagnostics Corporation, Indianapolis, IN) using the primers, Pep-1 AGT CTT TAG GGT CTT CTA CC and Pep-2 GGT GCC AAC CTA TGG AAC AG, which amplify a 176-basepair segment of the BK virus large T-antigen gene (15). BK virus-specific hybridization probes 5 TTG CCA TGA AGA TAT GTT TGC CAG TGA TGA FITC 3 and 5 LCRed640 GAA GCA ACA GCA GAT TCT CA 3 (TIB Molbiol LLC, Adelphia, NJ) were used for detection. These probes were designed to detect BK virus without detection of the related polyoma viruses, JC virus and SV-40. Reactions were carried out in a 20-lL volume that included 2 lL of Roche LightCycler FastStart DNA Hybridization Probe 10× reaction mix and 2 lL of sample DNA. The final concentrations of other components were 3.5-mM MgCl 2 , 0.5 lM of each primer and 0.2 lM of each probe. The reaction program consisted of denaturation for 7 min at 95◦ C followed by 45 cycles of denaturation at 95◦ C instantaneous, annealing at 52◦ C for 10 s and extension at 75◦ C for 7 s. Each reaction included positive controls consisting of 1000 copies of BK virus plasmid DNA (ATCC 45026) obtained from the American Type Culture Collection (Manassas, VA) and negative controls for amplification and DNA preparation. The sensitivity of the qualitative assay was 20–50 BK virus DNA copies per reaction. The validity and performance of our assay, including the lack of detection of JC virus and SV-40, have been confirmed in a multicenter international inter-laboratory comparison (manuscript in preparation).
Quantitative polymerase chain reaction BK positive samples were quantified retrospectively by repeat real-time PCR analysis of aliquots of extracted DNA that had been frozen at −70◦ C, using four copy numbers of control BK DNA (2 × 108 , 2 × 106 , 2 × 104 and 2 × 102 ). The quantitative standard was prepared from a plasmid (pBKV[35-1]) that contains the entire linearized BK genome (ATCC 45026) using standard techniques of plasmid growth and purification. The copy number of the standard was determined by spectrophotometry. The coefficient of variability for the quantitative BK LightCycler assay in our laboratory was less than 6% for results expressed as log 10 copies/mL. The lower limit of sensitivity of the quantitative PCR assay is 10 000 copies/mL, but it reliably detects 25 000 copies/mL and absolutely detects 5 log 10 copies/mL. Although the LightCycler was able to generate a number far below these levels through extrapolation from the standard curves, we chose to use the more conservative result from interpolation of the standard curves. Therefore, positive, but unable to quantitate, BK samples were thus assigned a median value of 2.5 log 10 copies/mL.
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Brennan et al. Management protocol Identification of BK viremia triggered discontinuation of AZA or MMF. If viremia failed to clear within 4 weeks, the calcineurin inhibitor dose was tapered to trough CyA levels of 100–200 ng/mL or trough FK506 levels of 3–5 ng/mL.
Allograft biopsy All patients with unexplained elevation of the serum creatinine underwent a kidney biopsy interpreted by a pathologist blinded to the treatment arms and BK virus status. At least two cores per biopsy were obtained with an 18-gauge biopsy needle. All specimens were examined with a dissecting microscope by the pathology technician in attendance for the presence of medullary and cortical tissue and evaluated with light microscopy as well as immunoperoxidase staining using a monoclonal Ab specific for BK virus (MAB8505; Chemicon, Temecula, CA). Acute rejection was defined by the Banff ‘97 criteria (16). All biopsy specimens were also evaluated for BK nephropathy by an independent pathologist outside the institution and blinded to the treatment regimen and BK virus status of the patients during and after the study.
Endpoints and outcomes Primary endpoints were incidence of BK viruria, viremia and nephropathy in each arm at 1 year. Also studied were the rates of acute rejection, patient and graft survival and renal function at 3, 6, 9 and 12 months.
Sample size and statistics Enrollment was based on an incidence of BK viruria of 45%. To find a difference of 25% with an alpha of 0.05 and power of 0.80, 45 patients in the CyA and 90 in the FK506 arm were to be enrolled. An interim analysis suggested the number enrolled should be increased to 200, as the observed difference in the incidence of viruria in the first 36 patients was 15%. Differences in the characteristics of patients were tested with Student’s t-test for continuous variables, Fisher’s exact test for binary categorical variables and an analysis of variance (ANOVA) for multiple categorical variables. Incidence comparisons were made with the Kaplan–Meier method. Multivariate analysis of incidence was performed with Cox proportional hazards regression. Subjects were censored from incidence calculations at the time of collection of their last specimen. Multivariate models were constructed using stepwise selection over the following donor, recipient and transplant characteristics and intermediate outcomes: donor—race, gender, age, body mass index, cold ischemia time, warm ischemia time, expanded criteria; recipient—race, gender, age, body mass index, cause of end-stage renal disease and pre-existing conditions (anemia, cardiovascular disease, hypertension and diabetes); transplant— individually HLA-A, B or DR mismatches, total HLA-A, B and DR mismatches, donor and recipient HLA identical, donor and recipient CMV seropairing, ureteral stent placement and immunosuppression; and intermediate outcomes: lymphocele, acute rejection and CMV disease measured as time-varying covariates beginning at the time of diagnosis. Forward and backward stepwise selection were used to construct all multivariate models due to the large number of factors considered relative to the sample size.
