The bronchiolitis obliterans syndrome (BOS) is the most significant factor limiting long-term survival after lung transplantation. Approximately 50% of lung transplant recipients will develop this airway-centered process of injury and fibroproliferative repair by 3 to 4 years after transplant, leading to a progressive loss of graft function and eventually death (1). Although to date alloimmune mechanisms have been blamed for the development of BOS, there is growing evidence that nonalloimmune mechanisms such as ischemia-reperfusion injury (2), gastroesophageal reflux disease (3), and viral infections (4–6) may have a role in the etiology. Bacterial infections and colonization of the allograft, particularly with Pseudomonas aeruginosa (PA) is common in recipients with BOS. To date this has not been considered to contribute to the development of the disease, but believed more likely to be a secondary phenomenon to airway injury in BOS itself. However, the nature of the association between Pseudomonas and BOS has not been previously characterized.
MATERIALS AND METHODS
All patients undergoing lung transplantation at the Freeman Hospital between January 2000 and December 2003 were included in the study. The study was approved by the Local Regional Ethics Committee. The results of all formal microbiological cultures of airway specimens, sputum, and bronchoalveolar lavage (BAL) were collected from this cohort. This included cultures of pretransplant sputum and BAL from recipient lungs at the time of excision. Recipients underwent routine bronchoscopy with BAL and transbronchial biopsy at 1 week, 1, 3, 6, and 12 months after transplant, and whenever clinically indicated. A BAL of 180 mL of sterile normal saline was performed in the right middle lobe or lingula and the first retrieved specimen was plated onto appropriate agar for microbiological culture. P. aeruginosa colonization was defined as more than 105 colony forming units observed on culture of routine or clinically indicated posttransplantation BAL.
Routine antibiotic prophylaxis and treatment protocols are summarized in the online only supplemental text. Standard immunosuppressive induction therapy with antithymocyte globulin was omitted in those colonized with Burkholderia cepacia or multidrug-resistant organisms. Standard triple-drug immunosuppressive therapy was used thereafter and not altered in the presence of PA colonization.
Recipient demographics are summarized in Table 1. Sixty-four (41.3%) of all transplanted patients became culture-positive for PA at some time after transplantation. Forty-four of these patients had persistence of pretransplant PA, which colonized the graft, and 20 became colonized de novo after transplantation. Of the 64 patients with PA colonization of the allograft, 47 (73.4%) had been transplanted for cystic fibrosis (CF). The pretransplant diagnosis in patients colonized de novo after transplant was chronic obstructive pulmonary disease in eight (40%), fibrotic lung disease in five (25%), and CF in four (20%). Prophylactic antibiotics had been administered for a median of 10 days (range 4–34) in these patients. Those in the upper limits of this range had all had a positive donor lavage culture. In patients developing de novo colonization of the allograft, the total number of isolates positive for PA ranged from 1 to 13 with a median of 2 per patient.
Thirty-two survivors of more than 90 days posttransplant (24.4%) developed BOS during a median follow-up of 824 days (95% confidence interval [CI] 308–1,727). Pretransplant colonization with PA was not associated with a significantly higher incidence of BOS within 2 years (20% vs. 14%, respectively, P=0.153). Recipient colonization with PA after transplant, however, showed a significant association with the development of BOS within 2 years of transplant (23.4% vs. 7.7%, P=0.006). This relationship was further explored in the subgroups with persistent pseudomonal colonization (i.e., with an existing pseudomonal reservoir present pretransplant that colonized the allograft) and those with de novo colonization of the allograft after transplantation. This revealed a statistically nonsignificant trend toward a greater overall incidence of BOS in patients with persistent colonization, with 10 of 44 patients (22.7%) developing BOS in this group, as opposed to only 14 of 91 (15.4%) of those remaining free of colonization (P=0.296). In contrast, 8 of the 20 patients (40.0%) with de novo colonization of the allograft developed BOS during follow-up (P=0.012 vs. those remaining culture-negative). Freedom from BOS analyzed by the Kaplan-Meier method correspondingly showed no difference for the group with persistent Pseudomonas as compared with those remaining free of PA after transplant (Fig. 1, log-rank P=0.42). In contrast, BOS-free survival time was significantly reduced in the 20 recipients with de novo PA colonization after transplant as compared with those with no PA (Fig. 1, Kaplan-Meier log-rank P=0.014). Eighteen lung transplant recipients developed postoperative graft colonization with PA and BOS. In 14 (78%) of these patients, PA colonization preceded BOS (Fig. 2). In the group as a whole, colonization with PA preceded the diagnosis of BOS by a median of 204 days (95% CI 115–492, Wilcoxon signed ranks P=0.002). To further explore the time course of colonization and onset of BOS, we performed an analysis using BOS 0p as an earlier physiological marker of the onset of BOS. Identification of de novo colonization preceded the diagnosis of BOS 0p in four of eight patients (50%), and by a median of 28 days (95% CI −156–604) in patients developing both these complications.
