Basiliximab

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, in part because its pathogenesis is poorly understood and treatment options are limited. To identify unique risk factors for BOS and death, we performed a retrospective cohort study on 259 consecutive adult lung transplant recipients over a 5-year period. The demographic and clinical characteristics of this population were analyzed for an association between BOS or death and potential risk factors, including community-acquired respiratory viral (CARV) infections, acute rejection, and cytomegalovirus pneumonitis. Respiratory syncytial virus, parainfluenza, influenza, and adenovirus accounted for 21 CARV infections. Univariate and multivariate time-dependent Cox regression analyses demonstrated that this CARV group was more likely to develop BOS, death, and death from BOS. Furthermore, these trends were more pronounced in patients with evidence of lower respiratory tract-CARV (lower-CARV) infections. Notably, the CARV and lower-CARV infections were risk factors for BOS, death, and death from BOS distinct from the risk attributable to acute rejection. Identification of CARV and lower-CARV infections as BOS and mortality risk factors has important clinical implications and may provide insight into disease pathogenesis and accelerate the development of novel treatment strategies to modify post-CARV BOS.

Keywords: lung transplantation; viruses; bronchiolitis obliterans; risk factors; graft rejection

Obliterative bronchiolitis (OB) is the fibrotic destruction of small airways that is the major cause of long-term graft failure, morbidity, and mortality in the lung transplant population (1, 2). Bronchiolitis obliterans syndrome (BOS), a clinical description of OB, is defined by the International Society of Heart and Lung Transplantation as graft deterioration manifested by persistent airflow obstruction not due to other causes (3). BOS is a frequent complication that is the cause of death in approximately one-third of patients after 1 year, and it affects one-half of lung transplant recipients alive at 5 years (1).

Prior studies have identified acute rejection (4-9), lymphocytic bronchitis (6-9), cytomegalovirus (CMV) pneumonitis (5, 6), single lung transplant (10), and anti-human leukocyte antigen antibody development (11, 12) as probable or potential risk factors (3, 13). Although these risk factors likely contribute to the pathogenesis of BOS in some patients, their occurrence does not fully correlate with the subsequent development of BOS in all patients (2). Unfortunately, the biologic mechanisms responsible for the development of BOS remain ill defined (2, 3), and current therapies to treat the associated pathophysiologic responses have not significantly altered the prevalence or progression of BOS during the last decade (10, 14). Identification of additional risk factors may provide further insight into disease pathogenesis for certain patients and provide the framework to design novel therapeutic strategies to prevent or delay the progression of BOS.

Previous studies have suggested that community-acquired respiratory viral (CARV) infections are associated with the development of BOS. Initial, uncontrolled pediatric and adult retrospective case series postulated an association between CARV infections and subsequent BOS (15-21). Recently, two cohort analyses have also supported this link (22, 23). A small prospective pediatric study correlated adenovirus infection with increased rates of OB, graft failure, and death (22). Similarly, a retrospective analysis of adults demonstrated that lower respiratory tract-CARV (lower-CARV) infections from influenza, parainfluenza, and respiratory syncytial virus (RSV) in the first year after transplantation were a risk factor for end-stage BOS, but not earlier stages of BOS (23). Although informative, these two studies did not examine the association between viral infection and all stages of BOS or the association between viral infection and death. More importantly, studies have not controlled for the effects of other established risk factors such as acute rejection. Therefore, we investigated the relationship of antecedent CARV infections to the development of BOS and death and whether the CARV risk was distinct from other BOS risk factors.

Accordingly, we designed a retrospective cohort study on 259 consecutive adult lung transplant recipients at Washington University School of Medicine/Barnes-Jewish Hospital (WUSM/ BJH) to investigate the role of RSV, parainfluenza, influenza, and adenovirus infections in BOS and death. We performed multivariate analysis with BOS stage 1 as the primary endpoint and BOS stage 2, BOS stage 3, death, and death from BOS as secondary endpoints.

