INTRODUCTION

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Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane regulator gene resulting in abnormal chloride ion transport across epithelia. Affected individuals have defective cyclic AMP-regulated chloride channel activity involving several organs, including the pancreas, biliary tract, gastrointestinal tract, and airways. Pathological findings in the lung include retention of secretions, harboring a variety of microorganisms, in the distal airways. When cultured from the respiratory tracts of CF patients, Staphylococcus aureus, Pseudomonas aeruginosa, and Burkholderia cepacia are associated with increased morbidity and mortality (22). Marked inflammation occurs in these airways, causing progressive bronchiectasis until the loss of functioning airways results in respiratory insufficiency and, eventually, death.

Investigators participating in the Epidemiologic Study of Cystic Fibrosis (ESCF) project began to collect microbiological, pulmonary, and nutritional data on CF patients at 180 North American sites in 1994 in order to describe the disease burden and course, current practice patterns, and safety and effectiveness of dornase alfa (Pulmozyme) in the general CF patient population. All CF patients were invited to enroll regardless of whether they were receiving this medication. Enrollment at the time of the survey included more than 18,000 of the estimated 20,000 to 30,000 CF patients living in North America. The present survey of microbiology practice was undertaken in August 1995 to determine, for each site, the frequencies of performance of sputum and throat cultures, the culture techniques used, and the frequency with which certain organisms are tested for antibiotic susceptibility. These microbiological practice patterns were then correlated with the reported prevalences of S. aureus, P. aeruginosa, and B. cepacia at the participating sites.

Coincident with this survey, the recommendations of a CF Foundation consensus conference on microbiological procedures for evaluation of infectious diseases in CF patients were published (8). This permitted a comparison of the current practices in the participating CF patient care sites with these guidelines as well as those previously published for CF specimens (20).

	MATERIALS AND METHODS

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Data on the microbiology practices of the ESCF sites were obtained by survey. Surveys were mailed to the 180 enrolled ESCF sites in August 1995. Survey part A, which dealt with clinical data and indications for specific cultures and sensitivities, was completed by the principal investigator and the study coordinator. Part B, which consisted of questions regarding specific culture media, was completed by, or in conjunction with, the director of the site's clinical microbiology laboratory. Completed surveys were blinded as to site and then forwarded for data management, processing, and analysis. Site confidentiality was maintained throughout the survey.

Of the 180 surveys sent, 142 (79%) were returned within 6 months. These 142 sites represent 15,328 enrolled patients.

The 2-year cumulative prevalences of S. aureus, P. aeruginosa, P. aeruginosa of mucoid phenotype, and B. cepacia were calculated from data on the case report forms submitted to the ESCF by each participating site for the period from 12 months prior to enrollment to 1 year after enrollment in the ESCF. On the ESCF case report form completed at the time of enrollment were recorded the presence or absence of these bacteria for the 12 months prior to enrollment, the source of the cultured sample (throat or sputum), and other clinical information. On the case report form completed at each subsequent encounter were recorded the presence or absence of these bacteria, the source, and clinical information at the time the patient visited the site. A report of at least one culture in each of two successive years (12 months prior to and 1 year following enrollment) was needed for the patient to enter the prevalence analysis. This analysis included patients who enrolled before March 1996. The predicted forced expiratory volume in 1 s (FEV₁) percentage was calculated from the FEV₁, gender, and height for those 6 years or older at the time of enrollment, using Knudson standards (13). The highest value reported for the period from 6 months prior to enrollment to 1 year after enrollment was used. The age used was that at the midpoint of the period. These data were available for 14,577 patients.

To assess the association between clinical and laboratory practices and observed bacterial prevalence rates, the sites were divided into two groups, those using a complete protocol and those using a partial protocol. To be categorized as using a complete protocol, a site had to report on its survey the use of the following elements: (i) the procurement and culture of throat swab specimens from patients who did not produce sputum; (ii) inoculation of specimens onto media selective for S. aureus, P. aeruginosa, and B. cepacia; (iii) performance of additional biochemical tests on, or referral to a reference laboratory for, organisms that the site's routine identification system failed to identify; and (iv) reporting of all isolates of S. aureus, P. aeruginosa, mucoid P. aeruginosa, and B. cepacia to the clinic. These sites were compared with those that did not employ one or more elements of this complete protocol. Because the selective media differed for each organism, a site might use a complete protocol for S. aureus but not P. aeruginosa or B. cepacia; hence the sites were categorized for each of these bacteria and the results were compared. To assess the association of culture frequency with observed prevalence, the culture data from only the year after enrollment were used. The chi-square test of independence was used to determine the significance of differences among the observed prevalences.

	RESULTS

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Clinical practices. Sixty-four percent of the sites sent 100% of their respiratory tract specimens from CF patients to an on-site laboratory for culture. Of the ESCF sites that sent out respiratory specimens for culture, 64% sent out 25% or less.

Microbiology laboratory practices. While clinicians at 96% of responding sites indicated on the laboratory request form that the respiratory cultures were from CF patients, only 65% of the laboratories, covering 72% of the patients, required a clinical diagnosis.

Association of clinical and laboratory practices with observed bacterial prevalences. The reported 2-year cumulative prevalences of several organisms of clinical interest in CF were significantly different for sites using complete protocols and those using partial protocols. For the purposes of this analysis, we focused on S. aureus, P. aeruginosa, and B. cepacia since patients whose airway secretions yielded these organisms upon culture have been documented to have increased airway obstruction compared with similar CF patients in whom these organisms have not been detected (1, 7, 12, 15, 16, 21, 23, 24).

