The study cohort (192 LTR) provided 1749 BAL specimens for analysis of CMV viral load and 1536 transbronchial biopsies for analysis of CMV pneumonitis and acute cellular rejection. The mean number of biopsies (8 vs. 8) and BAL specimens (9 vs. 9) collected from LTR with and without BOS was not significantly different. Patients diagnosed with BOS underwent significantly fewer pulmonary function tests than those without (18 [IQR: 11–30] vs. 24 [IQR: 15–38], p = 0.003). The median follow-up for the study cohort was 1015 days (IQR: 557–1702).
Incidence and time to CMV replication
During the study, 78/192 LTR (41%) had CMV DNA demonstrated in the BAL in the first 12 months following transplantation, with 44 LTR (23%) developing repeated episodes. CMV pneumonitis, defined histologically by the presence of CMV inclusions was diagnosed in 14 patients (8%). Median time to detection of CMV DNA in BAL was 270 days, with most replication detected (71/78 LTR) following the cessation of antiviral prophylaxis (median 133 days [IQR: 82–218 days]). Median viral load (in those positive) was 9410 copies/mL (IQR: 2700–77 300) (Table 2). Though CMV replication in the BAL was not observed in the low-risk LTR, it was more common in the high-risk LTR compared to the medium-risk LTR (61% vs. 46%, respectively; p = < 0.0001). There was no significant difference in time from cessation of antiviral prophylaxis to CMV detection (median 150 [IQR: 97–233] vs. 115 [IQR: 60–204] days posttransplant; p = 0.2), nor in the magnitude of CMV replication within the lung allograft (median 20 200 [IQR: 2830–73600] copies/mL vs. 6942 [IQR: 2405–77350] copies/mL; p = 0.47) (Table 3).
Table 2. Outcomes of study cohort
|Characteristic||n = 192 (%)|
|BOS patients (%)|| 73||(38)|
|Median time to BOS (days)||574|| |
|Acute rejection ≥ grade 2|| 89||(46)|
|Cytomegalovirus detected in BAL|
|At least one episode|| 78||(41)|
|Recurrent episodes|| 44||(23)|
|Time postcessation of prophylaxis, days||133||(IQR: 82–218)|
|Median viral load, copies/mL||9410||(IQR: 2700–77 300)|
|CMV pneumonitis, patients (%)|| 14||(8)|
|Death, patients (%)|| 70||(36)|
|Median time to death (days)||787||(IQR: 565–1159)|
|Follow-up, days||1029||(IQR: 557–1702)|
Table 3. Outcomes of study cohort by CMV serostatus
|Characteristic||Low risk n = 36||Medium risk n = 112||High risk n = 44||p|
|BAL CMV, patients (%)|
|At least one episode||0||51 (46)||27 (61)||<0.001*|
|Median time postcessation of prophylaxis (days) (IQR)||0||150||115||0.2*|
| ||(97–233)||(60–204)|| |
|Median viral load||0||6942||20200||0.47*|
| ||(2405–77350)||(2830–73600)|| |
|CMV pneumonitis, patients (%)||0||7 (6)||7 (16)||0.05*|
|Acute rejection ≥ A2, patients (%)||11 (31)||54 (48)||24 (55)||<0.001§|
|Bronchiolitis obliterans syndrome||7 (20)||53 (47)||13 (30)||0.005*|
There was no difference in the incidence of CMV replication detection between patients who did and did not receive basiliximab induction (39% vs. 41%, p = 0.88).
While CMV was not routinely tested for in the blood, 61% of episodes of CMV detected in BAL had a paired blood sample analyzed for CMV DNA. Of note of those concurrently analyzed for CMV DNA in BAL and blood, in 76% CMV replication was only detected in BAL.
CMV replication within the lung allograft as a predictor of BOS
At the end of the study 73/192 LTR fulfilled the diagnostic criteria for BOS. Median time to BOS was 574 days (IQR: 401–973). Seventy patients were deceased (median time to death: 787 days [IQR: 565–1159]). The incidence of BOS was significantly lower in low-risk (as defined by donor-recipient CMV serology) LTR compared to medium- and high-risk (19% vs. 47% vs. 30%; p = 0.005).
To characterize the relationship between CMV detection in the BAL and development of BOS, CMV detection was considered in two separate models. CMV detection in BAL was initially considered as a time-independent variable occurring at any point in the first 12 months following transplant. This was significantly associated with the development of BOS (HR 1.8 [1.1–2.8], p = 0.02). Secondly, CMV detected in BAL was considered more accurately as a time-dependent variable occurring at any time posttransplantation but prior to the attainment of BOS. This was significantly associated with an increased risk of BOS (HR 2.1 [1.3–3.3], p = 0.003) (Figure 1). Details of the univariate analysis are included as a supplement. After multivariable adjustment for other significant covariates (acute rejection score, CMV serostatus, transplant type and COPD), CMV detection in BAL remained a significant predictor of BOS (HR 1.9 [1.2–3.1], p = 0.007) (Table 5).
Table 5. Univariate and multivariate analysis development of BOS
|Time to BOS CMV variable type||Univariate model||Multivariate model1|
|HR (95% CI)||p||HR (95% CI)||p|
|CMV in first 12 months||1.8 (1.1–2.8)||0.02||1.5 (1.0–2.4)||0.08|
|CMV prior to BOS (time dependent)||2.1 (1.3–3.3)|| 0.003||1.9 (1.2–3.1)|| 0.007|
In light of the evolution of CMV prophylactic strategies and diagnostics we explored the association between CMV detection in BAL as measured by real-time quantitative PCR and the development of BOS in a large cohort of LTR, all of whom received 3–5 months of antiviral prophylaxis. The analysis showed that the detection of CMV DNA in the BAL was associated with the development of BOS irrespective of the magnitude of viral replication, the presence of tissue invasive disease (CMV-P) or whether viral replication was symptomatic or asymptomatic.
