Impact of Burkholderia dolosa on Lung Function and Survival in Cystic Fibrosis
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《美国呼吸和危急护理医学》
Clinical Research Program
Divisions of Respiratory Diseases and Infectious Disease
Infection Control Program
Department of Laboratory Medicine, Children's Hospital Boston, Boston, Massachusetts
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
ABSTRACT
Rationale: Chronic infection with Burkholderia cepacia complex bacteria in cystic fibrosis is associated with accelerated decline in pulmonary function and increased mortality. Clinical implications of the recently characterized genomovar VI, B. dolosa, are unknown.
Objectives: Characterization of impact of B. dolosa on pulmonary function and mortality in cystic fibrosis.
Methods: We compared patients chronically infected with B. dolosa (n = 31) with unmatched patients with B. multivorans (n = 24) and with age- and sex-matched control subjects without Burkholderia species (n = 58). We analyzed rates of pulmonary function decline (% predicted FEV1) using a random effects model assuming segmented linear trends. All available FEV1 measurements from 5 yr (median, 4.8) before until 2.5 yr (median, 1.5) after the first positive culture for Burkholderia (reference date) were analyzed. Survival was compared using the Kaplan-Meier method and proportional hazards model.
Measurements and Main Results: Baseline FEV1 and rate of decline were similar in the cohorts. Decline in FEV1 after the reference date accelerated in patients with B. dolosa (–2.3 percentage points/yr pre vs. –7.1 post, p = 0.002), but was unchanged in the B. multivorans and control patients (–2.3 vs. –0.8 post, p = 0.38, and –2.1 pre vs. –0.5 post, p = 0.20, respectively). The probability of dying within 18 mo of the reference date was 13, 7, and 3% for B. dolosa, B. multivorans, and control patients, respectively (B. dolosa vs. control hazard ratio, 10.8; 95% confidence interval, 1.3–92.8; p = 0.03).
Conclusions: B. dolosa chronic infection in cystic fibrosis is associated with accelerated loss of lung function and decreased survival.
Key Words: Burkholderia cepacia complex Burkholderia multivorans infection control
The major source of morbidity and mortality in people with cystic fibrosis is chronic endobronchial infection, most commonly with Pseudomonas aeruginosa and Staphylococcus aureus (1). Chronic infection with multiantibiotic-resistant gram-negative bacteria of the Burkholderia cepacia complex has been associated with accelerated decline in lung function and reduced survival (2–4). The clinical course after chronic infection is highly variable. Some patients have a "cepacia syndrome," characterized by recurrent fevers, bacteremia, necrotizing pneumonia, and a progressive decline resulting in death within weeks to months, whereas others experience little apparent change in clinical status (5–11). Patient to patient transmission of B. cepacia complex has been documented (12), and outbreaks among patients attending cystic fibrosis care centers have been noted (13, 14). For these reasons, the Cystic Fibrosis Foundation recommends stringent infection-control measures for patients chronically infected with B. cepacia complex, including segregation of these patients from each other as well as from patients with cystic fibrosis not chronically infected with B. cepacia complex and restrictions on social contacts (15).
Initial reports of the clinical sequelae of chronic infection with B. cepacia complex in patients with cystic fibrosis and subsequent formal epidemiologic studies (2–4) did not distinguish among different B. cepacia complex species, or genomovars, which had not been fully characterized at the time (6–10, 16). Ongoing refinements in Burkholderia taxonomy have revealed that there are at least 10 distinct species within the B. cepacia complex, each of which recently was assigned a formal binomial designation (17). The relative virulence and clinical impact of these different species remain unclear, complicating clinical decisions and prognostic advice given to patients and their families. B. cenocepacia (genomovar III) is the most prevalent species, accounting for half of all clinical isolates in the United States, and is the organism that has been associated most often with the "cepacia syndrome" (18.) B. multivorans (genomovar II) accounts for one-third of U.S. clinical isolates. This species is generally believed to be less virulent than B. cenocepacia by cystic fibrosis specialists (19), but the cepacia syndrome has been reported with this species (19–21). There are no published data regarding the clinical outcome associated with chronic infection by the recently characterized B. dolosa (genomovar VI) (22, 23).
The Cystic Fibrosis Center at the study hospital has had a policy regarding segregation of patients with cystic fibrosis chronically infected with B. cepacia complex since 1995. Respiratory tract cultures from the cystic fibrosis clinic population are obtained regularly using selective media. B. cepacia complex strains are speciated and genotyped through the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository (18). In 2001, surveillance methods were developed to aggressively track trends in chronic endobronchial infection. As genotyping methods improved and B. cepacia complex taxonomy was revised, review of surveillance cultures and retyping of all available isolates previously characterized as B. multivorans revealed an increasing incidence of the then newly described genomovar VI, B. dolosa (22), whereas the incidence of other B. cepacia complex isolates, including B. cenocepacia, remained constant. This review also revealed that B. dolosa had been present in the study hospital's clinic population as early as 1992, when a previously chronically infected patient transferred to this center. Ultimately, 36 patients were recognized as chronically infected with B. dolosa by July 1, 2004.