	DISCUSSION

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This survey describes the variation in the microbiology practices of 142 of 180 sites participating in a large multicenter study of CF. This variation in practice affects detection of these bacteria and, hence, the prevalence rates of microbes important in CF and must be taken into account when such rates are estimated and used to assess the impact of bacteria on disease status in CF patients. In particular, denominator data for epidemiological studies of the impact of H. influenzae, mycobacteria, yeasts, and fungi on disease progression in CF patients must be limited to sites performing routine culturing for the bacteria in question on selective media under optimal conditions.

All of the 142 sites request culturing of sputum produced by CF patients. However, if sputum cannot be produced, many sites do not request a throat swab culture. This is an important issue, since throat swab and sputum culture yields are similar for S. aureus and P. aeruginosa (9) and the frequency of detection of S. aureus and/or P. aeruginosa by throat swab culture in children under 2 years is associated with significantly increased morbidity and mortality in the following 10 years (12).

Ninety-six percent of the sites specify that the specimen is from a CF patient, although only 65% of the laboratories (representing 72% of the patients) require a clinical diagnosis. It is important that the diagnosis of CF be specified so that the laboratories are alerted to use appropriate media to enhance the detection of clinically important organisms (25).

Tablan et al. (20) showed that B. cepacia was isolated from simulated CF sputum by 95% of the laboratories using B. cepacia-selective medium (P. cepacia agar or oxidative-fermentative polymyxin B-bacitracin-lactose agar) but was detected by only 22% of the laboratories not using a selective medium. Our data show that the use of B. cepacia-selective media has increased significantly in the past 9 years, from 12% in Tablan's study to 73% in our study. Unfortunately, over half of the laboratories still do not carry out long-term incubation at a reduced temperature, conditions which may increase the yield of B. cepacia by allowing slower-growing colonies to become apparent. Further complicating matters, other organisms, such as Stenotrophomonas maltophilia, can grow on B. cepacia-selective media and be difficult to distinguish from B. cepacia, even with further biochemical testing (2). Early detection of B. cepacia in CF patients is extremely important with respect to both the individual patient and the CF population, since this organism appears to be spread from person to person among CF patients. Non-culture-based methods of identifying B. cepacia and P. aeruginosa, such as detection of species-specific DNA by PCR (4, 17), have been developed and may have more-widespread application in the future.

Routine culturing for organisms of clinical and prognostic importance in CF (S. aureus, P. aeruginosa, and B. cepacia) is carried out at most, but not all, sites. Most laboratories report these organisms routinely as being distinct from normal flora, and they almost all routinely report antimicrobial agent susceptibilities for these organisms. Mucoid Pseudomonas species are not reported as distinct from Pseudomonas species by over one-third of the laboratories. The presence of mucoid Pseudomonas species in respiratory secretions may have significant clinical implications (11, 14), making it potentially important to recognize these isolates as a distinct entity.

This survey indicates the degree to which practice currently follows the CF Foundation consensus conference recommendations. The sites follow these recommendations to various degrees. For example, 81% of the sites use protocols recommended for detection of P. aeruginosa, but only 65% follow the recommendation to use S. aureus-selective media for all CF patient sputum specimens. Less than half of the centers employ B. cepacia-selective culture media or incubate the cultures for extended periods, both of which improve the yield of this bacterium. These variations in laboratory practices contribute significantly to differences in the observed 2-year cumulative prevalences of several clinically important organisms in CF patients. These differences are most pronounced in the young. The contribution of each specific part of the complete protocol that accounted for the differences cannot be entirely determined because a site that completed one part of the protocol often completed the other parts as well and sample sizes allowing comparison of sites that used one part, with all other parts being equally employed or not employed, became too small. One specific part did, however, independently account for much of the variance: the more frequently cultures were obtained, the higher the observed prevalences for these bacteria. It appears likely that these differences in practice will affect estimates of the effect of these organisms on the clinical outcome.

This is the first large-scale survey of actual microbiological practices at a large number of CF patient care sites, representing over 14,000 CF patients. Several conclusions can be drawn from this survey. Substantial between-site differences exist in the protocols used for culturing the respiratory tracts of CF patients. This is particularly true for the use of throat swabs and thus affects the detection of S. aureus, P. aeruginosa, and B. cepacia most in the young CF patient. This results in significant differences in 2-year cumulative prevalences of bacteria between sites using complete protocols and those using partial protocols. Early detection of these organisms in this young CF patient population may significantly alter the therapy prescribed. In recent antibiotic trials, the degree of reduction of sputum bacterial colony counts correlated with the degree of reversal of airway obstruction in CF patients (18, 19). Hence, early detection of those microbes associated with greater airway obstruction at an earlier age in these patients (12) may motivate earlier appropriate antibiotic intervention and slow the development of airway obstruction in these patients. Finally, further communication among sites to routinely employ standard practices, as recommended by the CF Foundation and by a recent study by Burns et al. (3), will be valuable in improving the rates of detection of potentially pathogenic organisms associated with CF and may lead to improved infection control practices and allow for more-accurate measurement of the impact of new antimicrobial therapies in CF.