Cytomegalovirus infection post-pancreas― kidney transplantation ― results of antiviral prophylaxis in high-risk patients

Background: Cytomegalovirus (CMV) is a major pathogen affecting solid organ transplant (SOT) recipients. Prophylactic strategies have decreased the rate of CMV infection/disease among SOT. However, data on the effect of current prophylactic strategies for simultaneous pancreas–kidney (SPK) or pancreas after kidney (PAK) transplant remain limited. We report our experience of CMV prophylaxis in SPK/PAK recipients. Methods: A total of 130 post‐SPK/PAK patients were analyzed retrospectively for the rate of CMV and the risk factors associated with the acquisition of CMV. All patients received antiviral prophylaxis. The follow‐up period was one yr post‐transplant or until death. Results: The rate of CMV post‐SPK/PAK transplant was 24%, 44%, and 8.2% among the whole cohort, the D+/R− and the R+ groups, respectively. Median time of prophylaxis was 49 (0–254) d. In the whole cohort, risk factors for CMV infection/diseases were D+/R− CMV status (odds ratio [OR] = 16.075), preceding non‐CMV (infection caused by bacteria or fungi and other viruses) infection (OR = 6.362) and the duration of prophylaxis (OR = 0.984). Among the CMV D+/R− group, non‐CMV infection was the only risk factor for CMV disease (OR = 10.7). Conclusions: Forty‐four per cent (25/57) of the D+/R− recipients developed CMV infection/disease despite CMV prophylaxis. Current CMV prophylaxis failed to prevent CMV infection/disease in this group of patients.

cytomegalovirus; cytomegalovirus prophylaxis; mismatch; pancreas after kidney; simultaneous pancreas– kidney transplantation; simultaneous pancreas– kidney

Compared with other solid organ transplant (SOT), simultaneous pancreas–kidney (SPK)/pancreas after kidney (PAK) transplant recipients are at high risk of acute rejection. To offset the risk of graft loss, potent immunosuppressive therapy is used. The use of potent induction regimens for SPK/PAK recipients has increased between 1999 and 2008, from 42% of transplants to 81%. The most frequently used agents in 1999 were IL‐2 receptor antagonists, which were used in 31% of patients overall. In 2008, 41% of patients received rabbit antithymocyte globulin (thymoglobulin), 30% received an IL‐2 receptor antagonist, and 12% received alemtuzumab (Campath) [1] . Despite improved outcomes of SPK transplantation with modern immunosuppression, cytomegalovirus (CMV) remains an important pathogen [2] . Minimal data exist concerning the outcome of CMV infection in patients who have undergone SPK/PAK transplants.

This study highlights the incidence and characterizes risk factors for CMV infection in SPK/PAK transplant recipients while investigating the effect of duration of CMV prophylaxis on subsequent CMV infection.

One hundred and ninety‐seven consecutive patients who underwent combined pancreas–kidney transplantation at our institution between January 2000 and March 2009 were enrolled in the study. All patients were followed for one yr or until death. Thirty‐four percent (67/197) of patients were excluded as both donors and recipients were negative for CMV (D−/R−). Data for 130 patients were analyzed.

CMV prophylaxis: CMV prophylaxis was provided by ganciclovir (GCV) and/or valganciclovir (VGCV) for 3–6 months for the D+/R− group, while the D+/R+ and D−/R+ groups received prophylaxis for three months. The decision to treat D+/R− patients for three or six months was based on the patient's drug insurance program coverage. GCV initial dose was 5 mg/kg intravenously (IV) daily, followed either by oral GCV dose of 1 g three times/d or oral VGCV 900 mg/d, whenever the patient could tolerate oral medications. Doses of both GVC and VGCV were adjusted according to renal function. CMV antigen check was performed at the discretion of the treating physician.

Patients were also prophylaxed against Pneumocystis jiroveci (PJP) with sulfamethoxazole–trimethoprim (TMP/SMX), one single strength tablet orally, daily, or three times a week. Alternative regimens for patients intolerant of TMP/SMX were oral dapsone 50–100 mg daily or inhaled pentamidine 300 mg once a month. Duration for PJP prophylaxis was 6–12 months.

Fungal prophylaxis was provided with oral nystatin suspension, 500 000 units for swish and swallow, four times daily for six wk.

All the patients received cefazolin 1 g IV on their way to the operative theater. Patients allergic to penicillin received 1 g of IV vancomycin.

