PATIENT COHORT AND BASELINE CHARACTERISTICS
The study cohort (n=94) had 39 females (41.5%) and 55 (58.5%) males, with a mean age of 6.5 +/− 5.5 years (0.06 –18.3 yrs) at the time of transplantation. The mean follow up period from the time of transplantation to the completion of the study was 4.5 years (0.16 – 16.22 years) for a total of 420 patient years. Congenital heart disease (34/94, 36%) was the commonest indication for cardiac transplantation, followed by dilated cardiomyopathy (33/94, 35%), restrictive cardiomyopathy (13/94, 14%) and cardiac re-transplantation (12/94, 13%). All children were initially treated with a standard triple-drug immunosuppressive regimen consisting of cyclosporine or tacrolimus, prednisone, and azathioprine or mycophenolate mofetil. No induction therapy was used. The antiproliferative agent (azathioprine or mycophenolate) was discontinued in 41% of the patients, at an average of 1.5 years after transplantation, due to persistent leukopenia. The patients were then maintained on dual therapy with steroids and calcineurin inhibitors. The baseline characteristics of the viral PCR-positive and viral PCR-negative groups are shown in . Differences between the two groups included; the PCR-positive group had a higher mean recipient age (p=0.007) and weight (p=0.002) at transplant, higher mean donor age (p=0.004), higher number of retransplants (p=0.02), more patients with viral genome detected in the explanted heart (p=0.03) and more patients on LVAD/ECMO at transplant (p=0.05). There was no difference in the pattern of immunosuppression between the PCR+ and PCR− groups. The PCR+ and PCR− groups were similar with respect to the initial `triple-drug therapy' combination. There was no statistically significant difference in the two groups with respect to the percentage of patients in whom azathioprine or mycophenolate was discontinued and immunosuppression transitioned to a combination of prednisone and a calcineurin inhibitor alone. No adjustments in immunosuppression were made if virus was detected in the myocardium.
Baseline demographics and covariates in the PCR-positive and PCR-negative groups
Of the 94 patients in the study cohort, 24 underwent transplantation before the onset of the study in June 1999. Of these, 19 remained PCR-negative and 5 became PCR-positive during the course of the study. At their entry into the study, the mean time from transplantation was 5.19 years in the 19 PCR-negative patients and 3.84 years in the 5 PCR-positive patients. The average follow up period after transplantation was 4.99 years in the PCR-negative patients and 3.64 years in the PCR-positive patients.
VIRAL PCR RESULTS OF ENDOMYOCARDIAL BIOPSIES
PCR was performed on 928 serial EMBs from the 94 study patients. Viral genome was amplified from the myocardium of 39% (37/94) patients and 8.9% (83/928) of the biopsies. Parvovirus B19 genome was amplified most commonly and was found in 71.1% of all positive biopsies, from 24.5% (23/94) patients (62.1% of PCR-positive patients). Adenoviral genome was detected in 9 (9.6%) patients (24.3% of PCR-positive patients), EBV in 8 (8.5%) patients (21.6% of PCR-positive patients), CMV in 4 (4.3%) patients and enterovirus in 1 (1%) patient (). Eight patients (8.5%) had more than one virus type amplified, either in the same (3 cases) or follow-up EMBEMB (5 cases). Viral genome persisted for > 6 months in 40% (15/37) of the virus-positive patients; 14 of these patients were parvovirus B19 positive and 1 was EBV positive. Majority of patients with parvovirus-positive EMBs showed evidence of chronic persistence of viral genome with 61 % (14/23) positive for parvovirus > six months after the initial detection and in the subgroup where a follow up EMB was available more than a year after the initial parvovirus-positive EMB, 88% (14/16) showed persistence or recurrence of parvoviral genome > one year after the initial detection. A change in the infecting virus was noted over the study period () with increasing incidence of parvovirus B19 from 0% of the biopsies in 1999 to 18.6% in 2004 (p < 0.001, Fisher's exact) and decreasing incidence of adenovirus from 7.5% of the biopsies in 1999 to 0 % in 2004 (p < 0.001, Fisher's exact). Most of the viral endomyocardial infections were clinically silent and detected on routine surveillance biopsies. Only one patient with parvovirus positive biopsy developed aplastic anemia in our patient cohort.
