Abstract
Rationale: Stenotrophomonas maltophilia is one of the more common multidrug-resistant organisms isolated from the respiratory tract of patients with cystic fibrosis (CF), but the effect of chronic S. maltophilia infection on CF lung disease is unknown.
Objectives: To determine the impact of chronic S. maltophilia infection on lung disease in CF.
Methods: We developed a serologic assay specific for S. maltophilia and in a cross-sectional study, measured serum antibodies to S. maltophilia in patients with CF to determine if a definition of chronic S. maltophilia isolation based on culture results corresponded to an immunologic response (serologic study). We then used this validated definition to examine the effect of chronic S. maltophilia on the severity of lung disease in a retrospective cohort study using the Toronto CF Database from 1997–2008 (cohort study).
Measurements and Main Results: Serum antibody levels to S. maltophilia were measured in 179 patients with CF. Patients with chronic S. maltophilia had significantly higher mean antibody levels to S. maltophilia flagellin (P < 0.0001) and whole cell (P = 0.0004) compared with patients with intermittent or no S. maltophilia. The cohort study included 692 patients with an average follow-up of 8.3 years. In an adjusted log linear model, patients with chronic S. maltophilia infection had a significantly increased risk of pulmonary exacerbation requiring hospitalization and antibiotics compared with patients who had never had S. maltophilia (relative risk = 1.63; P = 0.0002).
Conclusions: Chronic S. maltophilia infection in patients with CF is associated with a specific immune response to this organism and is an independent risk factor for pulmonary exacerbations.
Stenotrophomonas maltophilia is one of the more common multidrug-resistant organisms isolated from the respiratory tract of patients with cystic fibrosis (CF). Epidemiologic studies suggest that it simply colonizes the CF lung and does not contribute to CF lung disease, but the effect of chronic S. maltophilia infection is unknown.
This study demonstrates that chronic S. maltophilia infection in patients with CF is associated with a specific immune response to this organism and is an independent risk factor for pulmonary exacerbations, suggesting that it may be a true pathogen in some patients with CF.
It is unclear whether S. maltophilia simply colonizes the airway or causes true infection and clinical decline in CF. Epidemiologic studies have not shown any association between S. maltophilia and survival or declining lung function in CF (4, 5). However, these studies focused on patients with CF who were positive at any point in time for S. maltophilia and did not make the distinction between chronic and intermittent isolation of S. maltophilia in the respiratory tract. As has been shown with methicillin-resistant Staphylococcus aureus and Aspergillus fumigatus, chronic infection with S. maltophilia may be more likely to cause clinical deterioration in CF (6–8).
The aim of this study was to determine the impact of S. maltophilia infection on lung disease in CF. Chronic infection with an organism can be defined by the number or percentage of positive cultures of respiratory specimens in a given year. This strategy has been validated for Pseudomonas aeruginosa infection (9). In addition, chronic exposure to an organism can stimulate an immune response and assessing the immune response can help to distinguish between transient colonization and infection. In the first part of this study, we developed a novel serologic assay specific for S. maltophilia and measured serum antibodies to S. maltophilia in patients with CF to determine if a definition of chronic S. maltophilia isolation based on culture results corresponded to an immunologic response. In the second part of the study, we used this validated definition to examine the effect of chronic S. maltophilia on the severity of lung disease as measured by pulmonary function tests and number of acute pulmonary exacerbations in a larger retrospective cohort study in patients with CF.
This was a cross-sectional study of patients with CF followed at The Hospital for Sick Children and St. Michael's Hospital (Toronto, ON, Canada). Patients were excluded if they could not produce sputum, were unable to perform reproducible spirometry, or had received a lung transplant. A serum sample was collected for each patient with routine clinical blood sampling in the CF clinic. Pulmonary function testing was performed on the same day as serum specimens were obtained in 94% of patients (within 3 mo in the remainder).
All subjects were classified according to their respiratory culture status in the previous 12 months (8): (1) chronic S. maltophilia, greater than or equal to two positive sputum or bronchoalveolar cultures for S. maltophilia in a given year; (2) intermittent S. maltophilia, one positive culture for S. maltophilia in a given year or a previous positive culture; and (3) never S. maltophilia, never having a positive culture for S. maltophilia. Subjects who never had S. maltophilia were divided into those who had chronic P. aeruginosa and those without chronic P. aeruginosa. These definitions were validated against the serologic results.