Concurrent with this heightened surveillance, a patient who had been recently chronically infected with B. dolosa developed a clinical course consistent with cepacia syndrome, with rapid decline in pulmonary function, persistent pan-resistant B. dolosa bacteremia, and death within 4 mo. Several other patients appeared to have a decline in pulmonary function roughly coincident with B. dolosa chronic infection, whereas others suffered no clear deterioration in clinical status. Therefore, we undertook a formal cohort study to clarify the clinical course of patients who had been chronically infected with B. dolosa, compared with patients with cystic fibrosis chronically infected with B. multivorans and those who had not been chronically infected with any B. cepacia complex species.
METHODS
An online supplement provides additional details on methods used in this study. All patients with cystic fibrosis monitored at the study hospital with a respiratory tract culture positive for B. dolosa or B. multivorans between January 1, 1992, and July 1, 2004, were considered for analysis. Respiratory tract culture specimens of sputum, bronchoalveolar lavage samples, and/or deep pharyngeal swab were plated to oxidative-fermentation agar supplemented with lactose, polymyxin B, and bacitracin, which is selective for Burkholderia species (24). Bacteria growing on this medium were screened using the oxidase test followed by biochemical testing for presumptive identification of B. cepacia complex. Definitive testing was performed by the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository (18). Genotyping of B. dolosa isolates was performed using repetitive extragenic palendromic polymerase chain reaction and pulsed-field gel electrophoresis (25). Inclusion in the study as a B. dolosa or B. multivorans case required at least two separate positive cultures for these species. Four patients met criteria for inclusion in the study both as B. dolosa and B. multivorans cases.
For each B. dolosa case, an attempt was made to identify two control subjects from the clinic with no history of a culture positive for B. cepacia complex, matched by sex and born within ± 1 yr of the case's birth date. Because of the small number of patients with B. multivorans, it was not possible to match this cohort to the B. dolosa cohort. For each patient with B. dolosa and B. multivorans, a reference date was defined as the date of the first culture positive for the species. The reference date for control subjects was defined as the corresponding B. dolosa case's reference date.
For inclusion in the study, patients were required to have at least one measurement of FEV1 before and one after their reference date. All FEV1 measurements are expressed as percent predicted (26). All available FEV1 measurements from 5 yr prior to 2.5 yr after the reference date were analyzed. Not all patients had the full extent of follow-up (see RESULTS). Preliminary analyses including FEV1 values beyond 2.5 yr after the reference date yielded unstable estimates, because only seven B. dolosa cases had available lung function measurements during this period.
FEV1 values as close as possible to a date 6 mo before the reference date were used to define a patient's baseline. This interval was chosen to allow for possible delay between chronic infection with Burkholderia and detection by culture.
A random-effects model assuming linear trends in FEV1 before and after the reference date was used. The segmented model assumed the "pre" and "post" lines joined on the reference date but that the slopes of the two lines could be different.
Statistical significance was set at p < 0.05, and two-sided p values are reported throughout. SAS version 9.0 software was used for all analyses (SAS Institute, Inc., Cary, NC).
RESULTS
A total of 36 patients were identified with positive cultures for B. dolosa before July 1, 2004. All patients with B. dolosa included in this study had multiple (> 3) positive cultures after first isolation, at time intervals ranging from a few weeks to a few months. These isolates had a distinct genotype (SLC6) (25), which differed from nine other B. dolosa strains in the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository. Two patients had no FEV1 data before the reference date, two had no FEV1 data after the reference date, and one patient had only a single positive culture and was excluded, leaving 31 cases for analysis (B. dolosa cohort). Thirty-six patients with B. multivorans were identified. One had no FEV1 data before the reference date, two had no FEV1 data after the reference date, one patient (who transferred to Boston from another institution) had incomplete culture data, resulting in an uncertain date of chronic infection, and eight had only a single positive culture, leaving 24 B. multivorans patients for analysis (B. multivorans cohort). As mentioned in METHODS, 4 of these 24 patients were also in the B. dolosa cohort. Fifty-eight non–B. cepacia complex control subjects were identified, two for each of 27 B. dolosa cases and one for each of four B. dolosa cases (non–B. cepacia cohort). Four control subjects had birth dates outside the ± 1-yr matching window (maximum, 3.0 yr).
