Abstract

METHODS

RESULTS

Cohort

A total of 250 consecutive patients who received a first lung transplant at UWMC between January 2007 and May 2012 were included. Table ​Table22 shows patient characteristics. There were no significant differences in demographics between patients with RVI and those without (data not shown). CLAD was diagnosed at a median time of 95 weeks (interquartile range [IQR], 53–157 weeks) post-transplantation. Supplementary Figure 1 shows the Kaplan–Meier curve for overall survival. Overall survival of the cohort was at or above that reported among lung transplantation centers in the United States. During the follow-up period, 85 patients (34%) died. One-year survival was 88%, and median time to death was 89 weeks (IQR, 22–144 weeks). At last follow up, mortality was higher for patients with CLAD: 22 (44%) vs 63 (31.5%) without CLAD. Figure ​Figure11 shows the Kaplan–Meier curve of the development of CLAD and of BOS and R-CLAD separately. The definition of CLAD requires documentation of a decrease in FEV1 of at least 20% for at least 3 weeks. However, to confirm that the functional decline was permanent, we also assessed later time points. Among the 50 patients diagnosed with CLAD, 31 (62%) had pulmonary function test (PFT) information for at least 6 months following CLAD diagnosis. In all of these patients, the drop in FEV1 values was sustained and continued to meet criteria for CLAD. Similarly, PFT values for those patients with <6 months of follow-up from CLAD diagnosis also had persistent spirometry findings consistent with CLAD.

Table 2.

Characteristics of Lung Transplant Recipients

Characteristic	All (n = 250) n (%)
Age, median (range), y	57 (18–71)
Female	106 (42.4)
Underlying diseasea
Idiopathic pulmonary fibrosis	78 (31.2)
Chronic obstructive pulmonary disease	67 (26.8)
Cystic fibrosis	52 (20.8)
Alpha-1-antitrypsin	12 (4.8)
Bronchiectasis	6 (2.4)
Cytomegalovirus Serostatus
R+	132 (52.8)
D+/R−	87 (34.8)
D−/R−	25 (10)
Unknown	6 (2.4)
Type of Transplantation
Bilateral	207 (82.8)
Year of Transplantation
2007–2009	139 (55.6)
2010–2012	111 (44.4)
Duration of Follow-up
Median (range), weeks	143 (0.3–338)
Developed chronic lung allograft dysfunction	50 (20.0)
Bronchiolitis obliterans syndromeb	33 (66.0)
Restrictive chronic lung allograft dysfunctionb	17 (34.0)
Died during study	85 (34.0)

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^a Frequencies are shown for the 5 most common diseases in the cohort. All other diseases appeared in 5 or fewer patients.

^b Percentage is out of total number of chronic lung allograft dysfunction patients.

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Figure 1.

Kaplan–Meier curve showing development of all chronic lung allograft dysfunction (CLAD), bronchiolitis obliterans syndrome (BOS), and restrictive CLAD (R-CLAD).

Respiratory Tract Infection

A total of 114 viral episodes were diagnosed in 79 patients (31.6%). Table ​Table33 details the characteristics of the RVI episodes. Median time to first RVI was 19 weeks (IQR, 9–63 weeks). Nine viral episodes had more than 1 virus isolated, 8 had 2 viruses (all had rhinovirus as 1 of the viruses), and 1 had 3 viruses for a total of 124 viruses isolated. Figure ​Figure22 is a Kaplan–Meier curve showing the time to development of first RVI following transplantation. Among the 50 patients with CLAD, 21 (42%) had 1 or more RVIs preceding CLAD diagnosis compared with 56 (28%) patients who remained CLAD-free during follow-up.

Table 3.

Details of Respiratory Virus Infection

Respiratory Virus Infection Characteristic	n (%)
Unique patients with respiratory virus infection	79
Respiratory viral episodes	114
Lower tract infectiona	97 (85.1)
Viral episodes per patient
1	50
2	25
≥3b	4
Respiratory virusc
Rhinovirus	42 (33.9)
Parainfluenza 1–4	21 (16.9)
Coronavirus	20 (16.1)
Influenza A, B	16 (12.9)
Adenovirus	10 (8.1)
Respiratory syncytial virus	10 (8.1)
Metapneumovirus	4 (3.2)
Bocavirus	1 (0.8)
Viral episodes with >1 virusa	9 (8.7)
Diagnosed by polymerase chain reaction	107 (93.9)d

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^a Percent of viral episodes (114).