METHODS

Study Design

Institutional review board approval for this study was obtained before initiation of data acquisition. A retrospective review of medical records was conducted on every adult patient (age > or = 18 years) at WUSM/BJH who underwent lung transplantation between January 1, 1996, and December 31, 2000. Data were accrued through July 1, 2002. Recipient sex, age, underlying lung disease, donor organ ischemic time, post-transplant bronchoscopy results, pulmonary function test data, survival status, and cause of death were obtained from medical records and computerized databases maintained by physicians and transplant coordinators in the lung transplant center at WUSM/BJH. Recipients who could not be assessed for BOS because of early death (

Standard Care of Lung Transplant Subjects

Pretransplant evaluation, surgical procedures, postoperative care, and immunosuppressive regimens were delivered by physicians in the lung transplant program at WUSM/BJC and did not change substantially throughout the study period (24, 25). For induction therapy, patients received equine antithymocyte globulin (5-15 mg/kg/day) for 3 days alter transplantation. In situations of CMV mismatch (donor +/recipient -) or early postoperative instability, patients received 20 mg of basiliximab on the day of surgery and on postoperative Day 4 or received no induction agent. Patients were maintained on triple-drug immunosuppression with corticosteroids, azathioprine, and cyclosporine, which were not routinely altered when a CARV infection was diagnosed. Tacrolimus replaced cyclosporine, and mycophenolate mofetil replaced azathioprine in cases of specific drug intolerance or toxicity. The medication dose was adjusted according to trough levels, and immunosuppression was gradually lowered at 6 months postoperatively in the absence of recent graft rejection.

In the first year after transplantation, patients underwent five surveillance bronchoscopies that were scheduled monthly during the first 3 months and again at 6 and 12 months. Clinically indicated bronchoscopies were conducted for new respiratory symptoms or signs (e.g., shortness of breath, new radiographie findings, > 10% decline from baseline FEV^sub 1^, and hypoxemia). After the first year, only clinically indicated bronchoscopies and follow-up bronchoscopies were performed to monitor for treatment responses. Tissue and bronchoalveolar lavage fluid from all bronchoscopies were analyzed for evidence of rejection and viral, bacterial, and fungal infections.

Diagnostic Definitions

Pulmonary function test data were used to establish post-transplant baseline and to diagnose the onset of BOS according to International Society of Heart and Lung Transplantation criteria (3). The date of BOS stage 1 was defined as the first of two FEV^sub 1^ measurements obtained at least 3 weeks apart that demonstrated a decrease in FEV^sub 1^ to 66-80% of the post-transplant baseline. The dates of BOS stage 2 and BOS stage 3 were similarly defined when FEV^sub 1^ values dropped to 51-65% and 50% or less, respectively. Acute rejection was diagnosed using standard histologic criteria according to the Lung Rejection Study Group (26). All grades of acute rejection, that is, any biopsy specimen of grade Al or more, were considered positive in this study regardless of the presence of symptoms or treatment. Donor organ ischemic time for bilateral transplants was the mean of right and left lung ischemic times. HLA-A, HLA-B, and HLA-DR antigen mismatches between donor and recipient were determined using the complement-dependent microlymphocytic assay (27). CMV pneumonitis was defined purely on a histologic basis by the presence of typical cytomegalic cells with characteristic intracellular inclusions or positive immunolabeling with a CMV-specific antibody (28). The cause of death was determined after expiration using available medical records.

Identification and Diagnosis of CARV Infection

To identify CARV infections, a computer-generated search of all virology records at WUSM/BJH from January 1, 1996, to July 1, 2002 retrieved positive reports for RSV, parainfiuenza, influenza, and adenovirus from bronchoalveolar lavage, bronchial wash, tracheal aspirate, sputum culture, and nasopharyngeal swab specimens. The date of CARV infection was defined as the date of specimen collection. Viral detection was performed using standard microbiologic techniques in the virology laboratory for WUSM/BJH. Virus-specific immunofluorescent labeling for RSV, parainfiuenza, influenza, and adenovirus were performed on cell cytospin preparations. If cells lacked immunofluorescent labeling, the cell-free supernatant was further tested for viral presence by incubation with rhesus monkey kidney cells, human lung fibroblasts (MRC-5), human embryonic kidney cells, and human epidermoid cancer cells (HEp-2). Specimens with cytopathic changes underwent viral identification as described previously here. Similar to published work, lower-CARV diagnosis required CARV infection and additional documentation of new respiratory decline manifested as shortness of breath, wheezing, radiographic changes, hypoxemia (Pa^sub O^sub 2^^ 10%) decline in FEV^sub 1^ (23).