The earliest and latest FEV1 measurements were a median of 4.8 and 1.5 yr before and after the reference date, respectively. The average numbers of FEV1 measurements per patient were 46.1, 40.1, and 31.8 in the B. dolosa, B. multivorans, and non– B. cepacia cohorts, respectively. When restricting to the highest FEV1 measurement in each quarter, the corresponding averages were 18.8, 15.6, and 17.5. Overall, the three cohorts were well balanced with respect to a variety of baseline characteristics (Table 1). In particular, the mean (± SD) baseline percent-predicted FEV1 was similar in the three cohorts: 78.9 (± 27.5), 81.9 (± 24.9), and 78.9 (± 24.2), respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts (p = 0.68). The B. multivorans cohort had a higher prevalence of pancreatic sufficiency and, although not statistically significant, the B. dolosa cohort had a lower prevalence of diabetes mellitus.
As shown in Table 2 and Figure 1, the rates of decline in percent-predicted FEV1 before the reference date were similar in the three cohorts (–2.3, –2.3, and –2.1 percentage points/yr, respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts). The rate of decline in lung function after the reference date accelerated significantly in the B. dolosa cohort (–2.3 pre vs. –7.1 post, p = 0.002) but was unchanged in the B. multivorans cohort (–2.3 pre vs. –0.8 post, p = 0.38) and in the non– B. cepacia cohort (–2.1 pre vs. –0.5 post, p = 0.20; Table 2). There were no significant departures from the assumption of linear changes over time.
Similar results were seen when the analysis was performed using only the highest FEV1 values in each 3-mo interval before and after the reference date (Table 2). Similar results were also seen in secondary analyses when the four patients who converted from B. multivorans to B. dolosa were excluded from the B. multivorans cohort, and when their FEV1 measurements after conversion were included in the analysis (data not shown).
With median follow-up for survival of 19 mo after the reference date, there were five, two, and one deaths, respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts. The respective Kaplan-Meier estimates of dying within 18 mo were 13, 7, and 3% (B. dolosa vs. non–B. cepacia hazard ratio, 10.8: 95% confidence interval, 1.3–92.8; p = 0.03; B. multivorans vs. non–B. cepacia hazard ratio, 3.8; 95% confidence interval, 0.3–42.8; p = 0.27). The wide confidence intervals in our hazard ratio estimates reflect the small numbers of deaths in each group. Survival analysis results were similar when survival times for patients who converted from B. multivorans to B. dolosa were not censored at the time of conversion.
DISCUSSION
We report the first epidemiologic study regarding the clinical implications of chronic infection with B. dolosa in cystic fibrosis. Acquisition of this organism was associated with an accelerated decline in lung function (Figure 1 and Table 2) and reduced survival. Conversely, we did not observe an accelerated decline in lung function in patients chronically infected with B. multivorans, and survival in these patients was intermediate to the other two cohorts, although not significantly different from either. These observations help explain seemingly conflicting clinical and epidemiologic reports concerning the prognosis of patients chronically infected with B. cepacia complex (2–11). All of these reports preceded our current appreciation that at least 10 distinct species comprise the B. cepacia complex. Our findings provide additional evidence that, as previously reported (27), B. cepacia complex species differ in their virulence and clinical implications, and underscore the importance of examining the clinical effects of individual strains.
We elected to match on age and sex but not on "baseline" FEV1 in our study design, allowing us to make the important observation that prior reduced lung function is not associated with acquisition of B. dolosa or B. multivorans (Figure 1 and Table 1). Severity of underlying cystic fibrosis has been noted to be a risk factor for acquisition of B. cepacia complex in some (6, 7), but not all (8), prior reports. These disparate findings may reflect differences between patient populations in host susceptibility factors or in the distribution and virulence of B. cepacia complex species.
We sought to determine if the worse clinical course in patients with B. dolosa was due to confounding by variables that have been shown to be associated with a poor clinical outcome in cystic fibrosis (baseline FEV1, weight-for-age z score, diabetes mellitus, pancreatic insufficiency, and the absence of S. aureus) (3). The cohorts were well balanced with respect to all patient characteristics (Table 1) except for pancreatic sufficiency and diabetes mellitus. It was difficult to evaluate possible confounding effects of these characteristics because they are relatively rare. However, we saw similar results when the analysis was repeated within the subgroups of patients with pancreatic insufficiency and in those without diabetes mellitus (data not shown), suggesting that cohort imbalances did not impact the main conclusions.
Our experience reinforces the importance of fully exploiting ongoing refinements in speciation of B. cepacia complex bacteria. B. dolosa was not recognized as a species distinct from B. multivorans during the initial stages of the outbreak in the study hospital, complicating identification of the increasing incidence of B. dolosa. It later became clear that this study hospital has the largest single group of patients chronically infected with B. dolosa (25). All B. dolosa isolates from this group of patients are identical by genotyping analysis. Interestingly, this strain, designated SLC6, was identified in the patient who transferred to the study hospital in 1992 and is the same strain implicated in the first reports of interpatient spread of B. cepacia in cystic fibrosis (28, 29). A review of the prevalence of this strain in the clinic population of the study hospital indicates a low-level endemic presence for almost a decade before the rapid increase seen over the last few years.
