Cytomegalovirus (CMV)infection continues to be a major opportunistic pathogen in solid organ transplant recipients (1, 2) Increasingly sensitive techniques now allow for the detection of subclinical infection that may have relevance to long-term graft function (3). Aside from acute CMV disease affecting any organ system, the virus has long been a suspected contributor to acute graft rejection as well as to cardiac allograft vasculopathy (CAV) (4–7). Studies in humans and animal models have shown that inhibition of viral replication by ganciclovir not only prevents acute CMV disease in a wide range of solid organ transplants, but also improves long-term outcomes by inhibiting the development of CAV (5) and bronchiolitis obliterans (8, 9). Prophylaxis of seropositive heart transplant recipients with a 28-day course of ganciclovir dramatically reduces the incidence of acute CMV disease (10) and has become a standard of care at many medical centers (11). However, this regimen was shown to be ineffective in CMV seronegative patients receiving a graft from CMV seropositive donors (R−/D+) who are at risk for primary infection. Longer prophylaxis with ganciclovir alone or combined with hyperimmune globulin (7) has been employed to control acute CMV disease in patients at highest risk of disease.
There is a paucity of data regarding the levels of active virus infection or the role that ganciclovir and/or CMV hyperimmune globulin (CMVIG) prophylaxis play in reducing subclinical viral loads. Furthermore it is unknown whether inhibition of subclinical infection has any impact on allograft outcomes such as acute rejection and CAV. Data from previous studies show that despite a 28-day course of prophylactic ganciclovir, (10) shed virus continues to be readily detected in seropositive transplant recipients. Moreover, virus may reactivate soon after completion of ganciclovir prophylaxis, indicating that viral replication may persist despite the absence of any overt signs or symptoms of acute CMV disease (12). The objective of this study was to compare the effect of standard antiviral prophylaxis employed in seropositive recipients (R+) with a more aggressive protocol administered to higher risk patients (R−/D+), on subclinical CMV infection and on allograft-related effects of CMV infection, such as acute rejection and CAV development during the first year after transplant.
PATIENTS AND METHODS
All consecutive patients undergoing first heart transplantation (HT) between January 2002 and February 2004 at our institution were considered for enrollment in this prospective cohort study. Study flow chart is depicted in Figure 1. Renal insufficiency requiring prolonged dialysis, contraindications to receive anti-CMV prophylaxis, and inability or unwillingness to provide signed informed consent represented exclusion criteria. In our center, patients younger than 18 years who are CMV seropositive before the transplant receive an aggressive regimen of anti-CMV prophylaxis similar to R−/D+ patients. The eight patients with these features were excluded from the final analysis because the combination of younger age and the positive CMV serology could have represented a confounding factor in evaluating the effect of the aggressive regimen. Two patients who died within the initial 30 days of transplant were also excluded from the final analysis. All the remaining 66 patients completed one year of follow up and were included in the final analysis. All patients gave informed consent to the protocol approved by our institutional review board for studies in human subjects. For descriptive purposes, we recorded occurrence of acute rejection and sampled for CMV DNA detection in peripheral blood also D−R− patients.
Patients considered to be at intermediate risk for CMV disease, CMV seropositive recipients (R+), received standard prophylaxis consisting of ganciclovir administered (i.v) at 5 mg/Kg/bid for the first two weeks after transplant, and 6 mg/kg once daily for the subsequent two weeks, for a total duration of 25±4 days (10). Patients considered to be at high risk for CMV disease, CMV seronegative recipients of hearts from seropositive donors (R−/D+), received aggressive prophylaxis consisting of i.v. ganciclovir administered as in the standard prophylaxis protocol, followed by maintenance with valganciclovir (450–900 mg daily, adjusted for renal function) for a total duration of (val)ganciclovir prophylaxis of 73±12 days, and CMVIG administered at 150 mg/kg i.v. 72 hr after transplant, then at 100 mg/kg at week two, four, six, and eight, then at 50 mg/kg at weeks 12 and 16. (Fig. 1).