Isolation of PA from airway samples, sputum, and BAL fluid is relatively common in lung transplant recipients. Many patients remain clinically well despite the presence of this organism and are often termed “colonized” even though the presence of PA in the airway is always pathological. The term infection in contrast, usually implies a systemic or local response of the airway to the pathogen, and it follows that the immune response to this organism varies greatly between patients and perhaps even over time within the same patient. In patients who were colonized with PA before transplant, colonization of the allograft after lung transplantation tends to occur early and is believed to be a result of spread from an upper airway reservoir. How the organism is able to get a foothold in the lung allograft that is structurally normal is unclear. We have previously demonstrated a decreased ciliary beat frequency in lung transplant recipients, which can contribute to decreased clearance of airway secretions (7). Defective iron homeostasis with a decreased availability of lactoferrin, a natural bacteriostatic agent of the innate immune system, has been implicated in the development of chronic infection in those with CF (8). Similar defects in iron homeostasis have been demonstrated in the transplanted lung, and may contribute (9, 10).
The BOS, representing chronic dysfunction of the lung allograft, is the most common cause of morbidity and mortality in lung transplant recipients after the immediate perioperative period. The development of allograft colonization with PA also bears some relation to the development of BOS and we have previously shown that quorum-sensing molecules can be detected in the lungs of stable lung transplant recipients that are culture-negative for PA (11). These novel intercellular signaling molecules are secreted by, among others, PA and Burkholderia cepacia and aid in establishing biofilm-type growth patterns in the lung. Our study has demonstrated that the development of allograft colonization with PA is significantly associated with the development of BOS. It would seem that colonization occurs significantly earlier than the onset of BOS by classical definition and it is therefore possible that PA may play a role in the development of the chronic inflammation and airway injury that contributes to BOS. Furthermore, there seems to be a significant difference in the impact of persistent pseudomonal colonization, representing spread of organisms from an upper airway reservoir, and de novo colonization of the allograft, on the development of BOS. Although the relatively small number of patients in the subgroup with persistent PA colonization make a type II error possible, this type of colonization did not seem to predispose to the development of BOS in our study. Prolonged infection with PA in patients with CF may induce a degree of tolerance, which together with impairments in innate immunity could result in a less florid immune response. Alternatively, allograft colonization in this setting may represent simple recolonization from the upper airway reservoir with little pathological impact on otherwise healthy airways. Growth of PA de novo may represent a true infection of damaged airways that had begun to lose epithelial integrity already by another process. In this way, de novo colonization with PA may be acting as an early sign of airway dysfunction before the objective measures of lung function are affected.
Forced expiratory volume in 1 sec may be a poor marker of early airway injury and damage at the cellular level, and therefore, lymphocytic bronchiolitis (a forerunner of obliterative bronchiolitis) or early obliterative bronchiolitis may already be present when de novo PA colonization occurs. The interaction between PA and the allograft airway is undoubtedly complex and further studies are needed to clarify the role of PA in the development of BOS. Whether PA initiates the airway injury that leads to the development of BOS, is simply a marker thereof, or a contributor to this process, this study demonstrates a strong association between these two complications of lung transplantation. Additionally, it raises the possibility that therapies that prevent de novo allograft colonization or aggressive early treatment thereof, may prove protective against the development of BOS.
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