Antiviral prophylaxis is largely effective in controlling CMV replication. CMV detection predominantly (90%) occurred following the cessation of ganciclovir or valganciclovir prophylaxis, a pattern that is well recognized (12,13). Compared to 3 months of oral ganciclovir prophylaxis, the use of oral 5 months of valganciclovir was associated with a reduced peak of CMV replication, as well as a reduced incidence of acute rejection. This association between augmented antiviral prophylaxis and reduced cellular rejection has been previously demonstrated in renal transplant recipients (14) and also identified in lung transplant recipients (15,16).
Despite a number of studies examining this, the link between CMV and BOS has not been uniformly observed (17–19). This inconsistency is potentially attributable to differences in LTR cohort sampling and follow-up, varying definitions of CMV infection and BOS and changes in viral prophylaxis regimens over time. There is however, an emerging consensus that CMV disease is associated with the development of BOS and mortality following lung transplantation (20–23). A recent analysis by Snyder et al. reported that treated CMV-P was a risk factor for the development of BOS and reduced survival following lung transplantation (22). The rate of CMV-P was significantly higher in their cohort compared to ours (19% vs. 8%); an observation that maybe related to the shorter courses of antiviral prophylaxis that the majority of Snyder cohort received (4 weeks of intravenous ganciclovir for medium risk serostatus and 14 weeks for high risk serostatus). In our own center prolonged antiviral prophylaxis has reduced the incidence of CMV-P from over 30% to less than 10% (8). The Snyder analysis adds weight to the studies that have reported a decrease in the incidence of BOS with the use of CMV prophylaxis (12,16,21) and support our findings, suggesting that CMV replication within the allograft may result in subsequent chronic allograft dysfunction.
In contrast to our results, a recent study by Manuel et al. (25) reported no increased risk of BOS in a cohort of patients with beta herpesvirus (CMV, HHV6 and HHV7) replication within the lung allograft. The discordant findings between the two studies may be related to differences in the two study populations. The Manuel study cohort, in contrast to our own, included a smaller number of patients (93 vs. 192), providing fewer BAL samples (581 vs. 1756), with reduced follow-up (777 vs. 1015 days). Additionally, their patients received intensive induction immunosuppression (Campath) and overall had a lower incidence of BOS at study completion.
Though our current study does not explore how CMV may lead to BOS, a recent paper by Weight et al. does provide some mechanistic insights. They demonstrated that episodes of CMV pneumonitis and infection lead to upregulation of the chemokines, CCL2 and CCL5, perpetuating inflammation and potentiating the development of allograft dysfunction (24). The low incidence of acute rejection and BOS seen in the absence of CMV replication in the “low-risk” (D−/R−) group would add weight to this argument and suggests that CMV replication has indirect effects on graft function that extend beyond those associated with direct graft infection.
The strengths of our study include a well-characterized cohort of consecutive LTR that received standardized management, surveillance bronchoscopy and follow-up at a single institution with an extended mean follow-up of 2.9 years, allowing adequate time for BOS development. Each patient had the presence of CMV in BAL prospectively analyzed and this was compared to the development of BOS strictly graded according to the ISHLT guidelines allowing for a precise objective determination of outcome. Our statistical analysis additionally recognized and adjusted for the time-dependent nature of CMV DNA replication in the BAL as a risk factor for the outcome of BOS and when considered and adjusted for other BOS risk factors in our final multivariate model, the effect of CMV persisted.
Limitations of the study include recognition that while the sample is representative of a large cohort of LTR we were not able to consider all the factors that could potentially influence BOS development. We were unable to fully consider the impact of respiratory viral infections and nonpulmonary CMV as no serial testing was conducted but rather the tests were completed as clinically indicated. Additionally, as it has not been our practice to repeat bronchoscopy unless clinically indicated, we are unable to assess viral clearance following treatment and as such cannot categorically rule out persistent infection.
Antiviral drugs adequately suppress CMV replication within the lung allograft during the period of prophylaxis. However, patients remain vulnerable to both clinical and subclinical CMV infection on their cessation. A recent randomized control trial by Palmer et al. showed that extending antiviral prophylaxis with valganciclovir decreased the incidence of CMV disease and infection (15) changing the course of late onset disease. This adds high level evidence in support of groups that have previously suggested that longer courses of antiviral prophylaxis are beneficial (16,26,27). Given that clinically apparent CMV antiviral drug resistance was not a significant issue in our patient group (and was also not evident in the Palmer cohort), we have moved to prolonged antiviral prophylaxis (beyond 5 months); a duration of therapy in keeping with that suggested in a recent position paper on the management of CMV in solid organ transplant recipients (28). Further studies will inform us whether this strategy will limit both the direct and indirect sequelae of CMV replication, namely further reduce the incidence of CMV replication, acute rejection and importantly BOS, the major factor limiting successful clinical outcomes following lung transplantation.
This analysis suggests the direct quantitative assessment of CMV replication by PCR within the allograft offers diagnostic and prognostic information beyond that offered by histological detection of CMV inclusions and provides new and essential information in the management of CMV replication following lung transplantation. Despite the evolution of and improvement in antiviral prophylactic strategies, our study demonstrates that CMV replication remains common following short course antiviral prophylaxis and is associated with BOS. These findings mandate us to further improve how we control CMV, the ubiquitous herpesvirus that, perhaps surprisingly, continues to influence the enduring success of lung transplantation.