Statistical Analysis

Patient information was tabulated in Excel 2002 (Microsoft Corp., Redmond, WA) and analyzed using SPSS 8.0 (SPSS Inc., Chicago, IL). To compare the demographic data and outcomes between groups, two-tailed Student's t tests of independent samples were used for continuous variables and two-sided chi-squared tests or two-sided Fisher's exact tests were used for categoric variables, the latter when expected cell size was less than five. Survival analysis used time-independent and time-dependent Cox regression models. For clinical events that occurred after lung transplantation, such as CARV infection and acute rejection, time-dependent models were constructed to avoid assignment of risk before their occurrence. Initial univariate Cox regression analysis identified potential predictors for the development of all stages of BOS, death, and death from BOS (p

RESULTS

Cohort Assembly and Baseline Comparison

Of the 259 patients that received lung transplants from 1996-2000 at WUSM/BJH, 31 patients were excluded because they could not be assessed for the development of BOS: 17 died within 3 months of transplantation, 7 developed anastomotic complications, 4 demonstrated postoperative restrictive ventilatory defects, and 3 lacked sufficient pulmonary function test data. In our study population of 228 patients, 21 cases of CARV infection were identified, 17 before the development of BOS stage 1, and 4 after BOS stage 1 but before BOS stage 2. The remaining uninfected patients constituted the no-CARV group; 211 for the BOS stage 1 endpoint and 207 for all other endpoints. The demographic and clinical information in the CARV and no-CARV groups was statistically indistinguishable in terms of sex, age, pretransplant disease, ischemic time, type of transplant, HLA mismatches, post-transplant baseline FEV^sub 1^, CMV pneumonitis, acute rejection of A1 or more, acute rejection of A2 or more, and length of follow-up time. The CARV group had a significantly higher proportion of patients with BOS stage 2, BOS stage 3, death, and death from BOS (Table 1). Collectively, the CARV and no-CARV groups had similar baseline clinical characteristics; however, the development of advanced BOS stages and incidences of death were greater in the CARV group.

Clinical Characteristics of CARV Infection Cases

The 21 patients diagnosed with CARV infection included 8 cases of RSV, 7 of parainfluenza, 4 of influenza, and 2 of adenovirus, with no more than 1 episode in any patient (Figure 1). Seventeen cases were identified from bronchoalveolar lavage or bronchial wash, three from nasopharyngeal swab, and one from a tracheal aspirate. Of the 17 cases found through bronchoscopy, 9 procedures were performed for clinical indications, 5 for scheduled surveillance, and 3 for follow-up of acute rejection. CARV infection and acute rejection were simultaneously diagnosed in four patients (three of four cases with influenza and one of eight with RSV). Within the CARV group, 15 of 21 patients (71%) had evidence of a lower-CARV infection (Figure 2). Presenting clinical characteristics included eight with new chest radiographic changes, six with decreased FEV^sub 1^, and four new instances of shortness of breath (13 cases with 1 finding, 1 case with 2 findings, and 1 case with 3 findings). For all patients with CARV infections, the average time from transplant to viral infection was 304 days (median 150, range 4-995). The average time from CARV infection to BOS stage 1 was 479 days (median 378, range 12-1245), from BOS stage 1 to stage 2 was 165 days (median 28, range 0-540), and from BOS stage 2 to stage 3 was 105 days (median 0, range 0-574). Interestingly, the CARV group demonstrated an accelerated progression from BOS stage 1 to stage 2 (CARV vs. no CARV, 165 vs. 307 days, respectively) and from BOS stage 2 to stage 3 (CARV vs. no CARV, 105 vs. 214 days, respectively). Three of eight recipients with RSV underwent treatment with inhaled ribavirin, and none of these three patients developed BOS, whereas four of five untreated RSV recipients developed BOS (p = 0.14).