Serologic responses were measured by an ELISA using three S. maltophilia antigens individually (flagellin [10], protease [11], and whole bacterial cell [12]) (see online supplement). Results were represented as the ratio of the average serum sample optical density to the average optical density value of the two negative controls (wells with pooled normal human serum) (ratio units).
Analysis of variance was used to assess if there were significant differences between groups. Correlation analysis was done using Pearson correlation. Multiple regression analysis was also done on FEV1 percent predicted and the mean antibody levels adjusting for age, P. aeruginosa, and Burkholderia cepacia complex infection.
This was a retrospective cohort study from 1997–2008 using the Toronto CF Database. Patients with chronic S. maltophilia were compared with those who had intermittent S. maltophilia and those who did not have S. maltophilia over the study period. The primary outcome measure was the rate of decline of FEV1 in percent predicted in patients in each of these three groups. The secondary outcome was the number of hospital admissions for pulmonary exacerbations requiring antibiotics per year. The same exclusion criteria and microbiologic classification were used as previously mentioned.
Two separate regression models were generated as used in a previous study (8). In the first, a hierarchical linear model was generated to look at the effect of chronic S. maltophilia on quarterly FEV1 allowing for a random effect and slope for each patient. The second model assessed the effect of chronic S. maltophilia on the number of hospitalizations for pulmonary exacerbation requiring antibiotics using Poisson regression. Multivariable analysis was performed to account for potential confounding variables (see online supplement). The Research Ethics Board at the Hospital for Sick Children and St. Michael's Hospital approved the studies.
Sera were obtained and S. maltophilia antibody levels were measured for 179 patients with CF. Patient characteristics at the time of serologic testing are summarized in Table 1. Patients with chronic S. maltophilia isolation were significantly older, had lower mean FEV1 percent predicted, and were not coinfected with B. cepacia complex. Figure 1 illustrates the mean antibody levels to S. maltophilia flagellin (Figure 1A), whole cell (Figure 1B), and extracellular protease (Figure 1C) in patients with chronic, intermittent, and in those who never had S. maltophilia (subdivided into those who had chronic P. aeruginosa and those without chronic P. aeruginosa). Patients with chronic S. maltophilia had significantly higher mean antibody levels to S. maltophilia flagellin (P < 0.0001) and whole cell (P = 0.0004), but not to protease, compared with patients with intermittent or no S. maltophilia. Mean antibody levels to flagellin and whole cell were 1.5 times higher in the chronic group compared with the intermittent group and approximately two times higher in the chronic group compared with the negative group. There was also no evidence of cross-reactivity of antibodies to S. maltophilia with antibodies to P. aeruginosa because mean antibody levels in patients with chronic P. aeruginosa infection were similar to those in patients who were S. maltophilia–negative.
Characteristic | Chronic Stenotrophomonas maltophilia (n = 37) | Intermittent Stenotrophomonas maltophilia (n = 33) | Never Stenotrophomonas maltophilia (n = 109) | P Value |
|---|---|---|---|---|
| Age, mean (range) | 25.5 (9.5–58.7) | 17.6 (5.4–56.6) | 22 (6.2–52.5) | 0.02 |
| Sex (male) | 21 (57%) | 13 (39%) | 57 (52%) | 0.31 |
| Pancreatic insufficiency | 32 (86%) | 31 (94%) | 93 (85%) | 0.43 |
| CFRD | 0 (0%) | 1 (3%) | 2 (2%) | 0.60 |
| BMI | 20.3 (13.5–26.9) | 18.7 (13.8–24.6) | 19.9 (12.7–27.8) | 0.16 |
| FEV1 mean (range) | 58.8 (23.9–97.3) | 71.3 (27.3–114.5) | 72.3 (27.4–120.5) | 0.01 |
| Pseudomonas aeruginosa positive | 15 (40%) | 14 (42%) | 59 (54%) | 0.25 |
| Burkholderia cepacia positive | 0 (0%) | 3 (9%) | 15 (14%) | 0.05 |
Figure 1. Mean antibody levels, with SEM, to Stenotrophomonas maltophilia (A) flagellin, (B) extracellular protease, and (C) whole bacterial cell in sera samples from patients with chronic S. maltophilia (Chronic SM), intermittent S. maltophilia (Intermittent SM), never S. maltophilia with chronic Pseudomonas aeruginosa (Never SM with Chronic PA), and never S. maltophilia without chronic P. aeruginosa (Never SM without Chronic PA). Antibody levels are reported as ratio unit, which represents the ratio of the average serum sample optical density to the average optical density value of the two negative controls (wells with pooled normal human serum). *P = 0.0004; **P < 0.0001 by analysis of variance comparing Chronic SM with Intermittent SM, with Never SM with Chronic PA, and with Never SM without Chronic PA.