Methylprednisolone 500 mg IV was administered intraoperatively for all patients. The dose was then tapered over the first five postoperative days. On day 6, methylprednisolone was replaced with a tapered dose of oral prednisone. Prednisone was tapered to 5 mg/d by 3–6 months post‐transplant. Some patients had steroids completely discontinued during the first year post‐transplant. A subsequent analysis of our experience revealed a high rate of acute rejection, and the majority of these patients were subsequently restarted on prednisone 5 mg/d. The majority of patients received induction therapy with thymoglobulin; some received basiliximab, while daclizumab, rabbit antithymocyte serum (RATS), RATS plus basiliximab, and the combination of thymoglobulin and basiliximab were used occasionally. Some patients received no induction.

The dose of thymoglobulin was 1.0–1.5 mg/kg/d, IV. The first dose was given postoperatively. A total dose of 7 mg/kg was provided for high‐immunologic risk patients, which included patients with a known donor‐specific antibody at the time of transplant, second transplant (including PAK), and panel reactive antibodies (PRA) >50%. For low‐immunologic risk patients, thymoglobulin was given daily until renal function improved to allow tacrolimus to be started, usually 3–5 mg/kg. All thymoglobulin doses were preceded by pre‐medication, which included acetaminophen 650 mg orally, diphenhydramine 50 mg IV, and methylprednisolone 100 mg IV. Patients receiving basiliximab were administered doses of 20 mg IV infusion within 2 h prior to transplant surgery and a second dose on day 4 post‐transplant. Maintenance immunosuppression was provided with tacrolimus and mycophenolate mofetil. Tacrolimus blood level was targeting a trough level of 10–15 ng/mL. Mycophenolate mofetil dose was 500 mg twice daily.

It was defined as per the American Society of Transplant (AST) criteria [3] .

It was defined as any infection (viral, bacterial, or fungal) that took place during the follow‐up period.

It was defined as absolute neutrophil count of <1500/microliters (<1.5 × 109/L) [4] .

Acute rejection in the renal transplant was usually based on a rise in serum creatinine and biopsy evidence of acute cellular or humoral rejection. Histologic diagnosis for kidney rejection was made according to Banff criteria [5] . Occasionally, patients were treated empirically without a biopsy. Low‐grade acute cellular rejection was treated with pulse methylprednisolone, followed by a steroid taper and increase in maintenance immunosuppression. More severe grades of acute cellular rejection or steroid‐resistant episodes were treated with thymoglobulin. Acute humoral rejection was treated with steroids, intravenous immunoglobulin, and plasma exchange. Thymoglobulin and/or rituximab were used in some cases.

The diagnosis of acute pancreatic rejection was made in some cases on clinical grounds, that is, a rise in serum amylase. Patients where rejection was suspected were pulsed with methylprednisolone 500 mg IV/day for three d, followed by a steroid taper and increase in baseline immunosuppression. Patients whose amylase remained elevated underwent percutaneous pancreatic biopsy. Biopsies were graded according to the Banff criteria for pancreas transplant biopsies [5] . Biopsy‐proven acute cellular rejection was treated with steroids and/or thymoglobulin. Acute humoral rejection was treated with steroids, thymoglobulin, plasma exchange, and intravenous immunoglobulin; rituximab was given in some cases.

It was defined as return to exogenous insulin use [6] or return to dialysis.

Data were obtained from inpatient and outpatient charts. Parameters collected include age, sex, date of transplant, type of induction, CMV status, duration of prophylaxis, medications used for prophylaxis, surgical procedure, onset of CMV infection/disease, onset of non‐CMV infection, onset and treatment of rejection, onset of organ failure, neutropenia, and death.

To determine the CMV status, CMV IgG testing was performed for all donors and recipients using the Abbott AxSYM CMV IgG enzyme immunoassay. For the diagnosis of CMV infection, the peripheral blood CMV pp65 leukocyte antigen was used utilizing the BioTest Clonab's CMV Monoclonal Anti‐HCMV pp65, as per manufacturer's instructions for both tests. CMV PCR test was not in use at our laboratory during the period of our study. Rejection was confirmed by histopathology in all patients except for two episodes.

Statistical analysis was performed with STATA 12 software (StataCorp LP, College Station, TX, USA). Statistical differences in categorical variables were determined using chi squared or Fisher's exact test as appropriate. Differences in continuous variables were determined using Student's t‐test, analysis of variance, or Mann–Whitney U‐test as appropriate for the distribution. Values are presented as mean ± SD deviation when normally distributed. To determine the impact of infection predictors, a stepwise regression model with backward and forward analyses was performed. Duration of prophylaxis was retained in the model despite the lack of statistical significance. Predictor variables included in the model were CMV serostatus, type of transplant, type of induction, duration of prophylaxis, preceding infection, neutropenia, preceding rejection episodes, and era effect. Retransplantation effect was not included as no patient was retransplanted during the follow‐up period. The model was evaluated using Hosmer–Lemeshow goodness of fit test. A p value of <0.05 was considered significant. Our study methodology was approved by the Research Ethics Board of our institution.