Medical Treatment and Study Procedures
The immunosuppressive regimen consisted of daclizumab (1 mg/kg given i.v.) administered at the time of transplant surgery and on alternate weeks for a total of five doses; (13) cyclosporine (3–5 mg/kg/day); prednisone initiated at one mg/kg/day, tapered to <0.1 mg/kg/day by the sixth postoperative month; and either mycophenolate mofetil (MMF) 1000–4000 mg daily, or sirolimus 1–4 mg daily (target trough levels of eight to 18 ng/ml). In patients treated with MMF, cyclosporine dose was adjusted to target blood trough levels between 200 and 300 ng/ml within month three after transplant, and 150 to 250 ng/ml thereafter. While in patients treated with sirolimus, target cyclosporine trough levels were 150 to 250 ng/ml during the first three months and 50 to 150 ng/ml thereafter. Use of sirolimus or MMF was equally distributed within the prophylaxis groups. Patients were monitored for acute rejection (AR) by surveillance endomyocardial biopsies performed at scheduled intervals after transplant: weekly during the first month; bi-weekly until month three; monthly until month six; and then at month nine and 12. Biopsies were graded accordingly to ISHLT classification, 1A, 1B, 2, 3A, 3B, and 4 (14). Patients with rejection grade ≥3A were treated with intravenous corticosteroids.
Unless contraindicated, all patients received therapy with statins for CAV prevention (15).
Allograft vascular disease during the first year after transplant was assessed by intravascular ultrasound (IVUS) imaging of the left anterior descending coronary artery at baseline (within 6 weeks following transplant), and at year one. Briefly, ultrasound images were obtained as previously described (16–18) using an automated pullback system. From these images, vessel area, lumen area, and intimal area (plaque) were measured, and an automated volumetric reconstruction of the coronary artery was performed, using the Simpson’s method (18). The change in each of these measurements comparing studies recorded at baseline and at year one for each individual patient was determined. Patients with serum creatinine ≥2.0 mg/dl or unwilling to undergo the procedure were excluded from IVUS evaluation.
Monitoring for CMV Infection: DNA Isolation, Nested PCR, and Real-Time PCR
Systemic CMV infection was monitored by analyzing CMV DNA in peripheral blood polymorphonuclear cells (PMN) using a qualitative, nested polymerase chain reaction (PCR) assay, followed by quantification of the positive samples by real-time PCR, as described in the supplementary methods.
Study Endpoints and Data Analysis
The primary study endpoint was occurrence of CMV infection, defined by the detection of a blood sample positive for CMV DNA by nested PCR (19). Because previous studies have shown that the risk for CMV disease is proportional to the CMV burden as assayed with PCR (20–22) we additionally analyzed the effect of prophylaxis on the incidence of a CMV burden >110 DNA copies/1×105 PMN, i.e. the 75th percentile of the distribution of all the positive samples tested.
Secondary endpoints were the occurrence of acute rejection, and changes in IVUS measurements of CAV. The effect of the two prophylaxis protocols on acute rejection was first evaluated by comparing the biopsy score during specified posttransplant periods (≤Month1, Months 2–3; 4–6, 7–9, and 10–12). Biopsy score was determined as previously described, by assigning a numerical value from zero to six to each rejection grade, and then calculating the mean biopsy score at each of the specified time points (23, 24). Subsequently, we evaluated survival free from moderate rejection, grade ≥3A, by using the Kaplan-Meier method of estimation.
Statistical analysis was performed using SPSS v11.0.2 statistical package. Continuous data are presented as mean values±standard deviations, and categorical variables as percentages. Differences between groups were assessed by Student’s t test, Mann-Whitney, chi-square tests, or log-rank tests as appropriate, with Hochberg corrections for multiple comparisons. Cox’s univariate and multivariate model was used to determine risk factors for events. Variables in the univariate model testing with a P value ≤0.1 were included in the multivariate model.
Finally, changes from baseline to Year one posttransplant in coronary artery lumen area, vessel area, and intimal area (measures of CAV) were compared between groups using Mann-Whitney test. A P value <0.05 was considered statistically significant.
Table 1 shows the baseline demographics including donor and recipient CMV serological status in the two prophylaxis groups. Except for donor/recipient serology and duration of (val)ganciclovir administration, the two groups of patients had similar demographical and clinical baseline characteristics. Note that patients in the aggressive group had a nonsignificant higher prevalence of sirolimus treatment, as compared with standard prophylaxed group.