Prevalence and outcome of viral endomyocardial infection in a pediatric cardiac transplant cohort
RISK FOR VIRAL ENDOMYOCARDIAL INFECTION
Patients were most prone to develop viral endomyocardial infection in the first year after cardiac transplantation; 59% (22/37) of the viral PCR-positive patients had their first viral PCR-positive EMB within the first year after transplantation. To get a more accurate estimation of how the time period from transplantation affected the risk for viral endomyocardial infection, we performed a Kaplan-Meier analysis of the `freedom from viral endomyocardial infection after transplantation' on the 70 patients (transplanted after 6/1/1999) on whom viral PCR status of all the EMBs performed after transplantation was known. The cumulative probability of developing viral endomyocardial infection was 35% (95% CI, 26%–48%) by the end of first year after transplantation and 63% (95% CI, 48%–78%) by the end of fifth year after transplantation ().
LONG-TERM GRAFT SURVIVAL
Graft survival was decreased (p<0.001, logrank) in the PCR-positive group compared to the PCR-negative group. The risk for graft loss in the PCR-positive group was 4.2 (p= 0.015, 95% CI 1.33–13.29) times that of the PCR-negative group, after adjusting for recipient age, recipient weight, recipient gender, donor age, retransplantation and acute rejection episodes, using Cox regression analysis. The median graft survival was 4.8 years in the PCR-positive group () compared to 12.4 years in the whole cohort. The median graft survival time in the PCR-negative patients could not be estimated due to lack of enough graft failures during the study period. Eleven (35.5%) of the PCR-positive patients and 11 (19.3%) of the PCR-negative patients lost their grafts (death or retransplantation) during the study period. The causes of graft loss are listed in Appendix 3
. After excluding patients with non allograft specific causes of mortality (malignancy, aplastic anemia and pulmonary vein stenosis), the PCR+ group still had a higher risk for premature graft loss (p=0.01, logrank). Persistence of viral genome in EMBs for longer than 6 months did not further increase the risk of graft loss.
ADVANCED TRANSPLANT CORONARY ARTERY DISEASE
Data on coronary arteries were not available in 7 of the 94 study patients, as they were within the first year after transplantation and had not undergone the first annual screening coronary angiography by the end of the current study. Of the remaining 87 patients on whom coronary angiography or coronary histopathology results were available, 33 (38%) were PCR-positive and 54 (62%) were PCR-negative. One patient developed advanced TCAD before detection of viral endomyocardial infection and was considered as PCR-negative for purposes of this analysis. Average follow up time after transplantation was 5.15 years in the PCR-negative patients compared to 3.87 years in the PCR-positive patients. Fourteen patients (16%) developed advanced TCAD; 7 (21.2%) of the PCR-positive and 7 (12.9%) of the PCR-negative patients. PCR-positive patients developed advanced TCAD prematurely compared to the PCR-negative group (p=0.001, logrank) and had a 8.3 times higher risk for developing advanced TCAD (p=0.001, 95% CI, 2.48–28.02) (). After adjusting for time from transplantation at entry into the study, being PCR-positive remained a risk factor for premature development of advanced TCAD (p= 0.002, logrank). The risk for developing premature advanced TCAD in the PCR-positive group was 6.8 (p=0.01, 95% CI 1.47–32.06) times that of the PCR-negative group, after adjusting for recipient weight, age and gender, donor age, re-transplantation, time from transplantation and acute rejection episodes, using Cox regression analysis. Persistence of viral genome in EMBs for longer than 6 months did not further add to the risk of developing advanced TCAD. The median time to developing advanced TCAD was 4.8 years in the PCR-positive group compared to 12.2 years in the whole cohort. The median time to developing advanced TCAD in the PCR-negative group could not be calculated due to insufficient number of events during the study period.
ACUTE GRAFT REJECTION
A total of 101 rejection episodes occurred in 62 of the 94 patients during the study period. No statistically significant association was found between viral endomyocardial infection and AR. Concomitant rejection was present in 8.3% of the PCR-positive biopsies and 8.6% of the PCR-negative biopsies.