Accurate, timely speciation and genotyping also will help clinicians provide appropriate counseling to patients chronically infected with individual species of the B. cepacia complex. This counseling must take into account the variable impact of B. cepacia complex species, and individual strains within species. Indeed, not all of the patients in the B. dolosa cohort had acceleration in the rate of decline in lung function. Similarly, there were variable rates of decline in patients in the B. multivorans and non–B. cepacia complex cohorts.
These discussions with patients must also take into account limitations of both the current study and the published literature regarding the clinical impact of B. cepacia complex. Because all of the strains in the study population were identical, and differed from other isolates referred to the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository, it is not known whether the results observed in the study population can be extrapolated to patients with other strains of B. dolosa. At present, there are too few patient isolates with B. dolosa from hospitals other than the study hospital in the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository to generate meaningful comparison data. It is also unclear how the clinical course of patients with B. dolosa compares with that of patients with the most prevalent genomovar, B. cenocepacia. The study hospital has only had eight patients with B. cenocepacia in the last decade, preventing meaningful comparison to the patients with B. dolosa. Published reports from other centers regarding mortality and the rate of decline in FEV1 in patients with B. cenocepacia are hampered by inclusion of patients with strains other than B. cenocepacia in some of the studies and/or conflicting data regarding outcomes (2–11).
This article suggests several avenues for future study. Because all B. dolosa–infected patients in this study were infected with the same strain, further work is needed to determine the microbial factors that may account for the apparent increased virulence of this strain, and to determine whether these factors are shared by all strains within this species. The importance of host susceptibility factors to B. cepacia complex, such as mannose binding lectin (30), and the nature and efficacy of the humoral and cellular response to chronic infection need to be evaluated in light of the variable clinical course of individual patients. The rapid increase in incidence of B. dolosa among the study hospital's patient population, during a time in which the incidence of other B. cepacia complex isolates remained stable, suggests increased transmissibility of the implicated strain. An exhaustive investigation failed to identify an environmental reservoir of B. dolosa in this hospital, or any other common source exposure for chronically infected patients. However, there was substantial overlap in dates of clinic visits and hospital stays between patients who were already chronically infected and those who were later chronically infected with B. dolosa. Of note, strain SLC6 contains neither B. cepacia epidemic strain marker nor capable pilin subunit gene, markers which were previously believed to be associated with B. cepacia complex strains with enhanced transmissibility (18).
Finally, current infection-control recommendations for patients with cystic fibrosis with and without B. cepacia complex should be interpreted in light of our findings suggesting increased transmissibility of at least this strain of B. dolosa despite seemingly appropriate infection-control measures based on Cystic Fibrosis Foundation guidelines. These recommendations currently state, in part, that patients with B. cepacia complex should be segregated from other patients and from each other (15). Cohorting of patients chronically infected with B. cepacia complex in a separate ward or clinic, without rigorous separation of these patients from each other, may have unintended consequences. Replacement of B. multivorans with more virulent B. cenocepacia has been reported (14, 31–33), and we observed four cases in which B. multivorans was replaced by B. dolosa. Conversely, eight patients not included in this analysis had only one culture positive for B. multivorans, and six additional patients who were included in the study eventually became culture negative for this species; only one patient with B. dolosa became culture negative. Cohorting of patients chronically infected with B. cepacia complex without separation is thus not a rational infection-control policy. Such a strategy potentially would expose patients chronically infected with relatively less virulent Burkholderia species to patients chronically infected with more virulent species (34). In addition, the use of standard precautions without contact precautions for patients with cystic fibrosis not chronically infected with methicillin-resistant S. aureus, B. cepacia complex, and multidrug-resistant P. aeruginosa (15) should be reevaluated. Standard precautions commonly stipulate that the use of barrier precautions (i.e., gown and gloves) be based on the type of anticipated exposure to respiratory secretions. For instance, gloves are recommended "for touching blood, body fluids, secretions, excretions, contaminated items" and "for touching mucous membranes and nonintact skin" and gowns are recommended "during procedures and patient care activities likely to generate splashes or sprays of blood, body fluids, secretions, excretions" (15). Because it is not possible to anticipate when health care workers might come in contact with respiratory secretions during an encounter with a patient with cystic fibrosis, or which organisms that patient might harbor at that time, we advocate the universal use of barrier precautions by all health care workers during every interaction with a patient with cystic fibrosis.
FOOTNOTES
These authors contributed equally to this article.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200503-344OC on November 4, 2005
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject matter of this manuscript.
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Divisions of Respiratory Diseases and Infectious Disease
Infection Control Program
Department of Laboratory Medicine, Children's Hospital Boston, Boston, Massachusetts
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
ABSTRACT
Rationale: Chronic infection with Burkholderia cepacia complex bacteria in cystic fibrosis is associated with accelerated decline in pulmonary function and increased mortality. Clinical implications of the recently characterized genomovar VI, B. dolosa, are unknown.
Objectives: Characterization of impact of B. dolosa on pulmonary function and mortality in cystic fibrosis.
Methods: We compared patients chronically infected with B. dolosa (n = 31) with unmatched patients with B. multivorans (n = 24) and with age- and sex-matched control subjects without Burkholderia species (n = 58). We analyzed rates of pulmonary function decline (% predicted FEV1) using a random effects model assuming segmented linear trends. All available FEV1 measurements from 5 yr (median, 4.8) before until 2.5 yr (median, 1.5) after the first positive culture for Burkholderia (reference date) were analyzed. Survival was compared using the Kaplan-Meier method and proportional hazards model.
Measurements and Main Results: Baseline FEV1 and rate of decline were similar in the cohorts. Decline in FEV1 after the reference date accelerated in patients with B. dolosa (–2.3 percentage points/yr pre vs. –7.1 post, p = 0.002), but was unchanged in the B. multivorans and control patients (–2.3 vs. –0.8 post, p = 0.38, and –2.1 pre vs. –0.5 post, p = 0.20, respectively). The probability of dying within 18 mo of the reference date was 13, 7, and 3% for B. dolosa, B. multivorans, and control patients, respectively (B. dolosa vs. control hazard ratio, 10.8; 95% confidence interval, 1.3–92.8; p = 0.03).
Conclusions: B. dolosa chronic infection in cystic fibrosis is associated with accelerated loss of lung function and decreased survival.
Key Words: Burkholderia cepacia complex Burkholderia multivorans infection control
The major source of morbidity and mortality in people with cystic fibrosis is chronic endobronchial infection, most commonly with Pseudomonas aeruginosa and Staphylococcus aureus (1). Chronic infection with multiantibiotic-resistant gram-negative bacteria of the Burkholderia cepacia complex has been associated with accelerated decline in lung function and reduced survival (2–4). The clinical course after chronic infection is highly variable. Some patients have a "cepacia syndrome," characterized by recurrent fevers, bacteremia, necrotizing pneumonia, and a progressive decline resulting in death within weeks to months, whereas others experience little apparent change in clinical status (5–11). Patient to patient transmission of B. cepacia complex has been documented (12), and outbreaks among patients attending cystic fibrosis care centers have been noted (13, 14). For these reasons, the Cystic Fibrosis Foundation recommends stringent infection-control measures for patients chronically infected with B. cepacia complex, including segregation of these patients from each other as well as from patients with cystic fibrosis not chronically infected with B. cepacia complex and restrictions on social contacts (15).
Initial reports of the clinical sequelae of chronic infection with B. cepacia complex in patients with cystic fibrosis and subsequent formal epidemiologic studies (2–4) did not distinguish among different B. cepacia complex species, or genomovars, which had not been fully characterized at the time (6–10, 16). Ongoing refinements in Burkholderia taxonomy have revealed that there are at least 10 distinct species within the B. cepacia complex, each of which recently was assigned a formal binomial designation (17). The relative virulence and clinical impact of these different species remain unclear, complicating clinical decisions and prognostic advice given to patients and their families. B. cenocepacia (genomovar III) is the most prevalent species, accounting for half of all clinical isolates in the United States, and is the organism that has been associated most often with the "cepacia syndrome" (18.) B. multivorans (genomovar II) accounts for one-third of U.S. clinical isolates. This species is generally believed to be less virulent than B. cenocepacia by cystic fibrosis specialists (19), but the cepacia syndrome has been reported with this species (19–21). There are no published data regarding the clinical outcome associated with chronic infection by the recently characterized B. dolosa (genomovar VI) (22, 23).
The Cystic Fibrosis Center at the study hospital has had a policy regarding segregation of patients with cystic fibrosis chronically infected with B. cepacia complex since 1995. Respiratory tract cultures from the cystic fibrosis clinic population are obtained regularly using selective media. B. cepacia complex strains are speciated and genotyped through the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository (18). In 2001, surveillance methods were developed to aggressively track trends in chronic endobronchial infection. As genotyping methods improved and B. cepacia complex taxonomy was revised, review of surveillance cultures and retyping of all available isolates previously characterized as B. multivorans revealed an increasing incidence of the then newly described genomovar VI, B. dolosa (22), whereas the incidence of other B. cepacia complex isolates, including B. cenocepacia, remained constant. This review also revealed that B. dolosa had been present in the study hospital's clinic population as early as 1992, when a previously chronically infected patient transferred to this center. Ultimately, 36 patients were recognized as chronically infected with B. dolosa by July 1, 2004.