Prophylaxis and CMV Infection
CMV DNA was detected in 56 out of 66 patients during the study period, with an estimated incidence of 87±4% at month 12. As depicted in Figure 2, patients treated with aggressive prophylaxis had a significantly longer freedom from CMV infection, that occurred less frequently than in those receiving standard prophylaxis (CMV infection estimated incidence: 73±10% vs. 94±4%, respectively; P=0.038). Note that the curves estimating survival free from CMV infection start to diverge after the first month from transplant, when anti-CMV prophylaxis is discontinued in the standard treated group. By multivariate analysis (Table 2), the aggressive prophylaxis regimen was found to be independently associated with a 28% reduction in relative risk for CMV infection (RR [95% CI]=0.72 [0.52 to 0.96]; P=0.02).
Despite CMV DNA titer varied widely across the study population, ranging from five to 4356 DNA copies/105 PMN, the majority of patients (70%) never reached the upper quartile of CMV titer distribution, i.e. 110 CMV DNA copies/105 PMN. However, the proportion of patients with this increased burden of CMV infection was higher among patients treated with standard prophylaxis (n=17, 38%) than in those treated with the aggressive protocol (n=3, 14%, P=0.043). Acute CMV disease, gastritis (1), colitis (1), and CMV syndrome (2), developed in four patients, all of whom were detected with a CMV titer >110 DNA copies/105 PMN, two in each treatment group. Symptoms developed after completion of the prophylaxis and all patients responded to treatment with intravenous ganciclovir.
Prophylaxis and Acute Rejection
Figure 2B depicts the time course of acute rejection analyzed as the mean biopsy score, comparing in aggressive versus standard prophylaxis groups. During the first month, when all the patients received CMV prophylaxis, there was no difference in rejection score comparing the aggressive and standard prophylaxis groups. During months two to six, however, rejection score did not rise in patients treated with the aggressive prophylaxis, remaining significantly lower than in patients treated with the standard protocol during months four to six (P=0.03). Note that during months seven to nine after transplant, (i.e., 4 to 6 months after prophylaxis discontinuation) rejection score in the aggressively treated patients peaked, reaching those who received only one month of anti-CMV prophylaxis. This trend suggests an effect of prolonged prophylaxis in delaying rejection occurrence.
This observation was confirmed when comparing the differences in the survival free from rejection ≥3A, after i.v. ganciclovir discontinuation (Fig. 2C). Patients receiving the prolonged and more aggressive treatment showed a rejection free survival longer than standard treated patients, reaching significance six months after i.v. ganciclovir discontinuation. Consistently with what depicted in Figure 2, occurrence of rejection ≥3A tended to increase to a similar level in both groups of patients by the end of the study follow-up. In Table 3, we report the univariate and multivariate predictors of risk for rejection grade ≥3A occurring after ganciclovir discontinuation. Multivariate analysis highlights the independent and probably synergic effect on the risk for rejection of cold ischemic time and anti CMV prophylaxis. While the aggressive prophylaxis regimen accounted for a 45% independent relative risk reduction for rejection, the risk increased 1.3 times per each increase of 30 min in cold ischemic time.
Along with a reduced and delayed occurrence of acute rejection, at the end of the follow-up patients treated with aggressive prophylaxis tended to have better metabolic profile in terms of a non-significant lower glucose (107±30 vs. 121±53 mg/dl; P=0.29) and lower creatinine (1.44±0.65 vs. 1.86±1.35 mg/dl; P=0.09) serum concentrations, as compared with patients who received standard prophylaxis.
For descriptive purposes we reviewed the occurrence of rejection ≥3A in the D−/R− patients, initially excluded from the study because not receiving any prophylaxis. These patients are at a negligible risk of developing CMV infection, and indeed, we never detected CMV DNA in any sample from these patients. Ten of the 12 transplanted reached the first year of posttransplant follow up, and none of them ever experienced any episode of ≥3A rejection.