[More] [Minimize]The relationship between antibody levels and FEV1 percent predicted was then examined. There was a significant inverse correlation between mean flagellin antibody levels and FEV1 percent predicted (P = 0.0061) (Figure 2) but not between mean whole-cell antibody levels and FEV1 percent predicted (P = 0.28) (data not shown). A multivariate regression analysis of mean flagellin antibody level and FEV1 percent predicted was performed adjusting for age, P. aeruginosa infection, and B. cepacia complex infection. Mean flagellin antibody levels were significantly inversely associated with FEV1 percent predicted (P = 0.0094).
Figure 2. Mean flagellin antibody levels and corresponding FEV1 percent predicted. Each dot represents a single patient.
[More] [Minimize]Using this validated definition of chronic infection, we then retrospectively compared patients with CF and chronic S. maltophilia with those who had intermittent S. maltophilia and with those who did not have S. maltophilia. Of 692 patients with CF included in the analysis, 49 (7%) were chronically infected with S. maltophilia at baseline. Baseline patient characteristics are summarized by S. maltophilia infection status in Table 2. Patients chronically infected with S. maltophilia were not significantly different in age, sex, pancreatic status, CF-related diabetes, or body mass index (BMI) compared with those who had intermittent S. maltophilia or never had S. maltophilia. There were no significant differences in the percentage of patients in the chronic, intermittent, or never S. maltophilia groups with respect to mucoid P. aeruginosa infection or B. cepacia complex infection. A greater percentage of patients with intermittent S. maltophilia infection were P. aeruginosa positive (67%) than patients with chronic S. maltophilia (46%) or patients who never had S. maltophilia (36%) (P = 0.01). In addition, patients with chronic S. maltophilia infection had a significantly lower mean FEV1 percent predicted (47.06%) than patients with intermittent S. maltophilia (78.76%) or patients who never had S. maltophilia (73.40%) (P < 0.0001).
Characteristic | Chronic Stenotrophomonas maltophilia (n = 49) | Intermittent Stenotrophomonas maltophilia (n = 22) | Never Stenotrophomonas maltophilia (n = 621) | P Value |
|---|---|---|---|---|
| Age, mean (range) | 17.4 (2.4–49.4) | 13.6 (3–39.2) | 17.1 (2.1–49.9) | 0.81 |
| Sex (male) | 63.2% | 48.1% | 55.7% | 0.44 |
| Pancreatic insufficiency | 89.5% | 88.5% | 82.8% | 0.44 |
| CFRD | 15.8% | 9.6% | 8.5% | 0.53 |
| BMI (range) | 18.7 (12.9–27.7) | 19.4 (14.1–30.4) | 20.1 (12.4–34.8) | 0.06 |
| FEV1 mean (range) | 53.8 (17.4–102.8) | 78.8 (55.6–101.9) | 74.3 (20–127) | <0.0001 |
| Pseudomonas aeruginosa positive | 46.9% | 67.3% | 36.8% | 0.01 |
| Mucoid P. aeruginosa | 18% | 28.8% | 21% | 0.15 |
| Burkholderia cepacia positive | 0% | 1.9% | 11.1% | 0.12 |
The mean number of years of patient follow-up was 8.3. In an adjusted model, the rate of decline in FEV1 percent predicted was −1.02% predicted per year for patients with chronic S. maltophilia (P = 0.3 compared with those who never had S. maltophilia); −0.94% predicted per year for patients with intermittent S. maltophilia; and −1.06% predicted per year for patients who never had S. maltophilia. Figure 3 illustrates the adjusted FEV1 percent predicted over time for patients with chronic, intermittent, and those who never had S. maltophilia infection, demonstrating similar rates of pulmonary decline.
Figure 3. Decline in FEV1 percent predicted over time for chronic Stenotrophomonas maltophilia (Chronic), intermittent S. maltophilia (Intermittent), and never S. maltophilia (Never) based on adjusted model.