Concurrent with this heightened surveillance, a patient who had been recently chronically infected with B. dolosa developed a clinical course consistent with cepacia syndrome, with rapid decline in pulmonary function, persistent pan-resistant B. dolosa bacteremia, and death within 4 mo. Several other patients appeared to have a decline in pulmonary function roughly coincident with B. dolosa chronic infection, whereas others suffered no clear deterioration in clinical status. Therefore, we undertook a formal cohort study to clarify the clinical course of patients who had been chronically infected with B. dolosa, compared with patients with cystic fibrosis chronically infected with B. multivorans and those who had not been chronically infected with any B. cepacia complex species.
METHODS
An online supplement provides additional details on methods used in this study. All patients with cystic fibrosis monitored at the study hospital with a respiratory tract culture positive for B. dolosa or B. multivorans between January 1, 1992, and July 1, 2004, were considered for analysis. Respiratory tract culture specimens of sputum, bronchoalveolar lavage samples, and/or deep pharyngeal swab were plated to oxidative-fermentation agar supplemented with lactose, polymyxin B, and bacitracin, which is selective for Burkholderia species (24). Bacteria growing on this medium were screened using the oxidase test followed by biochemical testing for presumptive identification of B. cepacia complex. Definitive testing was performed by the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository (18). Genotyping of B. dolosa isolates was performed using repetitive extragenic palendromic polymerase chain reaction and pulsed-field gel electrophoresis (25). Inclusion in the study as a B. dolosa or B. multivorans case required at least two separate positive cultures for these species. Four patients met criteria for inclusion in the study both as B. dolosa and B. multivorans cases.
For each B. dolosa case, an attempt was made to identify two control subjects from the clinic with no history of a culture positive for B. cepacia complex, matched by sex and born within ± 1 yr of the case's birth date. Because of the small number of patients with B. multivorans, it was not possible to match this cohort to the B. dolosa cohort. For each patient with B. dolosa and B. multivorans, a reference date was defined as the date of the first culture positive for the species. The reference date for control subjects was defined as the corresponding B. dolosa case's reference date.
For inclusion in the study, patients were required to have at least one measurement of FEV1 before and one after their reference date. All FEV1 measurements are expressed as percent predicted (26). All available FEV1 measurements from 5 yr prior to 2.5 yr after the reference date were analyzed. Not all patients had the full extent of follow-up (see RESULTS). Preliminary analyses including FEV1 values beyond 2.5 yr after the reference date yielded unstable estimates, because only seven B. dolosa cases had available lung function measurements during this period.
FEV1 values as close as possible to a date 6 mo before the reference date were used to define a patient's baseline. This interval was chosen to allow for possible delay between chronic infection with Burkholderia and detection by culture.
A random-effects model assuming linear trends in FEV1 before and after the reference date was used. The segmented model assumed the "pre" and "post" lines joined on the reference date but that the slopes of the two lines could be different.
Statistical significance was set at p < 0.05, and two-sided p values are reported throughout. SAS version 9.0 software was used for all analyses (SAS Institute, Inc., Cary, NC).
RESULTS
A total of 36 patients were identified with positive cultures for B. dolosa before July 1, 2004. All patients with B. dolosa included in this study had multiple (> 3) positive cultures after first isolation, at time intervals ranging from a few weeks to a few months. These isolates had a distinct genotype (SLC6) (25), which differed from nine other B. dolosa strains in the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository. Two patients had no FEV1 data before the reference date, two had no FEV1 data after the reference date, and one patient had only a single positive culture and was excluded, leaving 31 cases for analysis (B. dolosa cohort). Thirty-six patients with B. multivorans were identified. One had no FEV1 data before the reference date, two had no FEV1 data after the reference date, one patient (who transferred to Boston from another institution) had incomplete culture data, resulting in an uncertain date of chronic infection, and eight had only a single positive culture, leaving 24 B. multivorans patients for analysis (B. multivorans cohort). As mentioned in METHODS, 4 of these 24 patients were also in the B. dolosa cohort. Fifty-eight non–B. cepacia complex control subjects were identified, two for each of 27 B. dolosa cases and one for each of four B. dolosa cases (non–B. cepacia cohort). Four control subjects had birth dates outside the ± 1-yr matching window (maximum, 3.0 yr).
