In patients undergoing solid organ transplantation, cytomegalovirus (CMV) infection may cause life-threatening tissue invasive disease. However, the long-term adverse effects of CMV are predominantly mediated by its indirect effects (1, 2). CMV raises the risk of other infections, Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder, new-onset diabetes mellitus, and cardiovascular complications (1, 3–5). Early asymptomatic CMV infection and CMV disease have been shown to increase long-term mortality (6, 7).
The relation of CMV to acute rejection and chronic allograft damage is of paramount importance. Several authors have shown that CMV disease and, even asymptomatic infection in some cases, led to an increased incidence of acute rejection in renal transplant recipients (8, 9). The most compelling data supporting the effect of CMV on the development and progression of chronic allograft damage have emerged from studies after heart transplantation, where CMV represents a major cause of cardiac allograft vasculopathy (10, 11). However, evidence from patients undergoing renal transplantation is also currently available (12, 13).
Still, many controversies remain. At present, most centers use prophylaxis or preemptive therapy to prevent CMV infection. Both strategies are associated with a significant decrease in the incidence of CMV disease (14–24). Nonetheless, prophylaxis resulted in a reduction of acute rejection in only some randomized trials (18, 20). A direct comparison of prophylaxis and preemptive therapy yielded controversial results (7, 21, 22). It is unclear whether low-level CMV viremia controlled by preemptive therapy or whether the viremia developing despite the prophylaxis poses a risk for acute rejection. A minimal number of trials have explored the relationship between CMV and subclinical rejection (SCR) or interstitial fibrosis and tubular atrophy (IF/TA) diagnosed by protocol biopsy (25). Protocol biopsies have become an integral part of clinical practice in many centers (26, 27). The presence of SCR, IF/TA and, most importantly, a combination of both predicts subsequent renal function deterioration and graft failure (28–31).
In the prospective study, we evaluated the association of CMV viremia, as detected by routine monitoring using quantitative real-time polymerase chain reaction (PCR), with histologic findings in protocol biopsy at 3 months after renal transplantation. All patients received 3-month prophylaxis (valacyclovir or oral ganciclovir) or were managed by preemptive therapy.
MATERIALS AND METHODS
All consecutive patients undergoing kidney transplantation in the period from January 2003, when protocol biopsy at 3 months became a part of clinical practice in our center, through July 2007 were considered for inclusion. Enrolled were patients at risk for CMV (CMV donor/recipient serostatus of D+/R−, D+/R+, and D−/R+). Exclusion criteria included D−/R− serostatus, allergy to ganciclovir or acyclovir, severe leukopenia or thrombocytopenia, and inability to provide informed consent. Renal transplantation procedures were performed at the Charles University Teaching Hospital in Pilsen, Czech Republic. The study was approved by the local ethics committee and conducted in compliance with the Declaration of Helsinki. All patients signed informed consent before transplantation.
Immunosuppression and Clinical Acute Rejection Treatment
Immunosuppression was based on cyclosporine microemulsion (Neoral; Novartis, Basel, Switzerland). Recipients of second or third transplant and those with panel-reactive antibody of more than 60% received induction rabbit antithymocyte globulin (ATG Fresenius; Germany) and tacrolimus (Prograf; Astellas, Killorglin Co., Kerry, Ireland). Target trough levels of cyclosporine and tacrolimus were 200 to 300 and 10 to 15 ng/mL over the first 3 months and, subsequently, 100 to 150 and 5 to 10 ng/mL, respectively. Graft recipients from marginal donors (donation after cardiac death, 70 years older, and donors with hypertension and significant nephrosclerosis in biopsy) were treated with anti-interleukin 2R monoclonal antibody (Simulect; Novartis, Basel, Switzerland) and low-dose sirolimus (Rapamune; Wyeth Laboratories, UK) with target trough levels of 5 to 10 ng/mL or low-dose tacrolimus with target trough levels of 4 to 8 ng/mL. All groups were given mycophenolate mofetil (Cellcept; Hoffman-La Roche, Basel, Switzerland) at a dose of 2 g per day, and corticosteroids.