^b Three patients had 3 episodes, and 1 had 5 episodes.

^c Percent of viruses isolated (124).

^d Other 7 viral episodes diagnosed by fluorescent antibody, culture, or both.

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Figure 2.

Kaplan–Meier curve of the probability of respiratory virus infection following transplantation.

Association of RVI With Outcomes

We analyzed the relationship between several factors and CLAD in both univariate and multivariate analyses in a time-dependent manner, and modeled periods of risk of 3 months, 6 months, 12 months, and “ever” (ie, at last follow-up) for RVI. In both univariate and multivariate analysis, RVI at all modeled risk periods was independently associated with development of CLAD (Table ​(Table4).4). The association was stronger at shorter modeled periods of risk. We confirmed the previously reported association of acute rejection with development of CLAD (Table ​(Table4).4). Our secondary endpoint, the composite endpoint of CLAD and death, showed a similar trend as the primary endpoint in both univariate and multivariate analysis, with a higher hazard ratio (HR) of RVI at 3 months and progressively declining HR as time from RVI increased (HR, 4.85; 95% confidence interval [CI], 2.76–8.43; P < .01 vs HR, 2.75; 95% CI, 1.72–4.37; P < .01 at 3 months vs 12 months post-RVI, respectively). We also analyzed the association between gastroesophageal reflux disease (GERD) and CLAD using 2 methods, first requiring a positive pH probe test for GERD diagnosis and second including anyone with a clinical diagnosis of GERD. No statistically significant association was seen in univariate analysis (data not shown) with either definition of GERD; therefore, GERD was not included in the final models. We were also interested in whether 1 CLAD phenotype was more strongly associated with RVI than the other (R-CLAD vs BOS). While the numbers are small, precluding a formal analysis, overall, a higher percentage of R-CLAD patients had at least 1 preceding viral episode as compared with BOS (9/17 [52.9%] vs 12/33 [36.4%], respectively).

Table 4.

Univariate and Multivariate Analyses of the Development of Chronic Lung Allograft Dysfunction Modeled for Differing Risk Periods for Respiratory Virus Infection

Variable	HR (95% CI)	P Value	Adjusted HR (95% CI)	P Value
Respiratory virus infection (“ever”)
Age	1.01 (.99–1.03)	.22	1.01 (.99–1.03)	.27
Bilateral transplant	0.87 (.39–1.93)	.73	1.15 (.49–2.71)	.74
Rejection	2.33 (1.30–4.18)	<.01	2.16 (1.18–3.93)	.01
RVI	2.14 (1.20–3.82)	.01	1.92 (1.07–3.45)	.03
Cytomegalovirus pneumonia	1.44 (.74–2.80)	.28	1.16 (.59–2.29)	.67
Risk period following RVIa
3 mo	5.36 (2.20–13.04)	<.01	4.77 (1.91–11.64)	<.01
6 mo	3.75 (1.69–8.29)	<.01	3.37 (1.50–7.54)	<.01
12 mo	2.70 (1.34–5.41)	<.01	2.44 (1.20–4.96)	.02

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Abbreviations: CI, confidence interval; HR, hazard ratio; RVI, respiratory virus infection.

^a HR adjusted for age, bilateral vs single transplant, rejection, and cytomegalovirus pneumonia in multivariate analysis. However, HRs were similar to those for the no-risk period, so data not shown.

DISCUSSION

In a large contemporary cohort of LTRs who underwent symptom-guided testing for RVI using molecular diagnostic methods, we found high rates of RVI and an independent association between symptomatic RVI and CLAD. The association between RVI and CLAD was stronger with shorter modeled periods of risk but was also present when all follow-up time was considered. We demonstrate for the first time that the strength of the association of symptomatic RVI with CLAD is significantly influenced by time (eg, the proximity of the RVI episode with CLAD diagnosis). The stronger association in the first several months after RVI has important implications for future trials of antiviral agents, including powering trials for an endpoint of CLAD. This study also addresses several important limitations of prior studies, significantly extends our understanding of the role of RVI and CLAD, and provides a strong rationale for the conduct of large prospective studies to characterize the specific virologic determinants and mechanistic links between RVI and CLAD.