The earliest and latest FEV1 measurements were a median of 4.8 and 1.5 yr before and after the reference date, respectively. The average numbers of FEV1 measurements per patient were 46.1, 40.1, and 31.8 in the B. dolosa, B. multivorans, and non– B. cepacia cohorts, respectively. When restricting to the highest FEV1 measurement in each quarter, the corresponding averages were 18.8, 15.6, and 17.5. Overall, the three cohorts were well balanced with respect to a variety of baseline characteristics (Table 1). In particular, the mean (± SD) baseline percent-predicted FEV1 was similar in the three cohorts: 78.9 (± 27.5), 81.9 (± 24.9), and 78.9 (± 24.2), respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts (p = 0.68). The B. multivorans cohort had a higher prevalence of pancreatic sufficiency and, although not statistically significant, the B. dolosa cohort had a lower prevalence of diabetes mellitus.
As shown in Table 2 and Figure 1, the rates of decline in percent-predicted FEV1 before the reference date were similar in the three cohorts (–2.3, –2.3, and –2.1 percentage points/yr, respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts). The rate of decline in lung function after the reference date accelerated significantly in the B. dolosa cohort (–2.3 pre vs. –7.1 post, p = 0.002) but was unchanged in the B. multivorans cohort (–2.3 pre vs. –0.8 post, p = 0.38) and in the non– B. cepacia cohort (–2.1 pre vs. –0.5 post, p = 0.20; Table 2). There were no significant departures from the assumption of linear changes over time.
Similar results were seen when the analysis was performed using only the highest FEV1 values in each 3-mo interval before and after the reference date (Table 2). Similar results were also seen in secondary analyses when the four patients who converted from B. multivorans to B. dolosa were excluded from the B. multivorans cohort, and when their FEV1 measurements after conversion were included in the analysis (data not shown).
With median follow-up for survival of 19 mo after the reference date, there were five, two, and one deaths, respectively, in the B. dolosa, B. multivorans, and non–B. cepacia cohorts. The respective Kaplan-Meier estimates of dying within 18 mo were 13, 7, and 3% (B. dolosa vs. non–B. cepacia hazard ratio, 10.8: 95% confidence interval, 1.3–92.8; p = 0.03; B. multivorans vs. non–B. cepacia hazard ratio, 3.8; 95% confidence interval, 0.3–42.8; p = 0.27). The wide confidence intervals in our hazard ratio estimates reflect the small numbers of deaths in each group. Survival analysis results were similar when survival times for patients who converted from B. multivorans to B. dolosa were not censored at the time of conversion.
DISCUSSION
We report the first epidemiologic study regarding the clinical implications of chronic infection with B. dolosa in cystic fibrosis. Acquisition of this organism was associated with an accelerated decline in lung function (Figure 1 and Table 2) and reduced survival. Conversely, we did not observe an accelerated decline in lung function in patients chronically infected with B. multivorans, and survival in these patients was intermediate to the other two cohorts, although not significantly different from either. These observations help explain seemingly conflicting clinical and epidemiologic reports concerning the prognosis of patients chronically infected with B. cepacia complex (2–11). All of these reports preceded our current appreciation that at least 10 distinct species comprise the B. cepacia complex. Our findings provide additional evidence that, as previously reported (27), B. cepacia complex species differ in their virulence and clinical implications, and underscore the importance of examining the clinical effects of individual strains.
We elected to match on age and sex but not on "baseline" FEV1 in our study design, allowing us to make the important observation that prior reduced lung function is not associated with acquisition of B. dolosa or B. multivorans (Figure 1 and Table 1). Severity of underlying cystic fibrosis has been noted to be a risk factor for acquisition of B. cepacia complex in some (6, 7), but not all (8), prior reports. These disparate findings may reflect differences between patient populations in host susceptibility factors or in the distribution and virulence of B. cepacia complex species.
We sought to determine if the worse clinical course in patients with B. dolosa was due to confounding by variables that have been shown to be associated with a poor clinical outcome in cystic fibrosis (baseline FEV1, weight-for-age z score, diabetes mellitus, pancreatic insufficiency, and the absence of S. aureus) (3). The cohorts were well balanced with respect to all patient characteristics (Table 1) except for pancreatic sufficiency and diabetes mellitus. It was difficult to evaluate possible confounding effects of these characteristics because they are relatively rare. However, we saw similar results when the analysis was repeated within the subgroups of patients with pancreatic insufficiency and in those without diabetes mellitus (data not shown), suggesting that cohort imbalances did not impact the main conclusions.
Our experience reinforces the importance of fully exploiting ongoing refinements in speciation of B. cepacia complex bacteria. B. dolosa was not recognized as a species distinct from B. multivorans during the initial stages of the outbreak in the study hospital, complicating identification of the increasing incidence of B. dolosa. It later became clear that this study hospital has the largest single group of patients chronically infected with B. dolosa (25). All B. dolosa isolates from this group of patients are identical by genotyping analysis. Interestingly, this strain, designated SLC6, was identified in the patient who transferred to the study hospital in 1992 and is the same strain implicated in the first reports of interpatient spread of B. cepacia in cystic fibrosis (28, 29). A review of the prevalence of this strain in the clinic population of the study hospital indicates a low-level endemic presence for almost a decade before the rapid increase seen over the last few years.