As a rule, clinically suspected acute rejection was confirmed by core biopsy using the Banff ’05 classification (32). Rejection episodes were treated initially by high-dose intravenous methylprednisolone and switched from cyclosporine to tacrolimus. Steroid-resistant episodes were treated by antilymphocyte antibody (anti-CD3 monoclonal antibody or rabbit ATG).
CMV Prophylaxis and Monitoring
Patients were followed up prospectively, for 12 months posttransplant or until death or graft failure. In the period from January 2003 through October 2003, patients were randomized, as part of an earlier study, to 3-month prophylaxis with oral ganciclovir (Cymevene; Hoffman-La Roche, Basel, Switzerland) at a dose of 1 g three times per day or valacyclovir (Valtrex; Glaxo Wellcome, Dartford, UK) at a dose of 2 g four times per day with reduction depending on renal function (9). Later on, patients were randomized to preemptive therapy with valganciclovir or to valacyclovir prophylaxis (22). Preemptive group patients were monitored using quantitative real-time PCR from whole blood for CMV DNAemia detection once a week for 16 weeks and, subsequently, at months 5, 6, 9, and 12. At a viral load more than or equal to 2000 copies/mL, therapy with valganciclovir (Valcyte; Hoffman-La Roche, Grenzach-Wyhlen, Germany) was instituted at a dose of 900 mg two times per day within 7 days at the latest and continued until reaching clearance of CMV DNAmia (at least for 14 days). Doses were tapered based on renal function according to manufacturer’s instructions. In prophylaxis-treated patients, quantitative PCR was performed at weeks 2, 4, 6, 8, 10, and 12, and at months 4, 5, 6, 9, and 12. Asymptomatic CMV DNAemia was not treated in prophylaxis patients. CMV disease was treated with intravenous ganciclovir (Cymevene; Hoffman-La Roche, Basel, Switzerland) for a minimum of 3 weeks. The patients could be switched to valganciclovir. Mycophenolate mofetil was withdrawn in the presence of tissue invasive and recurrent CMV disease.
Quantitative real-time PCR was performed using a commercially available kit (RealArt CMV RG PCR Kit, Artus; Germany) according to manufacturers’ instructions on a Rotor-Gene 2000/3000 system (Corbett Research; Australia). DNA was isolated from 200 μL of whole blood using a commercially available kit (NucleoSpinBlood, Macherey-Nagel, Germany). The analytical detection limit in consideration of the purification (extraction volume, 0.2 mL; elution volume, 0.1 mL) according to the manufacturers’ assessments was consistently 50 copies/mL of whole blood. Physicians assessing the PCR results were blinded to the clinical data of patients.
Core biopsies were carried out at 3 months after transplantation using 18- or 16-gauge automated biopsy needles guided by ultrasound. Biopsy was repeated in cases where the specimen did not contain a single glomerulus. Tissues for light microscopy were fixed in 4% formaldehyde, embedded in paraffin using routine procedure. Five-micrometer thick sections were cut from the tissue blocks, and stained with hematoxylin-eosin, blue trichrome, and silver staining. In addition, immunohistochemistry was performed to detect C4d, BK polyoma virus, and CMV. Biopsy specimens were evaluated by a pathologist blinded to the patients CMV status and scored using the Banff ’05 classification (32). SCR was defined by histological finding of acute rejection (grade≥IA) or borderline changes. Patients with SCR of grade more than or equal to IA were treated by high-dose intravenous methylprednisolone. IF/TA was defined as ci+ct scores more than or equal to 2. Chronic allograft damage index was also calculated (28).