Results Randomization and immunosuppressive regimens From December 27, 2000 to October 29, 2002, 226 patients were considered for enrollment and 26 patients (11%) were ineligible (n = 6) or refused consent (n = 20). Of 200 enrolled subjects, 134 (67%) were randomized to receive FK506 and 66 (33%) to receive CyA and followed for 584
Table 1: Patient baseline demographics
Age (years) Gender Male Race Caucasian African American Others ESRD Diabetes Hypertension GN PCKD Others Donor Deceased Living BMI HLA mismatch CMV serostatus D−/R− D−/R+ D+/R− D+/R+ CIT
FK n = 134
CyA n = 66
p-values
44 ± 13
46 ± 13
0.30
86 (64%)
40 (61%)
0.73 0.99
106 (79%) 24 (18%) 4 (3%)
52 (79%) 12 (18%) 2 (3%)
30 (22) 24 (17) 46 (34) 13 (9) 21 (15)
18 (27) 15 (22) 14 (21) 6 (9) 13 (19)
79 (58%) 55 (41%) 25.9 ± 4.6 2.28
34 (51%) 32 (48%) 27 ± 5 2.48
26 (19%) 30 (22%) 31 (23%) 47 (35%) 13 ± 4.8
19 (28%) 13 (20%) 14 (21%) 20 (30%) 12 ± 4.3
0.41
0.59
0.13 0.41 0.52
0.38
FK = tacrolimus; CyA = cyclosporine; ESRD = end-stage renal disease; GN = glomerulonephritis; PCKD = polycystic kidney disease; BMI = body mass index; HLA = human leukocyte antigen; CMV = cytomegalovirus seropositivity; D = donor; R = recipient; CIT = cold ischemia time.
1 year. Demographic variables were similar between groups (Table 1). In the FK506 group, 69 (52%) received AZA and 65 (49%) received MMF. In the CyA group, 43 (65%) received AZA and 23 (35%) received MMF. There were 3 crossovers from CyA to FK506 in 3 women for hirsutism. One patient switched for interstitial nephritis and one for rejection. Three patients in the CyA arm were switched to sirolimus. Two were for CyA toxicity and 1 was for rejection. In the FK506 group, 2 patients were switched to sirolimus for FK506 toxicity and one for rejection. There were two crossovers from AZA to MMF for rejection. Patients were analyzed on an intent-to-treat basis. Analyzing them after the conversion did not impact the overall results. Incidence and timing of BK virus Of the 8400 total possible samples from recipients, 7177 (85%) were collected. Missing samples were explained primarily by the following: total non-compliance in 3 patients who were censored; 5 patients thrombosed their kidneys, early, and were censored; 1 elderly non-compliant patient died early; 1 patient had a severe rejection and lost the graft, early, and no further samples were collected, and finally most patients were unable to provide a urine sample when they presented for transplantation because of anuria. Thus we collected >90% of samples from analyzable patients. American Journal of Transplantation 2005; 5: 582–594
BK, Calcineurin Inhibitor and Preemptive Therapy
Figure 1: Overall cumulative incidence of BK viruria (yellow line) and viremia (red line). (A) Cumulative incidence of BK viruria (FK506 = teal line; CyA = magenta line) (B). Cumulative incidence of BK viremia (FK506 = teal line; CyA = magenta line) (C).
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The estimated incidence of BK viruria through 365 days post-transplant was 35% (Figure 1A). The baseline demographic characteristics did not affect incidence of BK viruria. BK viremia was detected in 23 patients. BK viremia never occurred in the absence of viruria and almost always followed BK viruria. The overall estimated incidence of viremia was 11.5% through 365 days (Figure 1A). Sustained BK viremia was detected in 11 (6%) patients. No BK nephropathy was seen at 12 months, and none had been seen at the last date of observation with a mean observation period of 32 ± 6 months. The median onsets of viruria and viremia were 40.5 (range: 0–415 days) and 60 days (range: 18–276 days) after transplantation, respectively. The median interval from the onset of viruria to the onset of viremia was 27 days (range: 0–147 days). Viruria always preceded viremia, with the exception of 1 patient with simultaneous onset at month 12. After onset of viruria, levels of BK viral DNA in the urine often increased rapidly, with a median interval from onset to peak level of 21 days (range: 0–325 days). BK viral levels in urine varied over a wide range. The median peak levels in recipients were 8.98 log 10 copies/mL (range:
