Cetrimide

Study objective: To investigate the effect of Pseudomonas aeruginosa infection on clinical parameters in Chinese patients with noncystic fibrosis and steady-state bronchiectasis.

Design: Prospective, cross-sectional clinicomicrobiological study with informed consent.

Setting: Consecutive outpatient recruitment from a specialist bronchiectasis respiratory clinic.

Patients: Outpatients (n = 100; 62 women; 55.1 [+ or -] 16.7 years old; [FEV.sub.1]/FVC 1.4 [+ or -] 0.7/2.1 [+ or -] 0.9 L), who had stable respiratory symptoms for more than 3 weeks.

Measurements and results: Respiratory pathogens isolated from the sputum were: Pseudomonas aeruginosa (33), Haemophilus influenzae (10), Moraxella catarrhalis (2), other Gram-negative bacilli (5), Streptococcus pneumoniae (6), Staphylococcus aureus (5), mycobacteria (3), and yeast (1). Clinical parameters in patients with positive isolation of P aeruginosa were compared with those without the organism in the sputum culture (non-P aeruginosa). In the P aeruginosa group, the [FEV.sub.1]/FVC ratio and sputum volume were lower (p [is less than] 0.005) and higher (p [is less than] 0.0001), respectively, than those of the non-P aeruginosa group. The [FEV.sub.1]/FVC ratio ([is less than] 60%) and sputum volume (grading [is greater than] 5) were independently associated with a positive sputum isolation of P aeruginosa with odds ratios of 3.1 (confidence interval [CI] 1.2 to 8.4; p [is less than] 0.01) and 4.7 (CI 1.6 to 13.3; p [is less than] 0.001), respectively.

Conclusions: P aeruginosa is the predominant respiratory pathogen isolated in the sputum of Chinese patients with steady-state bronchiectasis, and its isolation is associated with high sputum output ([is greater than or equal to] 75th quartile) and moderately severe airflow obstruction ([FEV.sub.1]/FVC [is less than] 60%). (CHEST 1998; 114:1594-1598)

Key words: bronchiectasis; Pseudomonas aeruginosa; sputum pathogens

Bronchiectasis is a debilitating disease with pathologic dilatation of the bronchial tree, and affected patients suffer from distressing regular sputum production and recurrent infective exacerbations. These patients also suffer from chronic colonization or infection of the usually sterile tracheobronchial tree, particularly with Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa.[1-4] In whites with idiopathic bronchiectasis, several studies[1-4] have suggested that P aeruginosa colonization is associated with extensive lung disease, severe airflow obstruction, and rapid decline in lung function parameters. However, little is known of the steady-state sputum microbiology in the nonwhite population. We have therefore performed this prospective study on the sputum bacteriology of patients in steady-state bronchiectasis and examined the clinical significance of isolating respiratory pathogens from the sputum culture of these patients.

MATERIALS AND METHODS

Study Design and Patient Recruitment

Consecutive patients with bronchiectasis, confirmed with high-resolution computed tomography, were recruited with informed consent into a baseline period (three consecutive weekly visits) to ensure that they were in steady-state bronchiectasis. The measurements of clinical and laboratory parameters were performed by a clinician and a technician who were unaware of the study protocol. A "steady-state" bronchiectasis was defined where there was an alteration of [is less than] 10% of a 24-h sputum volume, [FEV.sub.1], and FVC and where the deterioration in respiratory symptoms was absent at the three baseline visits. The study protocol had approval from the institutional ethics committee.

Parameters

At each visit, the patients were directly inquired as to the presence of respiratory symptoms, including cough, dyspnea, hemoptysis, sputum production, chest pain, and wheezing in the intervening period. Laboratory assessment included: 24-h sputum volume and sputum physical characteristics (purulence and viscosity scores). Lung function indexes were measured between 10 and 11 AM using standard protocols as recommended by the American Thoracic Society, with a pulmonary function analyzer (Model 2200; SensorMedics; Yorba Linda, CA).[5] The patients also underwent a full clinical evaluation and other investigations, including assessments of respiratory ciliary motility, serum immunoglobulin concentrations, and aspergillus precipitins; exclusion of tuberculosis; and other investigations as outlined by Cole[1] to determine the etiology of bronchiectasis.