The effect of CMV viremia detected within the first 100 days posttransplant on the incidence of SCR and IF/TA in protocol biopsy was evaluated. CMV viremia was defined by real-time PCR positivity. Tissue invasive CMV disease did not develop in any patient within the first 100 days, only three patients had CMV syndrome. These were evaluated together in the group with asymptomatic viremia. Variables assessed in the analyses included CMV status (viremia, no infection); strategy for prevention of CMV (ganciclovir prophylaxis, valacyclovir prophylaxis, preemptive therapy); previous acute rejection (including borderline changes); delayed-graft function (DGF); recipient characteristics (age, gender, cause of renal disease, previous transplantation, duration and modality of pretransplantation dialysis, diabetes, percentage of panel reactive antibody, human leukocyte antigen mismatches, and D/R CMV serostatus); donor characteristics (age, gender, donor source, expanded criteria donor, and cold ischemia); and baseline immunosuppressive regimen.
Demographic, immunologic, and clinical data between patients with and without CMV viremia were compared using the chi-square test for qualitative variables and the unpaired t test or the Mann-Whitney rank-sum test in nonparametric distribution for quantitative variables. Patient and graft survival and incidence of acute rejection were calculated using Kaplan-Meier curves, with the log-rank test used for comparison. Univariate and multivariate stepwise logistic regression analyses were used to determine the effect of CMV viremia and other covariates on SCR and IF/TA. Covariates with P less than 0.2 were included in multivariate analysis. A value of P less than 0.05 was considered significant. Statistical calculations were performed using the SAS system (SAS Institute Inc., Cary, NC).
Study Population and Incidence of CMV Viremia
Overall, 130 patients were considered for inclusion in the study (Fig. 1). Twelve patients were excluded: 11 met the exclusion criteria (nine patients were participating in another clinical trial and two patients had D−/R− serostatus) and one patient refused to participate. Protocol biopsy was performed in 107 patients. Reasons for not performing protocol biopsy were as follows: death (n=2), graft failure (n=5), contraindication because of acute pyelonephritis (n=2) or coagulopathy (n=1), and refusal by patient (n=1). In five cases, protocol biopsy failed to provide material containing a single glomerulus. A total of 102 patients were included into final analysis.
Within the first 100 days posttransplant, CMV viremia developed in 42 (41%) patients. Viremia of more than or equal to 2000 copies/mL was detected in 20 (21%) patients. The incidence of CMV viremia was significantly higher in patients managed by preemptive therapy compared with ganciclovir- or valacyclovir-based prophylaxis (91% vs. 7%; P<0.001). Mean time for the first CMV viremia was 35±19 days after transplantation. Peak viral load was 11,900±37,200 (median 1300) copies/mL of whole blood. Except for three patients developing CMV syndrome, all episodes of CMV viremia were asymptomatic. At the time of protocol biopsy, CMV viremia was present in only 15 (15%) patients. Basic characteristics of recipients and donors in patients with and without CMV infection are given in Table 1. The incidence of biopsy-proven acute rejection (grade≥IA) was significantly higher in the group with CMV viremia, whereas the difference in borderline rejection was nonsignificant. Apart from shorter time on pretransplant dialysis and a higher proportion of male donors in patients with CMV viremia, there were no significant differences between the groups.
Protocol Biopsy Histologic Findings
The mean number of glomeruli per biopsy was 9±7. In 60 (59%) biopsies, the number of glomeruli was more than or equal to 7. There was no difference between patients with and without CMV infection in the number of glomeruli per biopsy (9±7 vs. 9±6; P=0.139). SCR was observed in 30 (29%) patients showing borderline changes in 21 (21%), and acute rejection in 9 (9%) cases. IF/TA, predominantly mild (grade I), was present in 29 (28%) patients. Other histologic findings included calcineurin inhibitor toxicity (n=20), donor-related vascular nephrosclerosis (n=15), polyoma virus-associated nephropathy (n=2), and transplant glomerulopathy (n=1).
The incidence of SCR (26% vs. 32%; P=0.706) and IF/TA (35% vs. 25%; P=0.393) was similar in patients with CMV viremia and those without CMV infection (Table 2). There were also no differences in acute and chronic scores using the Banff classification. The mean chronic allograft damage index score was 1.70±1.69 and 1.33±1.56 in the groups with and without CMV infection, respectively (P=0.338). Among the 15 patients with CMV viremia at the time of biopsy, SCR and IF/TA was found in 6 (40%; P=0.150) and 4 (27%; P=0.735), respectively. According to retrospective power analysis, 220 patients would be required to show significant difference in the incidence of SCR in patients with CMV viremia compared with those without CMV viremia at the time of biopsy.
Risk Factors for SCR
CMV viremia did not affect the incidence of SCR in protocol biopsy compared with patients without CMV infection (odds ratio [OR] 0.77; 95% CI 0.32–1.84; P=0.551). Similarly, CMV serostatus of D+/R− and the mode of CMV disease prevention were not associated with SCR (Table 3). Univariate analysis identified the following variables significantly associated with increased risk for SCR: acute rejection (OR 3.00; 95% CI 1.25–7.23; P=0.014), expanded criteria donor (OR 2.63; 95% CI 1.07–6.42; P=0.034), and sirolimus-based immunosuppression (OR 6.75; 95% CI 1.84–24.75; P=0.004). The presence of diabetes before transplantation had a protective effect (OR 0.14; 95% CI 0.02–1.14; P=0.066).
In multivariate stepwise logistic regression analysis, acute rejection (OR 3.34; 95% CI 1.35–8.30; P=0.009), and sirolimus-based immunosuppression (OR 6.13; 95% CI 1.13–23.61; P=0.008) were independent predictors of SCR, whereas diabetes was associated with a reduced risk (OR 0.12; 95% CI 0.01–0.99; P=0.049) (Table 3).
Risk Factors for IF/TA
In univariate analysis, development of CMV viremia within the first 100 days had no effect on the incidence of IF/TA (OR 1.50; 95% CI 0.63–3.57; P=0.360). A major effect was likewise not demonstrated with CMV serostatus and the mode of CMV disease prevention. However, CMV viremia with viral load of more than or equal to 2000 copies/mL significantly increased the risk of IF/TA (OR 3.00; 95% CI 1.05–8.60; P=0.041) (Table 4). Variables characteristic for lower-quality donors and ischemia-reperfusion injury were associated with increased risk for IF/TA: donor age (OR 1.60; 95% CI 1.14–2.24; P=0.007), DGF (OR 5.78; 95% CI 2.10–15.88; P<0.001); a similar trend was noted in cases involving transplantation of grafts from expanded criteria donors. Use of induction therapy with antilymphocyte antibodies was also associated with the finding of IF/TA in protocol biopsy; however, induction was part of the immunosuppressive protocol in transplantation from expanded criteria donors.
The effect of CMV viremia of more than or equal to 2000 copies/mL was confirmed in multivariate analysis (OR 3.83; 95% CI 1.21–12.14; P=0.023). DGF (OR 6.02; 95% CI 2.07–17.52; P=0.001) and donor age (OR 1.53; 95% CI 1.13–2.35; P=0.009) were also independent predictors of IF/TA (Table 4).
Biopsy Classification and Renal Function
There were no differences in 1-year patient and graft survival rates between patients with SCR and IF/TA compared with those without SCR and IF/TA. There was no death within the first posttransplant year. Graft failure occurred in only one patient because of severe IF/TA. Protocol biopsy revealed concomitant SCR, IF/TA (grade I), and donor- related vascular nephrosclerosis.
Serum creatinine levels were not significantly increased in patients with SCR before and at the time of protocol biopsy. However, compared with patients without SCR, there was progressive deterioration of serum creatinine after biopsy reaching levels of 180±97 vs. 141±45; P=0.015 at 1 year. In the group with SCR, calculated glomerular filtration rate was decreased as early as 1 month posttransplant (Table 5). By contrast, renal function (serum creatinine and glomerular filtration rate) in protocol biopsy in patients with IF/TA significantly deteriorated in the early postoperative period, with the differences persisting during the first posttransplant year (Table 5). No significant differences in renal function were observed between patients with and without CMV viremia even if the level of viremia was considered.