Prophylaxis and Cardiac Allograft Vascular Disease
Of the 66 patients enrolled in the study, paired baseline and month 12 follow-up IVUS studies were available in 28 (42%). In the overall patient population, a significant increase in intimal volume (from119±82 to 139±95 mm3, P=0.02) was accompanied by decreases in both vessel volume (from 734±219 to 639±180 mm3, P<0.01) and lumen volume (from 615±182 to 500±148 mm3, P<0.01). This is consistent with vessel shrinkage and negative remodeling, as previously described (25, 26). However, negative remodeling was not evenly distributed across the two patients groups. We observed that despite similar increases in intimal volume over time (14.3±43.5 vs. 33.1±49.6 mm3; P=0.3), vessel and lumen volumes significantly decreased only in patients treated with standard prophylaxis (–126±111 and –140±106 mm3 respectively; P<0.01), but not in patients treated with aggressive prophylaxis (–28±122 and –61±96 mm3 respectively; P≥0.1). Similarly, analyzing the absolute change in vessel, lumen and intimal volumes from baseline to follow-up IVUS, we found that patients treated with standard prophylaxis showed a significantly greater reduction in vessel and lumen volumes as compared with those treated with the aggressive protocol (Fig. 3).
To exclude the effect of possible confounders in the association between anti-CMV prophylaxis and CAV development, we assessed the differences of other CAV risk factors between the two groups of patients. We found no significant differences in terms of cold ischemic time, donor age, donor/recipient gender mismatch, and baseline lipid panel between the nine patients treated with the aggressive protocol and the 19 treated with the standard protocol (all P values >0.3). However, we found a trend towards a lower concentration in LDL cholesterol assayed at the end of follow up in the patients treated with the aggressive prophylaxis, as compared to the standard group (82±39 vs. 119±73 mg/dl, respectively, P=0.09).
The salient findings from this study are that aggressive anti-CMV prophylaxis 1) delays CMV infection onset and lessens CMV viral burden, 2) delays the onset and partially reduces the incidence of acute rejection, and 3) attenuates cardiac allograft vasculopathy. Notably, the group of patients conventionally considered to a greatest risk for acute CMV disease, R−/D+, have lower rates of subclinical infection compared to R+ patients, presumably due to the aggressive antiviral therapy they receive.
Results of clinical trials of ganciclovir have shown that although shedding in urine is reduced and delayed, shed virus continues to be readily detected in transplant recipients despite a 28-day course of therapy (10). Studies in which valganciclovir or oral ganciclovir have been administered for up to three months have demonstrated significant protection from acute CMV disease, but few have examined the impact of reactivation of virus following completion of prophylaxis (12). Our observations indicate that aggressive long-term antiviral prophylaxis is effective in inhibiting activation of virus in high risk, R−/D+ patients, rendering them less susceptible to active virus infection than the R+ patients who received ganciclovir for only four weeks. In addition, R−D− patients, who did not have a predetermined risk for CMV infection, and thus did not receive any antiviral prophylaxis, remained always free from both CMV infection and acute rejection. Conversely, short-term prophylaxis in R+ patients leaves them at greater risk for subclinical CMV infection, as compared to D+R−, and this in turn is associated with increased rates of acute rejection and faster CAV progression. These observations provide further evidence in support of a pathophysiological role for CMV infection in acute rejection and CAV, underscoring the importance of effective inhibition of asymptomatic CMV infection, distinct from CMV disease prevention.