Assessment of Sputum Physical Characteristics

Sputum was collected over a 24-h period by patients at home from 9 AM on the day before to 9 AM on the day of visit and stored in clear sterile plastic (60 mL) pots at 4 [degrees] C. Patients were instructed to completely empty the contents of their mouth before they expectorated into the sputum pots to ensure that there was minimal contamination by saliva. The 24-h sputum volume was determined as the mean of a collection of 3 consecutive days and graded as 1, 2, 3, 4, 5, and 6 for concentrations of 0 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, and [is greater than] 50 mL, respectively. The sputum purulence was determined by a scoring system modified from Lam et al,[6] Deneuville et al,[7] and Tsang et al.[8] The score (expressed as 0, 1, 2, 3, 4, 5, 6, 7, and 8 for the following descriptions: absence of sputum, completely transparent, almost transparent, translucent but colorless, opaque and milky white, gray, pale green, moderately green, and dark green sputum, respectively) was taken as the highest on three random aliquots selected from the center of a 24-h specimen. Sputum viscosity was assessed by using an orange stick to "lift" the center of a 24-h specimen, and the results were recorded using a 4-point scoring system (0, 1, 2, and 3 for no sputum, mild, moderate, and severe, respectively). All evaluations were done by a single observer who was blinded to the patient identity of the samples. The reproducibility of the sputum purulence and viscosity scores have been validated in a previous pilot study (Ho and Tsang, unpublished data).

Isolation of Sputum Bacteria

Patients received chest physiotherapy (at least 15 rain of expectoration-aiding maneuvers) before being instructed by a research physician to produce fresh sputum for microbiological evaluation. Fresh sputum was collected in sterile clear plastic pots between l0 and 11 AM after the patients emptied their mouth. Sputum specimens with or without blood stains were sent directly to the microbiology laboratory for processing and inoculation onto the culture media within 2 h of collection. The quality of each specimen was screened by Gram staining of a carefully selected portion of the sputum. Criteria used for acceptance were [is greater than] 25 leukocytes per field (magnification, x 100) and [is less than] 10 squamous epithelial cells per field (magnification, x 100).[9] Acceptable specimens were homogenized by using Sputasol (Model SR089A; Oxoid; Basingstoke, UK), which contained 0.1% dithiothreitol, 0.78% sodium chloride, 0.02% potassium chloride, 0.112% disodium hydrogen phosphate, and 0.02% potassium dihydrogen phosphate, according to the manufacturer's recommendation. The microbial densities of various bacteria were determined by inoculating the media with 10 [micro]L of Sputasol-treated specimen using a standard plastic loop. The bacterial densities in the sputum specimens were determined semiquantitatively by streaking four quarters of the bacteriologic agar as described previously.[10] Predominant growth was defined as the bacterial pathogen(s) present in excess of five colonies in the second, third, or fourth streak quarters.[10] Standard microbiological procedures were employed to identify all the sputum bacteria and classify them into pathogens (P aeruginosa and species, H influenzae, Moraxella catarrhalis, other Gram-negative bacilli, S pneumoniae, S aureus, mycobacteria, and yeast) or commensal bacteria (Neisseria sp, [Alpha]-hemolytic streptococci, diptheroids, and coagulase-negative staphylococci). The following enriched and selective media were used for isolation of bacteria: blood agar (Oxoid CM271 [Oxoid] with 5% defibrinated horse blood), MacConkey agar, chocholate agars supplemented with 18.9 U/mL bacitracin (Sigma; St. Louis, MO), mannitol salt agar (Oxoid CM85), and cetrimide-nalidixic acid agar (Oxoid CM559 and SR102). The blood agar plate was incubated under anaerobic condition. All other plates were inocubated at 37 [degrees] C in 5% [CO.sub.2] and the selective plates with negative results were reincubated and re-examined daily for 4 days before disposal.

Statistical Methods

Preliminary examination of lung function and sputum indexes in subgroups of patients with different sputum bacteria was made using analysis of variance with Tukey's procedure, which allowed for multiple comparisons. The clinical significance of a positive sputum culture for P aeruginosa was specifically examined by comparing the findings between patients with and patients without a positive sputum culture for the organism. Multipleregression analysis was used to test the hypothetical relationship between a positive culture of P aeruginosa and various clinical parameters. The 75th quartile for sputum volume and the 25th quartile for the [FEV.sub.1]/FVC ratio for the 100 patients were, respectively, 32.5 mL/d and 57%. Therefore, a daily sputum volume of [is greater than] 30 mL and an [FEV.sub.1]/FVC ratio of [is less than] 60% were therefore used as the cut-off values in the regression analysis. The statistical analysis was performed (SAS Institute; Cary, NC).[11] A p value of [is less than] 0.05 was taken as indicative of statistical significance.

RESULTS

Patient Demography and Clinical Details

Between September 1996 and July 1997, 100 consecutive patients with bronchiectasis with apparently stable symptoms and sputum production (mean age [+ or -] SD, 55.1 [+ or -] 16.7 years; 62 women) were recruited. Of the patients recruited, 33 were current smokers, 6 were ex-smokers, and 61 were nonsmokers. The other patient characteristics, including etiology of bronchiectasis, current medications, duration from onset of regular sputum production, spirometry, 24-h sputum volume grading, and sputum purulence and viscosity scores, are shown in Table 1. None of the patients had alteration in their regular medications nor other additional antibiotic therapy throughout the baseline period.

(*) Median (interquartile range). Refer to text for details of sputum score and grading system.

Sputum Bacteriology and Physical Assessment

The following bacteria were isolated: commensals (n = 35), P aeruginosa (n = 33), H influenzae (n = 10), S pneumoniae (n = 6), and S aureus (n = 5). Twenty-five of the 33 (75.8%) P aeruginosa isolates were mucoid strains. Other infrequently isolated pathogens included M catarrhalis (n = 2), Aeinetobaeter sp (n = 1), Citrobaeter sp (n = 1), Escherichia coli (n = 1), Pasteurella multicida (n = 1), Stenotrophomonas maltophilia (n = 1), Mycobacterium chalonae (n = 2), Mycobacterium avium-intracellulare (n = 1), and Torulopsis sp (n = 1).

The results of the sputum bacteriology study are shown in Table 2. None of the patients had more than one pathogenic bacteria isolated from their sputum. The patients were not significantly different in age, gender distribution, spirometry, 24-h sputum volume grading, sputum purulence, and viscosity scores among the commensal, H influenzae, S pneumoniae, S aureus, or other pathogen group. However, patients with P aeruginosa isolated from their sputum had more severe airflow obstruction ([FEV.sub.1]/ FVC ratio) and higher sputum volume than those who had commensals only (p [is less than] 0.05). No significant difference between the P aeruginosa group and noncommensals were found (p [is greater than] 0.05). The frequency of etiology for bronehiectasis was not significantly different among the subgroups (data not shown).

Data shown are mean ([+ or -] SD) or frequency of occurrence, except for the sputum volume, purulence, and viscosity scores, which are median (interquartile range). See text for details for sputum volume grading and purulence and viscosity scores. Other pathogens were M catarrhalis (n = 2), Acinetobacter sp (n = 1), Citrobacter sp (n = 1), Escherichia coli (n = 1), Pasteurella multicida (n = 1), S maltophilia (n = 1), Mycobacterium chalonae (n = 2), M avium-intracellularae (n = 1), and Torulopsis sp (n = 1). (*) p < 0.05 compared with the commensal group.

Association of Clinical Parameters With Isolation of P aeruginosa

The clinical significance of airway infection with P aeruginosa was specifically examined by comparing the clinical parameters between patients with and patients without a positive sputum culture for the organism (Table 3). The [FEV.sub.1]/FVC ratio and sputum volume grading were significantly different between the P aeruginosa and non-P aeruginosa patient groups (p [is less than] 0.005 and p [is less than] 0.001, respectively). Two clinical parameters, including a daily sputum output of [is greater than] 30 mL and an [FEV.sub.1]/FVC ratio of [is less than] 60%, were found to be independently associated with the isolation of P aeruginosa from sputum with odds ratios of 3.1 (confidence interval, 1.2 to 8.4; p [is less than] 0.01), and 4.7 (confidence interval, 1.6 to 13.3; p [is less than] 0.001), respectively. There was no significant interaction between these variables. The other clinical parameters were not associated with isolation of P aeruginosa, commensals, or other pathogens (p [is greater than] 0.05).

Data shown are mean ([+ or -] SD) or frequency of occurrence, except for the sputum volume, purulence, and viscosity scores, which are median (interquartile range). The non-P aeruginosa group included all patients with other pathogens as well as those with commensals only in their sputum culture. See text for details for sputum volume, purulence, and viscosity scores.

(*) p < 0.005.

([dagger]) p < 0.0001.

DISCUSSION

The relative frequencies of bacteria isolated in this study among Chinese with bronchiectasis were different from those reported in sputum studies from the West.[1-5,12,13] In studies performed on white patients in the 1970s and 1980s, H influenzae, S pneumoniae, and S aureus were the predominant bacteria isolated. The relative frequencies of these organisms were reported to be 83%, 35%, and 29%, respectively.[14] Although there was a trend toward more P aeruginosa (9.3%) and less H influenzae (22%), S pneumoniae (8%), and S aureus (5%) in more recent studies, the relative frequencies of P aeruginosa and H influenzae are still, respectively, lower and higher than those reported in our study.[l-5,12-13] During infective exacerbation, several studies have also shown that P aeruginosa was an important pathogen in Chinese patients with bronchiectasis.[2,5]

The differences in the bacteriology between Chinese and whites could be due to ethnic or other factors. For patients with mixed colonization or infection, it is true that recovery of S pneumoniae and S aureus may be masked by a heavy growth of P aeruginosa, leading to falsely low isolation rates for the two organisms. For this reason, in our study we enhanced the growth of the former by incubating the blood agar plate anaerobically. P aeruginosa is a strict aerobe and thus will be inhibited. For the latter organism, its recovery was facilitated by the inclusion of a mannitol salt agar. The observed difference between our findings and others in terms of the types and frequencies of isolation of organisms is therefore unlikely to be a laboratory artifact. Overprescription of oral antibiotics without antipseudomonal activity could be another factor that contributes to the predominance of P aeruginosa in our patients. Among local practices outside the hospital, the use of antibiotics for bronchiectasis during the steady state appears to be common. This point warrants clarification with further studies, because it has potential implications for the use of antibiotics during the steady state because the transient benefit obtained with antibiotic therapy might be offset by selection for P aeruginosa. In both animal and human studies, there is increasing evidence that P aeruginosa colonization or infection will lead to accelerated disease progression. To complicate the issue further is the fact that, once the infection is established, it is often impossible to eradicate the bacteria even with intensive antibiotic chemotherapy.

Our study and the findings of others suggest that P aeruginosa colonization or infection was associated with advanced disease in both whites and Chinese with noneystic fibrosis bronchiectasis, although it remains unclear whether P aeruginosa is the cause or the result of advanced disease. In our study of what we believed to be the largest reported series of Chinese bronchiectaties, P aeruginosa colonization or infection was found to be associated with moderately severe airflow obstruction (odds ratio 3.1) and high sputum output (odds ratio 4.7). It has been suggested that patients with bronchiectasis infected by P aeruginosa have more extensive and severe bronchiectasis on high-resolution computed tomography than those without P aeruginosa infection. According to Evans et al,[15] [FEV.sub.1] and FVC were both lower in P aeruginosa-colonized than in noncolonized whites with bronchiectasis. Furthermore, it has been suggested that lung function indexes were lower at the time of initial P aeruginosa colonization and that decline in lung function is faster in those colonized by P aeruginosa than those colonized by other organisms. In Japanese patients with chronic bronchitis, it has also been suggested that P aeruginosa infection plays an important role in patients who subsequently develop bronchiectasis,[16]