CARV and Lower-CARV Infections Are Distinct BOS and Death Risk Factors Separate from Acute Rejection

To examine the hypothesis that CARV infections are a BOS risk factor, we chose BOS stage 1 as our primary endpoint. We studied BOS stage 2, BOS stage 3, death, and death from BOS as secondary endpoints to investigate whether CARV infections may also represent a risk for BOS progression and mortality. We used univariate Cox proportional hazards regression to determine whether individual variables were associated with an increased risk for BOS and to guide subsequent multivariate analysis. Time-dependent models were constructed for events that occurred at different times after transplantation to avoid assignment of risk to subjects before their occurrence. This method was chosen as the patients with CARV infections accrued approximately 28% of their total follow-up time before CARV diagnosis (6,393 out of 22,443 patient days). Similarly, time-dependent Cox models were generated for acute rejection, CMV pneumonitis, and lower-CARV infection. Univariate analysis of predictors for BOS stage 1 revealed that lower-CARV infections and acute rejection were significant risk factors (lower-CARV infection hazard ratio [HR] = 2.86, 95% confidence interval [CI], 1.32-6.19, p = 0.008; acute rejection HR = 2.03, 95% CI, 1.22-3.37, p = 0.006). CARV infection and single lung transplant showed a trend toward development of BOS stage 1 (CARV HR = 1.84, 95% CI, 0.92-3.66, p = 0.08, and single lung transplant HR = 1.55, 95% CI, 0.95-2.52, p = 0.08). Sex, age, pretransplant disease, ischemic time, HLA mismatches, and CMV pneumonitis were not predictors of BOS stage 1 development and were thus excluded from subsequent multivariate analysis (p > or = 0.10). Similar univariate analysis for the secondary endpoints BOS stage 2, BOS stage 3, death, and death from BOS all demonstrated that CARV infection, lower-CARV infection, acute rejection, and type of transplant (for the death endpoint only) were all significant predictors, whereas sex, age, pretransplant disease, ischemic time, HLA mismatches, and CMV pneumonitis were not significantly associated with these endpoints (Table 2).

To determine whether CARV infections and the subset of lower-CARV infections were BOS and death risk factors separate from other variables, a series of multivariate Cox regression models were constructed for each stage of BOS, death, and death from BOS as endpoints. Multivariate Cox regression models determined that CARV and lower-CARV infections were unique BOS stage 1 risk factors distinct from other candidate variables. CARV infection and acute rejection were found to be significant predictors of BOS stage 1 (CARV infection, HR = 2.05, 95% CI, 1.03-4.17, p = 0.04; and acute rejection HR = 1.78, 95% CI, 1.05-3.01, p = 0.03) (Model 1, Table 3). Inclusion of only lower-CARV infections with acute rejection revealed a stronger association with subsequent BOS stage 1 development (lower-CARV infection, HR = 3.03, 95% CI, 1.40-6.56, p = 0.005; acute rejection, HR = 1.81, 95% CI, 1.08-3.04, p = 0.02) (Model 2, Table 3). We repeated these multivariate analyses for our secondary endpoints, and again, CARV and lower-CARV infections were significant risk factors for the advanced stages of BOS and death (Table 3). Interestingly, we observed a progressively higher HR for each increment in BOS stage, suggesting that CARV infection may accelerate BOS progression. CARV and lower-CARV infection remained significant risk factors for BOS stage 1 and death when including type of transplant as a candidate variable (p = 0.04 and 0.006, respectively). Taken together, our results demonstrated that CARV infections, especially those involving the lower respiratory tract, were associated with a significant risk for the subsequent development of BOS stage 1 and death and that this viral risk was distinct from the risk attributable to acute rejection.

DISCUSSION

BOS has remained the most significant cause of graft failure and mortality after lung transplantation partly because the pathophysiologic mechanisms of disease are poorly defined and treatment options are limited (1-3). This retrospective cohort study demonstrated that CARV infections in the adult were associated with an increased risk of subsequent BOS and death. Our findings estimated that a patient who developed a CARV infection after lung transplantation had twice the risk of developing BOS stage 1, and if the infection involved the lower respiratory tract, the risk was tripled. Moreover, these risks from CARV infections were distinct from the risk attributable to other risk factors, such as acute rejection. In addition, CARV infection was also a significant risk factor for more advanced stages of BOS and death. Identification of CARV infections as a unique BOS risk factor has important clinical implications and suggests that antiviral strategies aimed to prevent or treat viral infection may provide a strategy to modify the onset or progression of post-CARV BOS.