[More] [Minimize]The effect of chronic S. maltophilia infection on the number of pulmonary exacerbations requiring hospitalization and antibiotics per year was evaluated using a log linear model. In an unadjusted model, patients with chronic S. maltophilia infection (relative risk [RR] = 2.5; P < 0.0001) and patients with intermittent S. maltophilia infection (RR = 1.85; P = 0.0002) had a significantly increased risk of pulmonary exacerbation compared with patients who had never had S. maltophilia. Nonsignificant variables were then removed and a reduced model was generated adjusting for age, pancreatic insufficiency, P. aeruginosa, BMI, and baseline FEV1 percent predicted. In this model, patients with chronic S. maltophilia infection still had a significantly increased risk of pulmonary exacerbation (RR = 1.63; P = 0.0002) compared with patients who had never had S. maltophilia but patients with intermittent S. maltophilia infection no longer had a significantly increased risk (RR = 1.18; P = 0.28) (Table 3). Patients with pancreatic insufficiency (RR = 2.44; P = 0.01), P. aeruginosa infection (RR = 1.61; P < 0.0001), lower BMI (RR = 1.05; P = 0.02), and lower baseline FEV1 percent predicted (RR = 1.02; P < 0.0001) also had a significantly increased risk of pulmonary exacerbation.
Variable | Relative Risk | SE | P Value |
|---|---|---|---|
| Chronic Stenotrophomonas maltophilia | 1.63 | 0.13 | 0.0002 |
| Intermittent S. maltophilia | 1.18 | 0.16 | 0.28 |
| Pancreatic insufficiency | 2.44 | 0.35 | 0.01 |
| Pseudomonas aeruginosa | 1.61 | 0.11 | <0.0001 |
| Lower BMI | 1.05 | 0.02 | 0.02 |
| Lower baseline FEV1 percent predicted | 1.02 | 0.003 | <0.0001 |
To our knowledge, this is the first study demonstrating that patients with CF with chronic S. maltophilia infection have a specific immune response to S. maltophilia that is associated with lower lung function. We have also shown that chronic S. maltophilia infection is an independent risk factor for pulmonary exacerbation requiring hospitalization and antibiotic therapy. This association was not observed in patients with intermittent S. maltophilia infection. These data suggest that chronic exposure of the respiratory tract of patients with CF to S. maltophilia may predispose to tissue invasion, resulting in rising antibody levels to S. maltophilia, pulmonary inflammation, and exacerbations.
Chronic infection can be defined in different ways. Multiple studies have used definitions based on number of positive cultures in a given year and shown that chronic, but not intermittent, methicillin-resistant S. aureus and A. fumigatus infection in patients with CF are associated with more severe lung disease (6–8). The original definition of chronic P. aeruginosa infection in patients with CF was based on both the presence of repeated positive respiratory tract cultures and rising P. aeruginosa antibody titers (13). The group in Leeds subsequently validated a definition of different stages of P. aeruginosa infection based on the percentage of positive cultures and showed that chronic infection was associated with increased disease severity and higher anti–P. aeruginosa antibody results (9). Several investigators have developed serologic assays to detect antibodies to P. aeruginosa in patients with CF and have shown that higher titers are associated with a worse clinical status, validating their own specific definitions of chronic infection (14, 15). In our study, the microbiologic classification of two or more positive cultures in a year as chronic infection was also supported by the selective immune response demonstrated in this group.
The immune response to whole-cell S. maltophilia and flagellin in patients who are chronically infected was approximately two-fold that seen in patients who never had S. maltophilia, suggesting that these patients are truly infected and not simply colonized (16). The serologic response to whole-cell S. maltophilia suggests that antibodies may be recognizing epitopes exposed on the bacterial surface (17). However, only the antibody response to flagellin, the main structural component of flagellae, correlated with lower FEV1 percent predicted. Flagellae are highly immunogenic and are a common, conserved feature of reference and clinical isolates (10). Even though S. maltophilia, like P. aeruginosa, may lose its motility in the CF lung over time (18), antibodies to gene products expressed early in the pathogenesis of infection, such as flagella, persist (13). In addition, S. maltophilia flagellae are antigenically distinct from those of P. aeruginosa, which prevented cross-reactivity with antibodies to this common CF pathogen in our study (10). Unlike serologic assays for P. aeruginosa in patients with CF, however, this serologic assay did not detect increased antibody levels to S. maltophilia protease (13, 19). Clinical strains of S. maltophilia are known to produce extracellular alkaline serine protease (11) and protease production may play a role in the development of acute fulminant hemorrhagic pneumonia caused by S. maltophilia in patients who are immunocompromised (20). In patients with CF, however, chronic infection with S. maltophilia may be associated with adaptive, phenotypic changes in the bacteria, such as loss of protease production (21).