Our finding of predominant isolation of P aeruginosa, which constituted 51% of all pathogenic isolates, might be important for empirical antibiotic prescription of our patients during infective exacerbation. In our locality, all the antipseudomonal penicillins, cephalosporins, and quinolones remain highly active against P aeruginosa. If the clinician anticipates the isolation of P aeruginosa, then quinolones appear to be a good empirical choice as an oral antibiotic. Therapy for S pneumoniae is more problematic due to the rapid emergence of penicillin-resistant pneumococci. In Hong Kong, the rate of penicillin-resistant pneumococci rose dramatically from 18% in 1993 to 57% in 1996. Resistance of S pneumoniae to other drugs was also very high, being 45% for amoxycillin-clavulanate, 50% for cefuroxime, 66% for erythromycin, and 43% for ofloxacin (unpublished data). In a recent Asian surveillance program, alarming rates of penicillin-resistant pneumococci were also reported in Korea (73.4%), Japan (67.7%), Thailand (63.1%), and Vietnam (53.4%).[17,18] The rapid emergence of drug-resistant pneumococci is probably due partly to the widespread use of antibiotics in the community for common respiratory tract infections, although the precise reasons for this must be defined by large-scale, community-based studies. At present, high-dose amoxycillin is our treatment of choice for S pneumoniae if the minimal inhibitory concentration of the isolate is less than 2 [micro]g/mL. In the future, the newer quinolones, such as clinafloxacin and trovafloxacin, could play more important roles because they appear to be highly active in vitro. Furthermore, it would be of interest to assess our patients longitudinally and to determine whether or not sputum bacterial pathogens in steady state correlate with those in acute exacerbation, thereby defining the value of our data in empirical prescription of antibiotics in exacerbation of bronchiectasis.

ACKNOWLEDGMENTS: We thank Dr. Ian Lauder for his expert statistical advice and Mr. Raymond Leung for technical support. We are grateful to the patients who participated in this study.

REFERENCES

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[9] Bartlett JG, Ryan KJ, Wilson WR. Laboratory diagnosis of lower respiratory tract infections. In: Washington JA II, eds. Cumulative techniques and procedures in clinical microbiology. Washington, DC: American Society for Microbiology, 1987; 1-18

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[11] SAS Institute. Statistical Analysis System user's guide (statistics version). 5th ed. Cary, NC: SAS Institute, 1995

[12] Hill SL, Morrison HM, Burnett D, et al. Short term response of patients with bronchiectasis to treatment with amoxycillin given in standard or high doses orally and by inhalation. Thorax 1986; 41:559-565

[13] Hill SL, Briton D, Johnson M, et al. Sputum and serum pharmacokinetics of loracarbef (LY163892) in patients with chronic bronchial sepsis. J Antimicrob Chemother 1994; 33:129-136

[14] May JR. The chemotherapy of chronic bronchitis and allied disorders. 2nd ed. London: English Universities Press, 1972

[15] Evans SA, Turner SM, Bosch BJ, et al. Lung function in bronchiectasis: the influence of Pseudomonas aeruginosa. Eur Respir J 1996; 9:1601-1604

[16] Nagaki M, Shimura S, Tanno Y, et al. Role of chronic Pseudomonas aeruginosa infection in the development of bronchiectasis. Chest 1992; 102:1464-1469

[17] Song JH, and the Asian Network for Surveillance of Resistant Pneumococci (ANSORP) Study Group. Prevalence of drug-resistant pneumococci in 11 Asian countries. In: Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, DC: American Society (or Microbiology, 1997; Abstract C48, p. 54

[18] Ho PL, Yuen KY, Yam WC, et al. Changing patterns of susceptibilities of blood, urinary and respiratory pathogens in Hong Kong. J Hosp Infect 1995; 31:305-317

(*) From the University Departments of Microbiology (Dr. Pakleung Ho), Medicine (Drs. Ip, Lam, Chu-sek Ho, Yuen, and Tsang), and Paediatrics (Dr. Chan), Queen Mary Hospital, The University of Hong Kong, Pokfulam Road, Hong Kong. Manuscript received January 15, 1998; revision accepted June 29, 1998.

Correspondence to: KWT Tsang, MD (with Honors), MRCP (UK), FCCP, University Department of Medicine, Queen Mary Hospital, Pokfulam Road, Hong Kong

Pak-leung Ho, MB; Kwok-ning Chan, MD; Mary S.M. Ip, MD, Wah-kit Lam, MD, FCCP; Chu-sek Ho, MB; Kwok-yung Yuen, MD; and Kenneth W.T. Tsang, MD

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