Our prospective study is the first to furnish information about the effect of CMV infection on SCR and IF/TA in a group of patients monitored systematically for the presence of CMV viremia using the sensitive method of real-time PCR, with CMV prophylaxis or preemptive therapy approach were additionally used in all patients. Hence, the treatment was consistent with current clinical practice (2). The main finding is the fact that CMV viremia within the first 3 months posttransplant is not an independent risk factor for SCR in protocol biopsy at 3 months. In contrast, CMV viremia with viral load of more than or equal to 2000 copies/mL although effectively suppressed by preemptive valganciclovir therapy, increased the risk of IF/TA by more than three times. The mode of CMV disease prevention, that is, preemptive therapy compared with universal ganciclovir- or valacyclovir-based prophylaxis, was not associated with increased risk for SCR or IF/TA.
Although the role of CMV disease and asymptomatic CMV infection in the pathogenesis of acute rejection and chronic allograft damage has been confirmed in several studies, the body of data regarding the relation of CMV to SCR and IF/TA, as detected by protocol biopsy is most limited (8–12, 33). An earlier study showed the presence of CMV in situ in renal biopsy specimens was associated with arteriolar intimal thickening in protocol biopsy at 6 months (25). However, the finding applied only to patients with a previous episode of acute rejection. Nonetheless, even CMV pp65 antigenemia, although symptomatic in most cases and documenting CMV disease, was not associated with an increased risk for acute or chronic histopathologic changes. The incidence of IF/TA was not affected by in situ CMV detection. Our study differed in several key issues. Biopsies were performed at 3 months when incidence of SCR is high and IF/TA is also documented in 20% to 40% of patients. In contrast, analysis at 6 months will reliably identify only chronic changes as the incidence of SCR falls dramatically (26, 30). The authors made investigations for the presence CMV pp65 antigenemia only in cases with clinically suspected CMV disease. CMV prophylaxis or preemptive therapy was not provided (25). The regular examination using PCR CMV in our patients made it possible to detect even asymptomatic CMV viremia. Some data suggest that the impact of CMV disease and asymptomatic CMV viremia on acute rejection is different (9).
Although acute rejection may lead to CMV reactivation because of enhanced immunosuppressive therapy and release of proinflammatory cytokines, CMV disease and asymptomatic CMV infection during the first 3 months posttransplant are independent risk factors for subsequent episode of acute rejection (8, 34). However, the associations between CMV and acute rejection may be modified by the use of prophylaxis or preemptive therapy. Delayed asymptomatic CMV viremia developing after the completion of prophylaxis did not raise the risk of acute rejection (9). Preemptive therapy has been criticized for allowing early CMV viremia with a potential risk of indirect effects of CMV. On the other hand, its proponents point out that controlled low-level viremia facilitates the recovery of CMV-specific T-cell responses minimizing the risk for late CMV disease and its adverse sequels (15, 35). Detectable CMV-specific CD4 T cells at 1 month after heart transplantation had a protective effect on the development of acute rejection and cardiac allograft vasculopathy (11). Consequently, preemptive therapy need not be associated with the risk of acute rejection. Randomized trials comparing prophylaxis with preemptive therapy have produced conflicting results. Although the incidence of acute rejection in two trials using (val)ganciclovir-based prophylaxis was comparable (7, 21), a trial of valacyclovir-based prophylaxis documented a lower incidence of biopsy-proven acute rejection with prophylaxis (22).