Several prior studies have assessed the association between RVI and BOS (Table ​(Table1).1). Magnusson et al found an association between BOS and RVI (odds ratio, 3.0; 95% CI, 1.3–6.8; P = .008) in a retrospective cohort study [6]. However, they only considered LRTIs based largely on surveillance BALs and their cohort was small (38 LTRs, 14 with RVIs). While they followed the LTRs for 10 years for outcomes, RVI episodes were only recorded for the first year post-transplantation. Khalifah et al also reported an association between RVI and BOS in univariate and multivariate analyses in a retrospective cohort study [2], but they assessed only 4 viruses using nonmolecular methods, did not distinguish BOS and R-CLAD or use blinded reviewer assessment of CLAD, and included relatively few cases of RVI [22]. Billings et al found no relationship between RVI and CLAD overall, but a subanalysis demonstrated an association with high-grade BOS only for LRTI and had many of the same limitations as that of Khalifah et al [9]. In contrast to the above studies, Milstone et al reported no association; however, the sample size was small (only 4 BOS cases, 17 RVIs) [8]. Thus, these prior studies were relatively heterogeneous and had 1 or more significant limitations that might explain discrepancies with the present study.

Our study had important strengths. We examined a large cohort of patients (among the largest to assess this issue) and diagnosed RVI using modern molecular diagnostic techniques that detected a large number of respiratory viruses. We used time-dependent multivariate analyses that controlled for factors associated previously with CLAD and also modeled differing periods of risk to provide insight into mechanisms the underlie the relationship between RVI and CLAD. Importantly, the primary endpoint (CLAD) was assigned using recently developed consensus definitions and by 2 blinded transplant pulmonologists. We also acknowledge certain limitations, including the retrospective single-center study design. Despite comprehensive medical records and close follow-up, we cannot exclude missing data. Thus, our rates of RVI should be considered minimal estimates because patients may have been diagnosed outside our medical center. We were not able to assess all the factors that have been linked to CLAD, including pseudomonas pneumonia and/or colonization [22–24] and small conidial forms of Aspergillus [22]. However, there is no consensus that these are proven risk factors for CLAD. Our study size was not large enough to permit assessment of risk of CLAD associated with specific viral organisms, nor was it large enough to assess differences between the 2 CLAD phenotypes, BOS and R-CLAD.

Although we found an independent association of symptomatic RVI with CLAD, it is possible that RVI could simply be a marker for patients who are otherwise predisposed to CLAD, rather than a cause of CLAD. There are several lines of evidence suggesting that RVI might be an underrecognized contributor (rather than marker) in a substantial proportion of CLAD cases. As summarized in Table ​Table1,1, RVI has been linked to CLAD in several prior studies [2, 6–10, 25–29]. RVI is relatively common in LTRs, and a significant proportion of cases detected by sensitive methods can be asymptomatic. Importantly, both symptomatic and asymptomatic RVI has been linked to CLAD. Additionally, in vitro and animal studies have identified specific biologically plausible mechanisms through which RVI could mediate CLAD [30, 31]. RVI-mediated regulatory T-cell dysfunction leading to augmentation of innate, allo-, and auto-immune pathways are possible mechanistic pathways to explain a causal link between RVI and CLAD. Finally, a small clinical trial of a novel antiviral (siRNA) for a specific RVI (RSV) in LTRs demonstrated reduced risk for CLAD in the group randomized to active RSV therapy [32]. In total, these epidemiologic and experimental data are consistent with a causal association between RVI and CLAD.

In summary, our study addresses many of the limitations of prior studies and adds significantly to the body of evidence linking RVI to development of CLAD. This association is relevant as RVI could represent a distinct, diagnosable, and potentially treatable major cause of allograft dysfunction and loss in LTRs. If the association were confirmed in future prospective studies, at a minimum, it could identify specific patients at increased risk for development of CLAD who could then be entered into trials of preventive or treatment strategies. With the development of new antivirals for several respiratory viruses [32–35], it will be possible to directly test the hypothesis that RVI causes CLAD by means of interventional controlled trials. Prior to embarking on such studies, it is imperative to carefully characterize the virologic determinants and mechanisms that underlie CLAD in order to aid in the design of such interventional trials. While our study strongly suggests an association between symptomatic RVI and CLAD, we propose a large, multicenter, prospective natural history study with careful virologic and clinical assessments and mechanistic studies as a logical next step. A better understanding of the role of respiratory viruses in the pathogenesis of CLAD through future prospective studies is critical in devising rational strategies to improve long-term patient and graft survival in LTRs.

Supplementary Data

Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.

Notes

References