In the context of previous work describing BOS risk factors, our study is the first to demonstrate that CARV infections were associated with an increased risk of developing BOS stage 1, BOS stage 2, death, and death from BOS and the first to separate the contribution of these infections from the risk attributable to acute rejection and CMV pneumonitis. Previous studies have not analyzed BOS stage 1 or death as endpoints; thus, direct comparisons to our study are difficult. A recent report used time-dependent univariate analysis to demonstrate that lower-CARV infections in the first year after transplant were associated with BOS stage 3 but somewhat unexpectedly not BOS stage 2 (23). In agreement with multiple published reports (4-9), we demonstrated that the presence of acute rejection was a significant risk factor for BOS. CMV pneumonitis was not found to be a significant risk for BOS in our analysis, a common finding in both adult and pediatric lung transplant recipients (7, 8, 27, 28). Taken together, the consistency of our results with published data supports the fidelity of our study that demonstrates an association between CARV infections and all stages of BOS and death.

As with any retrospective study, there are inherent limitations in our work related to availability of data, study design, and selection bias. Our data acquisition was complete (256 of 259 patients [98.8%]), with an average follow-up time of 3 years, and comparisons were made between consecutive transplant recipients that were subjected to identical diagnostic definitions and outcome assessment during the entire study period. Although performed in a retrospective manner, this longitudinal analysis identified an association between CARV infections and BOS and strongly suggests that a causal relationship between CARV infection and BOS may exist.

The methods of viral identification used in the study period probably missed numerous CARV infections in our cohort because of relatively infrequent screening and the use of relatively low sensitivity viral detection assays during the study period. Our proportion of post-transplant patients that developed a CARV infection was similar to previously published reports (9% vs. 8-21%) (17-19). However, all of these studies, including ours, likely underestimated the true incidence of respiratory viral infections because of infrequent screening and the relatively low sensitivity of viral detection with immunolabeling and culture compared with polymerase chain reaction-based assays (22, 29-31). In fact, a 6-month prospective study in 93 adult lung transplant patients used a weekly symptom screening protocol coupled with conventional and polymerase chain reaction-based viral detection assays to identify 31 respiratory viral isolates from these patients (0.66 infections per patient year compared with 0.02 infections per year in our study) (31). Although the impact of the missed viral infections cannot be assessed in our study, the detection of a significant association between CARV infections and BOS despite our limitation in viral detection is noteworthy and suggests that the true role of viruses in BOS pathogenesis is still under appreciated.

We may have modified the risk from CARV infection if a selection bias was present in our viral testing methods. If only the patients with more severe, BOS-predisposing CARV infections were identified because of a higher likelihood of lower respiratory tract involvement, and greater viral burden, the estimated risk could have been inflated. Nevertheless, our methods of viral detection reflect standard clinical practice and can serve as an effective method of identifying a group of patients at high risk for post-CARV BOS.

In several patients, CARV infections occurred before establishment of the patient's post-transplant maximum FEV^sub 1^ value. Because this post-transplant maximum value serves as the baseline for the eventual diagnosis of BOS, these early-CARV infections may have confounded the diagnosis of BOS. The direction and magnitude of this effect are uncertain, as the relationship between post-transplant baseline FEV^sub 1^ and the subsequent rate of FEV^sub 1^ decline has not been established. We anticipate the error introduced from this possible confounding effect was minimal given that post-transplant baseline FEV^sub 1^ was not significantly different in the patients with CARV infections and that post-transplant baseline FEV^sub 1^ was not a BOS risk factor in previous studies (6, 32). Finally, the study was limited to four viruses, and the primary end point chosen was stage 1 BOS rather than histologically proven OB. Accordingly, extrapolation of the CARV infection risk to other respiratory viruses or the subsequent development of OB is not justified at this time.