The ability of S. maltophilia to cause infection has been well characterized in vitro. Clinical strains of S. maltophilia have been shown to adhere to, form biofilm on, and invade CF airway epithelial cells, albeit at low levels (22). Clinical S. maltophilia strains have direct cytotoxic effects on a variety of cells (23) and are highly immunostimulatory, eliciting significant IL-8 expression by airway epithelial cells, and tumor necrosis factor (TNF)-α production by macrophages (18, 24). In two separate studies using a mouse model of pneumonia, S. maltophilia, similar to P. aeruginosa, caused pneumonia with a significant associated inflammatory response, mediated primarily by neutrophils (18, 25). Compared with P. aeruginosa, S. maltophilia induced substantially more TNF-α in the murine lung. TNF-α, a potent proinflammatory cytokine that induces neutrophil activation, seems to be important in the pathogenesis of S. maltophilia infection because significantly fewer TNFR1 null mice compared with wild-type mice developed pneumonia and bacteremia (18). Thus, although S. maltophilia does not readily invade, causing sepsis and increased mortality (18), it does contribute significantly to airway inflammation.
There are few studies, however, investigating the effect of S. maltophilia on clinical outcomes in CF (26–28). The largest epidemiologic study using the CF Foundation National Patient Registry found no association between S. maltophilia and short-term survival (3 yr) or declining lung function (4, 5). However, only short-term outcomes were assessed, antibiotic treatment of S. maltophilia infection was not taken into account, and the effect on pulmonary exacerbations was not determined. In addition, the authors focused primarily on patients with CF who were positive at any point in time for S. maltophilia. To determine the impact of repeated S. maltophilia detection on lung function, they defined chronic infection as S. maltophilia detected for 2 or more years (not necessarily consecutive). This could include a patient with one out of four cultures positive 1 year and one out of four cultures positive the following year, which is not a validated definition of chronic infection in CF (9). Our study adjusted for S. maltophilia antibiotic therapy, had twice as long a follow-up time, and used a validated definition of chronic infection. Similar to the study by Goss and coworkers (4), chronic S. maltophilia infection was not associated with an increased rate of decline in FEV1 percent predicted in patients with CF. The length of patient follow-up may still not have been long enough and the rate of decline in FEV1 (∼1% per year) may have been too slow to detect the effects of chronic infection on pulmonary function. It is possible that these effects occur predominantly in younger patients before the onset of severe bronchiectasis, but our study is underpowered to detect this because of the small number of pediatric patients with CF with chronic S. maltophilia. Chronic S. maltophilia infection, however, was a risk factor for hospitalization for pulmonary exacerbation requiring antibiotic therapy even after adjusting for other factors associated with increased lung disease severity, such as age, P. aeruginosa infection, and baseline FEV1 percent predicted. This risk was similar to that seen for P. aeruginosa infection and suggests that S. maltophilia may be playing a role as a pathogen in patients with CF.
There are several limitations to this study. Antibodies to whole cell S. maltophilia were measured using the laboratory ATCC strain and strain-specific immune responses may not have been detected (29). In addition, serologic responses in patients with CF were measured at one point in time with corresponding cross-sectional clinical data. It is possible that higher S. maltophilia antibody levels are associated with lower FEV1 because chronic S. maltophilia occurs more frequently in patients who are sicker. Similarly, the increased risk of hospitalization for pulmonary exacerbation in patients with chronic S. maltophilia may simply reflect an increased intensity of therapy in an already severely affected patient population. Antibiotic use is a known risk factor for the isolation of S. maltophilia in the respiratory tract of patients with CF (30–33). Increased treatment of pulmonary exacerbations may select out S. maltophilia in the airways of patients with CF who are older and have lower lung function. Studies that examine patient cohorts before and after the acquisition of infection are more appropriate in determining the effect of organisms on lung function but require large sample sizes (6, 34). Therefore, to address the question of whether chronic S. maltophilia directly results in worsening lung function, prospective longitudinal studies examining the change in immune responses over time and corresponding clinical outcomes are needed.
This study shows that patients with CF with chronic S. maltophilia mount a specific immune response to this organism that is associated with worse lung function, suggesting that this represents a true infection. Whereas previous studies have suggested that S. maltophilia simply colonizes the CF airways and does not affect pulmonary function, we have demonstrated for the first time that chronic S. maltophilia infection is an independent risk factor for pulmonary exacerbation requiring hospitalization and antibiotics. Further studies are required to define the role of S. maltophilia in pulmonary exacerbations in patients with CF.
The authors gratefully acknowledge Drs. Mary Corey and Annie Dupuis for their statistical support and assistance with the Cystic Fibrosis Database. They also acknowledge Dr. Niels Hoiby for his expertise and advice in the development of the serologic assay.
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