VIRUS SUBTYPE AND OUTCOME
After adjusting for viral subtypes using Cox regression, adenoviral (p=0.03) and EBV (p=0.002) endomyocardial infections were associated with decreased graft survival (). The risk for graft loss was 4.1 (95% CI: 1.13–14.73) times higher in the adenovirus-positive patients and 8.5 (95% CI: 2.18–33.56) times higher in the EBV-positive patients compared to the PCR-negative patients. However, the predicted graft survival for parvovirus endomyocardial infection was similar to that of PCR-negative patients. This was confounded by the fact that the majority (17/23, 74%) of the parvovirus positive patients received IVIG therapy after a PCR-positive biopsy, which could have had a beneficial effect on the outcome. After adjusting for viral subtypes using Cox regression EBV (p=0.03) and Parvovirus (p=0.005) endomyocardial infections were associated with premature development of advanced TCAD. The risk for advanced TCAD was 7.5 (p=0.03, 95% CI: 1.76–45.07, cox) times higher in the EBV-positive patients and 7.1 (p=0.005, 95% CI: 1.78–28.02, cox) times higher in the Parvovirus-positive patients compared to the PCR-negative patients. No statistically significant association was detected between AR and individual virus subtypes in our study cohort.
EFFECT OF IVIG THERAPY ON THE OUTCOME OF PATIENTS WITH PCR-POSITIVE BIOPSIES
Of the 37 patients with viral PCR-positive EMBs, 20 (54%) were treated with IVIG following a PCR-positive biopsy. The remaining 17 (46%) who did not receive IVIG therapy served as treatment-naïve controls. The mean time to IVIG treatment was 16.2 days from the PCR-positive biopsy (0 –136 days). The number of IVIG doses that aa patient received ranged from 1 to 3 doses (mean 1.6, median 1), with 7 patients receiving more than one IVIG dose due to multiple PCR-positive biopsies. The baseline characteristics of the IVIG-treated patients and IVIG-untreated controls are given in . Due to the surge in parvovirus-B19 positive EMBs in 2003 and 2004, the years during which IVIG therapy was administered for PCR-positive viral endomyocardial infection, the IVIG-treated PCR+ group had a disproportionately higher number of parvovirus positive patients compared to the IVIG-untreated PCR+ group. The other viral subtypes were not significantly different in the two groups. Three (15%) of the 20 IVIG-treated patients and 8 (47%) of the 17 IVIG-untreated patients suffered graft loss. Graft survival from the date of transplant (p=0.09, logrank) (), and graft survival after becoming PCR-positive (p=0.06, logrank) () trended to be better in the IVIG-treated group compared to the IVIG-untreated group. The 3 year survival after becoming PCR-positive was 86% (95% CI, 54% – 96%) in the IVIG-treated patients, compared to 33% (95% CI, 8% – 62%) in the IVIG-untreated patients (p=0.03, logrank).
Baseline characteristics of the IVIG-treated and IVIG-untreated viral PCR-positive patients
Kaplan-Meier analysis of the utility of IVIG-therapy in pediatric cardiac transplant patients with viral endomyocardial infection
Freedom from advanced TCAD, measured from the time of transplant, was better in the IVIG-treated patients (p=0.05, logrank) (). The onset of advanced TCAD after becoming PCR-positive was delayed in the IVIG-treated patients compared to the untreated control group (p=0.03, logrank) (). The 3 year freedom from advanced TCAD after the first PCR-positive biopsy was 82% (95% CI, 24% – 97%) in the IVIG-treated patients compared to 45% (95% CI, 11% – 75%) in the IVIG-untreated patients (p=0.03, logrank). IVIG therapy did not affect the number of AR episodes after becoming PCR-positive. In addition, it did not seem to help in the clearance of viral genome. Viral genome was amplified from the follow-up biopsy in 48% of the IVIG-treated biopsies and 37% of the IVIG-untreated biopsies (chi square, NS). A valid comparison of `virus-free time' after a PCR-positive biopsy could not be performed as the follow-up biopsies were obtained at varying time intervals.