Although, in our series, patients with CMV viremia showed a markedly higher incidence of biopsy-proven acute rejection (45% vs. 13%), there were no differences in the incidence or severity of SCR. Consistent with earlier data, a previous episode of acute rejection and sirolimus-based immunosuppression without calcineurin inhibitors predicted SCR in protocol biopsy (26, 36). We did not demonstrate lower incidence of SCR in patients treated with tacrolimus compared with cyclosporine as described previously (26, 36). This may be explained by selection of immunologic high-risk patients to tacrolimus-based regimen in our center. Why CMV viremia had no effect on risk of SCR is difficult to explain. A major factor may be the time of protocol biopsy. CMV viremia developed approximately 5 weeks posttransplant and was effectively suppressed by preemptive therapy with valganciclovir. CMV viremia was present in only 15% of patients at the time of protocol biopsy. There was a trend toward higher incidence of SCR in patients with CMV viremia at the time of biopsy as compared with those with negative PCR CMV results (40% vs. 18%; P=0.150). The effect in biopsy at the end of month 3 may thus be negligible in the whole CMV viremia group.
In our series, the presence of IF/TA was associated with DGF and older donor age. These are variables reflecting lower graft quality at the time of transplantation with increased susceptibility to ischemia-reperfusion injury (29). Given the high proportion of expanded criteria donors, donor-related factors could have been expected to dominate. Although IF/TA was not affected by low-level CMV viremia or the mode of CMV prevention, preceding episode of CMV viremia with levels above 2000 copies/mL increased the risk significantly. It accords with previous report of the same incidence rates of acute rejection, but improved long-term graft survival in patients receiving prophylaxis (7). Likewise, the association of early CMV infection and disease with reduced graft survival suggests that CMV does play a role in chronic allograft damage (6). Unrecognized transient inflammation within the allograft mediated by episodes of viremia with more than or equal to 2000 copies/mL may explain the emergence of IF/TA later on (34). Regardless, the time of 3 months posttransplant, when protocol biopsies were performed, is likely to be too short to evaluate an effect of CMV viremia on the development of IF/TA. Long-term trials with protocol biopsy in a later period are warranted to provide a definitive answer.
The advantages of our study include prospective follow-up and monitoring of CMV activity in all patients. However, the limitations of our data should be pointed out. The proportion of CMV D+/R− patients was—just as in the European population—relatively low, and a relevant assessment of the importance of CMV viremia would have required a larger sample size. Furthermore, the study did not assess CMV viremia not developing until the completion of prophylaxis. Although late asymptomatic CMV viremia did not raise the risk of acute rejection, its role in the progression of IF/TA has not been investigated (9). The definition of SCR included borderline changes and not solely findings with grade more than or equal to IA according to the Banff classification. The same definition has been used by a host of authors (29, 30). Considering the substantial interobserver variation in categorizing SCR further into “acute” and “borderline” rejection, exclusion of findings with borderline changes would have been controversial (37). In addition, some data have suggested that not only diffused cortical infiltrates with tubulitis, but also nonspecific infiltrates may be indicative of ongoing allograft damage (31). A subanalysis of our data, when evaluating only grade more than or equal to IA SCR, did not document an effect of CMV viremia (data not shown).
Our findings are important from a clinical point of view. SCR, IF/TA and, most importantly, a combination of both, are associated with deteriorated renal graft function and a reduced graft survival (26–30). Although within a period of 12 months posttransplant, our patients with SCR and IF/TA showed deteriorated renal function with progressive worsening between months 3 and 12, primarily those with SCR. Our study raises the question of optimal cut-off value for CMV viremia to start preemptive therapy whereas 2000 copies/mL used in our patients was adequate to avoid CMV disease, it was associated with increased incidence of IF/TA in protocol biopsy.
In conclusion; CMV viremia within the first 3 months after renal transplantation in patients treated by CMV prophylaxis or managed by preemptive therapy is not associated with an increased incidence of SCR; however, a subgroup of patients with viremia of more than or equal to 2000 copies/mL is at increased risk of IF/TA in protocol biopsy at 3 months. Further studies are warranted in patients at risk of primary CMV infection and to assess the effect of late CMV viremia. Protocol biopsy at a later posttransplant (>6–12 months) period would allow better assessment of the associations with IF/TA or chronic vascular changes.
The authors thank Gabriela Kucerova and Eliska Cagankova for their assistance in data collection.
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