Previous observations in human and animal studies have provided insight into potential mechanisms responsible for the association between CARV infections and BOS. A high proportion of post-transplant patients with active influenza and parainfluenza infection at one institution was simultaneously diagnosed with acute rejection (61 and 82%, respectively) (20, 21). Our study population demonstrated that 19% of total patients with CARV infections and 75% of influenza patients were simultaneously diagnosed with acute rejection. These results suggest that CARV infection may initiate inflammatory cascades that trigger acute rejection, an established risk factor for BOS. Of note, this association between CARV infection and acute rejection also raises the possibility that viral infection may masquerade histologically as acute rejection. In rats receiving orthotopic left lung transplants, infection with murine parainfluenza type I (Sendai virus) resulted in lumen obliteration with persistent allograft epithelial cell injury, enhanced immune cell accumulation, delayed viral clearance, and chronic bronchiolar scarring (33, 34). We have previously demonstrated that Sendai viral infection of mice resulted in an acute bronchiolitis/bronchitis accompanied by acute airway epithelial cell injury and induction of IFN-[gamma], interleukin-12 p40, regulated upon activation and normal T cell expresses and secreted, and intercellular adhesion molecule-1 (35-38). Interestingly, these acute inflammatory mediators are also increased in human lung transplant allografts with acute rejection (39-41) and BOS (42). Additionally, our murine paramyxoviral bronchiolitis/bronchitis model produced a chronic phenotype characterized by alteration in airway epithelial cell gene expression, mucous cell metaplasia, and airway hyperreactivity that persisted for 1 year after viral clearance (37). Whether respiratory viral infection in the setting of lung transplantation can produce chronic alterations that initiate or accelerate BOS is currently being investigated.

The biochemical mechanisms responsible for the variability in onset of post-CARV BOS are unknown. We speculate that respiratory viral infections, especially with the Paramyxoviridae members, can initiate both acute and chronic responses that may trigger the development and/or acceleration of OB progression in lung transplant recipients. Biologic plausibility for this proposal is provided by observations from another respiratory disease. Respiratory viral infection with RSV (a Paramyxoviridae member) can result in a chronic asthma phenotype that persists for many years (43). The initial infection may result in viral-dependent injury of airway epithelial cells and expression of epithelial cell injury-response genes. In turn, these injury-response genes could provide signals that initiate immunologic or nonimmunologic pathways that result in the eventual deposition of collagen in the airway. Further research is ongoing to characterize the interplay between viral-dependent acute injury of the airway epithelial cells, injury-response gene expression, the subsequent chronic cellular responses, and host factors that may account for the relatively long interval between CARV infection and BOS.

The described association between CARV infections and BOS stage 1 has important clinical implications. To minimize the incidence of infections, we recommend that lung transplant patients avoid sick contacts and receive appropriate CARV vaccination and chemoprophylaxis when indicated. Viral testing should be performed with all surveillance bronchoscopies and during clinically indicated situations with more sensitive and comprehensive testing methods. In the event of a CARV infection, patients should be considered for antiviral treatment on an individual case basis and receive continued monitoring for evidence of post-CARV respiratory decline. In the absence of prospective data aimed to further establish a causal link between CARV infection and BOS, we recommend a heightened awareness of the potential detrimental consequences of CARV infections in lung transplant patients.

In conclusion, this work demonstrated that CARV infections were a significant risk for both development and progression of BOS, distinct from the risk attributable to acute rejection. Identification of the association between CARV infections and BOS stage 1 provides important clinical implications for the management of lung transplant recipients. In turn, examination of this association may allow for valuable insight into the basic mechanisms that mediate disease pathogenesis and accelerate the development of novel treatment strategies aimed to prevent or treat post-CARV BOS.

Acknowledgment: The authors thank S. Brody, M. DeBaun, L. Danziger, and M. Schoolman for discussion and review of the article; B. Banks and J. Lange for searching the virology records; and A. Aloush, J. Fassler, C. Miller, and L. Roldan for assistance in chart reviews.

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Anthony P. Khalifah, Ramsey R. Hachem, Murali M. Chakinala, Kenneth B. Schechtman, C. Alexander Patterson, Daniel P. Schuster, Thalachallour Mohanakumar, Elbert P. Trulock, and Michael J. Walter

Divisions of Pulmonary and Critical Care Medicine, Biostatistics, and Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri

(Received in original form October 3, 2003; accepted in final form April 29, 2004)

Supported by a grant from the Doris Duke Charitable Research Foundation (A.P.K. and M.J.W.) and National Institutes of Health HL56543 (T.M.).

Correspondence and requests for reprints should be addressed to Michael J. Walter, M.D., Division of Pulmonary and Critical Care Medicine, Campus Box 8052, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: mwalter@im.wustl.edu

Am J Respir Crit Care Med Vol 170. pp 181-187, 2004

Originally Published in Press as DOI: 10.1164/rccm.200310-1359OC on May 6, 2004

Internet address: www.atsjournals.org

Copyright American Thoracic Society Jul 15, 2004
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