The lower incidence of acute rejection in the patients receiving aggressive CMV prophylaxis, coincident with a lower incidence of CMV infection, is consistent with an extensive body of clinical and preclinical data implicating CMV infection in the pathophysiology of acute rejection and graft loss (27, 28). However, most studies have correlated acute rejection with CMV disease, and none have addressed the question of whether subclinical infection increases the risk for acute rejection. Some investigators have suggested that activation of the virus occurs as consequence of the augmented immunosuppression used to treat acute rejection, rather than a direct effect of the virus in stimulating host immune responses directed against alloantigens of the graft (28). In kidney transplant patients recipients, CMV disease was found to have an independent effect on graft loss that was additive with acute rejection, (27) raising the question of whether persistence of low levels of CMV viremia over time could contribute to acute rejection and graft loss. During the first month after transplant, when both groups of patients were receiving intravenous ganciclovir, acute rejection rates were identical, paralleling similar viral DNA levels (Figs. 2 and 3). Although low rejection rates during this early time period may be explained by the immunosuppressive drugs administered to both groups of patients, subsequent higher rejection rates in the standard prophylaxis group cannot be accounted for by differences in immunosuppressive drugs. Rather, a protective effect of aggressive CMV prophylaxis is supported by the results of the multivariate analysis indicating this as an independent predictor of reduced risk for rejection ≥3A. Our observations are consistent with clinical trials in which antiviral efficacy of prophylaxis regimens frequently demonstrate concomitant reduction in acute rejection rates (12, 29, 30). A number of mechanisms have been proposed to explain increased acute allograft rejection occurring as a consequence of subclinical CMV infection. These include viral upregulation of adhesion molecules and of MHC class II molecules on multiple cell types in the graft, through increased cytokine expression, particularly γ-interferon (31). CMV-infected endothelial cells demonstrate enhanced expression of several cell surface molecules that are involved in the adhesion of leukocytes, such as ICAM-1, VCAM-1, VAP-1, and E-selectin (32, 33). These effects of CMV are of direct relevance not only to acute rejection but also to allograft vascular disease, which is the major determinant of patient and graft survival after heart transplantation.
In the current study, we found that patients receiving aggressive CMV prophylaxis developed less graft vascular disease than patients on standard prophylaxis. This is relevant to solid organ transplantation in general because it is apparent that despite the advances in immunosuppressive regimens that have reduced acute rejection rates, there has been little impact on the rates of chronic graft loss (34). Pathological change that are common to chronic allografts diseases include vascular disease and fibrosis, (35) and CMV infection has been implicated in the pathophysiology of these abnormalities. We have previously reported that prophylactic administration of ganciclovir during the initial 28 days after heart transplant is associated with decreased incidence of CAV compared to placebo. Despite a decrease in CAV associated with this short-term prophylaxis, the incidence of CAV remains unacceptably high, raising the possibility that ongoing subclinical infection may leave patients at risk for virally mediated vascular injury. The results of the current prospective study document those patients receiving longer and more aggressive prophylaxis experience significantly less coronary artery shrinkage. Notably, this protection from negative remodeling occurred in parallel with lower rates of subclinical infection. These results confirm a previous study where, in a setting of anti-CMV preemptive strategy, we found that patients with infectious burden needing therapy developed negative coronary remodeling (26). Whereas these findings cannot ascribe a causal role for CMV infection in coronary artery shrinkage, they do provide evidence in support of a role for the virus in the pathophysiology of CAV, suggesting that all heart patients should receive therapeutic strategies aiming to reduce as much as possible viral replication, such as more aggressive and prolonged prophylaxis.
The design of the current study does not permit the benefit of aggressive prophylaxis to be assigned to longer administration of (val)ganciclovir, CMVIG or their combination (5). In addition, we cannot exclude that the documented anti-inflammatory effect of CMVIG (36) may have influenced acute rejection occurrence and CAV development independently from the anti-CMV effect. However, others and we clearly documented that agents with anti-inflammatory properties decrease CMV infectious ability (37, 38). Thus, we may speculate that therapeutic interventions driven to reduce inflammatory activation may improve clinical outcome by reducing CMV activity, and then acute rejection and CAV development.
This is a prospective longitudinal cohort study, not a randomized clinical trial, and therefore bias due to nonrandom assignment to treatment groups cannot be excluded. However, this study provides compelling support for a randomized clinical trial to definitively address the role of subclinical CMV in acute rejection and cardiac allograft vasculopathy.
This study provides evidence that patients receiving aggressive prophylaxis consisting of a longer course of ganciclovir together with CMVIG experience lower rates of subclinical infection, acute rejection and CAV. These observations occur in parallel with lower rates of viremia, suggesting that amelioration of ongoing subclinical infection may contribute to improved outcomes after heart transplantation. These observations support the body of data that suggest a pathophysiological role for CMV in acute rejection and CAV, distinct from the direct effects of the virus in causing acute CMV disease.
The authors are grateful to Eric Wong, Roxanne Daniels, and Daniel Chang for their precious help in collecting data and processing samples.
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