One hundred and thirty patients formed the study cohort, and their demographic data are displayed in Table [NaN] . Median age at transplantation was 42 (range 27–58) yr. Fifty‐eight percent (75/130) of patients were males. Of the 130 patients, 100 of 130 (77%) underwent SPK, while the rest had PAK transplants. CMV infection/disease was noted in 24% (31/130) of our cohort. Thirty‐two percent (10/31) of cases had CMV infection, while 68% (21/31) had CMV disease. Acute viral syndrome (AVS) was observed in 86% of CMV diseases patients, while the remainder had gastrointestinal disease. The rate for CMV infection/disease among SPK recipients was 24% (24/100). Eight percent (8/100) of the patients had infection, and 16% (16/100) had CMV disease. In comparison, the rate of CMV in PAK recipients was 23% (7/30). Seven percent (2/30) of PAK patients had infection, and 17% (5/30) had disease.

Demographics of the cohort included in the study

1 SPK, simultaneous pancreas–kidney; CMV D+/R−: CMV donor positive/recipient negative; CMV R+: CMV recipient positive.

2 Either the combination of thymoglobulin or basiliximab or daclizumab or rabbit antithymocyte serum (RATS) or RATS plus basiliximab, or no induction.

Among the CMV primary mismatches (D+/R−), 44% (25/57) developed CMV infection/disease, in contrast to 8% (6/73) of the R+ population. Median time to onset of CMV infection/disease was 34 d (0–254), 65 d (0–97), and 33 (0–254) d post‐discontinuation of antiviral prophylaxis, for the whole cohort, the R+ group, and the D+/R− group, respectively.

Rejection occurred in 16% (21/130) of patients, accounting for 28 episodes of rejection. Thirty‐three percent (7/21) of those who experienced rejection subsequently developed CMV infection/disease, with median of 125 d (32–166) post‐rejection. Those seven patients had their rejection episodes treated with methylprednisone alone in 43% (3/7) of patients. Two patients (29%) required surgical removal of the transplant. Thymoglobulin alone and OKT3 were each administered in one patient, respectively.

Neutropenia during CMV prophylaxis was noted in 23% (30/130). It was associated with GCV in 4% (5/117) and in 30% (25/83) during VGCV prophylaxis. Thirty percent (9/30) of the neutropenic patients subsequently developed CMV.

Thirty‐nine episodes of non‐CMV infections were noted in 22% (29/130) of patients. Seventeen episodes were caused by viruses (10 episodes by Polyomavirus, 6 episodes by EBV, and one episode by Parvovirus B 19), 17 episodes were caused by bacteria (14 episodes by Pseudomonas aeruginosa, two episodes by Acinetobacter calcoaceticus–baumannii complex, and one by Enterococcus faecium), and five were caused by fungal pathogens (yeast not otherwise specified caused two episodes and Candida krusei, C. tropicalis, and C. albicans caused one episode each). The majority of non‐CMV infections were in SPK group (76% [22/29]). Fifty‐two percent (15/29) of patients who had non‐CMV infections were observed in the D+/R− group. CMV occurred along with non‐CMV infection in 45% (13/29) of patients. In 69% (9/13) of those patients, non‐CMV infection was diagnosed at median time of 98 d (14–244) before CMV infection/disease, and in the remaining patients (31% [4/13]), the diagnosis of non‐CMV infection was made on the same day as CMV. Those who had non‐CMV infection before the onset of CMV had the following organisms isolated: EBV from blood in 44% (4/9) of patients, Candida spp. (C. albicans, C. tropicalis, and C. krusei) from surgical sites in 22% (2/9) of patients, polyoma virus from the urine in 22% (2/9) of patients, and P. aeruginosa from the urine in 11% (1/9) of patients.

Mortality occurred in 2% (3/130). The three patients who died had kidney and pancreatic graft failure (GF), and none of them had rejection or CMV I/D. Median time from combined GF to death was 67 d (0–296).

The median duration of CMV prophylaxis was 104 d (2–365). Thirty‐nine percent (50/130) of patients received prophylaxis with GCV alone, 11% (14/130) received VGCV alone, and 51% (66/130) received the combination of both drugs sequentially. Among the D+/R− group, the median duration for prophylaxis was 105 (2–365) d.