Accurate, timely speciation and genotyping also will help clinicians provide appropriate counseling to patients chronically infected with individual species of the B. cepacia complex. This counseling must take into account the variable impact of B. cepacia complex species, and individual strains within species. Indeed, not all of the patients in the B. dolosa cohort had acceleration in the rate of decline in lung function. Similarly, there were variable rates of decline in patients in the B. multivorans and non–B. cepacia complex cohorts.
These discussions with patients must also take into account limitations of both the current study and the published literature regarding the clinical impact of B. cepacia complex. Because all of the strains in the study population were identical, and differed from other isolates referred to the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository, it is not known whether the results observed in the study population can be extrapolated to patients with other strains of B. dolosa. At present, there are too few patient isolates with B. dolosa from hospitals other than the study hospital in the Cystic Fibrosis Foundation B. cepacia Research Laboratory and Repository to generate meaningful comparison data. It is also unclear how the clinical course of patients with B. dolosa compares with that of patients with the most prevalent genomovar, B. cenocepacia. The study hospital has only had eight patients with B. cenocepacia in the last decade, preventing meaningful comparison to the patients with B. dolosa. Published reports from other centers regarding mortality and the rate of decline in FEV1 in patients with B. cenocepacia are hampered by inclusion of patients with strains other than B. cenocepacia in some of the studies and/or conflicting data regarding outcomes (2–11).
This article suggests several avenues for future study. Because all B. dolosa–infected patients in this study were infected with the same strain, further work is needed to determine the microbial factors that may account for the apparent increased virulence of this strain, and to determine whether these factors are shared by all strains within this species. The importance of host susceptibility factors to B. cepacia complex, such as mannose binding lectin (30), and the nature and efficacy of the humoral and cellular response to chronic infection need to be evaluated in light of the variable clinical course of individual patients. The rapid increase in incidence of B. dolosa among the study hospital's patient population, during a time in which the incidence of other B. cepacia complex isolates remained stable, suggests increased transmissibility of the implicated strain. An exhaustive investigation failed to identify an environmental reservoir of B. dolosa in this hospital, or any other common source exposure for chronically infected patients. However, there was substantial overlap in dates of clinic visits and hospital stays between patients who were already chronically infected and those who were later chronically infected with B. dolosa. Of note, strain SLC6 contains neither B. cepacia epidemic strain marker nor capable pilin subunit gene, markers which were previously believed to be associated with B. cepacia complex strains with enhanced transmissibility (18).
Finally, current infection-control recommendations for patients with cystic fibrosis with and without B. cepacia complex should be interpreted in light of our findings suggesting increased transmissibility of at least this strain of B. dolosa despite seemingly appropriate infection-control measures based on Cystic Fibrosis Foundation guidelines. These recommendations currently state, in part, that patients with B. cepacia complex should be segregated from other patients and from each other (15). Cohorting of patients chronically infected with B. cepacia complex in a separate ward or clinic, without rigorous separation of these patients from each other, may have unintended consequences. Replacement of B. multivorans with more virulent B. cenocepacia has been reported (14, 31–33), and we observed four cases in which B. multivorans was replaced by B. dolosa. Conversely, eight patients not included in this analysis had only one culture positive for B. multivorans, and six additional patients who were included in the study eventually became culture negative for this species; only one patient with B. dolosa became culture negative. Cohorting of patients chronically infected with B. cepacia complex without separation is thus not a rational infection-control policy. Such a strategy potentially would expose patients chronically infected with relatively less virulent Burkholderia species to patients chronically infected with more virulent species (34). In addition, the use of standard precautions without contact precautions for patients with cystic fibrosis not chronically infected with methicillin-resistant S. aureus, B. cepacia complex, and multidrug-resistant P. aeruginosa (15) should be reevaluated. Standard precautions commonly stipulate that the use of barrier precautions (i.e., gown and gloves) be based on the type of anticipated exposure to respiratory secretions. For instance, gloves are recommended "for touching blood, body fluids, secretions, excretions, contaminated items" and "for touching mucous membranes and nonintact skin" and gowns are recommended "during procedures and patient care activities likely to generate splashes or sprays of blood, body fluids, secretions, excretions" (15). Because it is not possible to anticipate when health care workers might come in contact with respiratory secretions during an encounter with a patient with cystic fibrosis, or which organisms that patient might harbor at that time, we advocate the universal use of barrier precautions by all health care workers during every interaction with a patient with cystic fibrosis.
FOOTNOTES
These authors contributed equally to this article.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200503-344OC on November 4, 2005
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject matter of this manuscript.
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