The R+ group on the other hand received prophylaxis for a median duration of 103 (12–283) d. In both the univariate (UV) and multivariate (MV) models of CMV infection/disease, 180 d of prophylaxis was noted to decrease the risk by 40% (odds ratio [OR] = 0.59, CI = 0.13–2.64). However, this variable was not statistically significant. Surprisingly, CMV prophylaxis >180 d increased the risk of CMV disease in R+ recipients (OR = 34.5, CI = 1.54–71.9). The same observation was noted in a recent meta‐analysis [7] .

Practice change over time was most evident in the introduction of immunosuppression and induction drug regimes change [1] . Therefore, study period was divided into two groups: the first was from the year 2000 to 2004 and it included 61 patients. Induction therapy during this period was mainly with lymphocyte non‐depleting agents (daclizumab or basiliximab). The second period was from 2005 to 2009 with total number of 69 patients. Induction agents during that era were mainly with lymphocyte‐depleting agents (thymoglobulin, RATS, basiliximab plus thymoglobulin, or basiliximab plus RATS) [8] . Eighteen percent of patients (11/61) in the first era had CMV I/D, whereas 29% (20/69) of those in the second era had CMV I/D (p = 0.15, CI 0.81–4.3). The era effect had no influence on CMV I/D rate. We also did an exclusive analysis for the effect of induction therapy on the rate of CMV I/D; 69% (90/130) of the cohort received induction therapy with lymphocyte‐depleting agents, and the rest (31% [40/130]) received induction with non‐lymphocyte‐depleting agents. CMV I/D occurred in 23% (21/90) of the first group and in 25% (10/40) of the second group. Our analysis showed that changing induction therapy over years had no effect on CMV I/D rate (p = 0.84, CI = 0.4–2. 2).

For the whole cohort, the risk factors for CMV infection/disease were CMV mismatch (OR 16.075, 95% CI [4.821–53.598]), preceding non‐CMV infection (OR 6.362, 95% CI [1.98–20.438]), and duration of prophylaxis (OR 0.984, CI [0.972–0.996]). Each additional day of prophylaxis reduced the rate of CMV infection/disease by 2%. Rejection and the presence of neutropenia were not significant risk factors for CMV; they were associated with OR of 3.478, 95% CI (0.897–13.489), and OR of 3.32, 95% CI (0.98–11.163), respectively. Among the CMV donor positive/recipient negative (CMV D+/R−) group, preceding non‐CMV infection was the only risk of subsequent CMV disease (OR 10.7 [95% CI: 1.93–59.93]). Type of transplant (SPK vs. PAK), presence of neutropenia, induction regimen, and preceding rejection were not associated with the development of CMV infection/disease in our cohort (Tables [NaN] and [NaN] ).

Multivariate analysis of risk factor for CMV infection/disease among the whole cohort of simultaneous pancreas–kidney

3 OR, odds ratio; p, probability; CI, confidence interval; CMV MM, CMV mismatch.

Multivariate analysis of risk factor for CMV disease among the CMV MM group of simultaneous pancreas–kidney

4 OR, odds ratio; p, probability; CI, confidence interval.

Risk factors for CMV infection/disease post‐SOT have been extensively reported. Risk of CMV is highest among D+/R− SOT patients. Such patients lack cellular and humoral immunity to CMV [9] . As well, the net state of immunosuppression appears to be an important determinant for the development of CMV infection [10] . Data on CMV infection/disease and risk factors associated with CMV are sparse for SPK/PAK. We evaluated the risks of CMV infection/disease and found that CMV serostatus and non‐CMV infection were the most significant risk factors for subsequent CMV infection/disease. More interesting, non‐CMV infection remained an important risk factor in the CMV D+/R− group. Bacterial or fungal infection has been postulated to be one of the factors that influence CMV reactivation, and it has been associated with a higher incidence of CMV disease after SOT [11] .

Historically, the rate of CMV infection/disease can be as high as 70% among the CMV D+/R− SOT in the absence of prophylaxis. Among the CMV D+/R− SPK/PAK transplant, the rate of CMV is 6–29% with prophylaxis and 45–65% without prophylaxis [11] . The outcome of our analysis is in keeping with these data. In our study, despite prophylaxis, 44% (25/57) of the CMV D+/R− patients developed CMV infection/disease, while 11% (6/57) had only infection and 33% (19/57) were diagnosed with CMV disease.

As for the duration of CMV prophylaxis, a multicenter randomized trial showed that, in CMV D+/R kidney transplant recipients, extending VGCV prophylaxis from 100 to 200 d decreased the incidence of CMV infection/disease and prolonged the time to onset of viremia [12] . That effect was shown in our whole cohort (OR = 0.984, CI = [0.972–0.996]), but not among the CMV D+/R− group. Each additional day of prophylaxis resulted in a 2% reduction in the risk of CMV infection/disease.

Twenty‐three percent (27/118) of those who received ≤180 d of CMV prophylaxis developed CMV, whereas CMV was noted in 33% (4/12) of those who received ≥180 d of prophylaxis (p = 0.37). The median onset of viremia from discontinuation of prophylaxis was similar among the two groups. It was 50 (6–80) d and 49 (0–254) d for the ≤180 and ≥180 d of prophylaxis, respectively. The total number of patients who received ≥180 d of CMV prophylaxis, however, was small (12 patients) compared with those who did not (118 patients), which did not allow for a robust comparison. However, the median onset of CMV from discontinuation of CMV prophylaxis was shorter for the D+/R− than the R+ group (33 d [0–254] vs. 65 d [0–97]) emphasizing the more aggressive nature of CMV infection/disease in the R− individuals.

Traditionally, organ allograft type does influence CMV susceptibility, irrespective of the immunosuppressive protocol employed. The risk of CMV is highest in lung transplant recipients and lower in liver and kidney transplantation. This may be due to the degree of immunosuppression and/or the viral load present in the transplanted allograft [9] . In our cohort, the ratio for SPK to PAK recipients was 100:30. CMV occurred in 24% (24/100) and 23% (7/30) of the patients, respectively, despite the fact that there was CMV mismatch in 47% (47/100) of SPK and 30% (10/30) of the PAK recipients. Transplant procedure had no influence on CMV rate among our cohort.

Our study possesses several limitations. One is that it is a retrospective, single‐center study design with the usual disadvantages associated with this type of study. The data are also heterogeneous, due to the fact that the data were collected over almost a decade (January 2000–March 2009), during which strategies for immunosuppression and rejection therapy underwent significant modification. Moreover, surgical technique changed during this time from portal to systemic venous drainage of the pancreas. Nonetheless, these data were captured electronically. The presence or absence of CMV infection/disease was based on laboratory assessments and histopathology reports, which are unlikely to be biased over time. Moreover, to assess changes in strategies for immunosuppression and rejection therapy, these variables were included in the MV model to minimize disparities and our UV analysis for era effect was not significant.

The results of this study will add to the body of knowledge of CMV infection in pancreas transplantation [2] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] . Furthermore, our study is unique in including non‐CMV infection and neutropenia in the analysis of risk factors for CMV. We noted that preceding non‐CMV infection is an independent risk factor for CMV among SPK transplantation recipients. We have also excluded the very low‐risk CMV status (D−/R−); therefore, our study is the only one that represents the actual CMV rate among susceptible SPK and PAK recipients.

Our study is not alone in addressing the efficacy of CMV prophylaxis among SPK transplantation recipients. All previous studies, with some exceptions [13] , [18] , [21] , had a maximum period of prophylaxis of three months, and in some [14] , [16] , [18] , [19] , prophylaxis was provided with acyclovir with or without GCV or VGCV. As a result, the efficacy of prophylaxis and the reporting of the incidence rates in these studies may have been influenced by the shorter duration and type of prophylaxis, especially in high‐risk recipients. Our study reflects today's practice, because 79% (103/130) of the cohort received CMV prophylaxis for more than three months and all the patients received GCV, VGCV, or the combination of both as both drugs are considered to be bioequivalent [22] . Another feature of our study, which is also generalizable to current practice in pancreas transplantation, was our use of induction therapy. The majority of our patients received lymphocyte‐depleting therapy with thymoglobulin. Ours is among the few studies [2] , [15] , [17] , [20] that have included induction therapy in the analysis of risk factors for CMV infection/disease and found no added risk of the development of CMV.

Based on our data, one may conclude that the duration of CMV prophylaxis (≥90 d) may be insufficient to prevent CMV infection/disease in SPK and PAK patients. Moreover, although fewer in number, >180 d of CMV prophylaxis in D+/R− patients also failed to prevent CMV infection/disease. Future studies directed toward optimal prophylactic strategies are warranted.

The authors have contributed to the manuscript as follows: Shahid Husain participated in research design; Samira M Fallatah, Max A Marquez, Shahid Husain, and Coleman Rotstein participated in the writing of the manuscript; Samira M Fallatah, Max A Marquez, Fateh Bazerbachi, Jeffrey R Schiff, Mark S Cattral, Ian D McGilvray, Andrea Norgate, Markus Selzner, and Shahid Husain participated in the performance of the research; Max A Marquez and Shahid Husain participated in data analysis.