Smoking, season, and detection of chlamydia pneumoniaeDNA in clinically stable COPD patients

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality throughout the world [1]. By 2020, the World Health Organization predicts that COPD will become the 5^th (currently 12^th) most prevalent disease worldwide, and the 3^rd (currently 6^th) most common cause of death [2]. Smoking is the major identified risk factor, and approximately 15% of smokers develop COPD [1]. Smoking cessation reduces the rate of decline in forced expiratory volumes, but no pharmacological intervention has been shown to modify the progression of disease. Bacterial and viral infections of the respiratory tract trigger acute exacerbations of chronic bronchitis, and may contribute to disease progression [3].

A role for Chlamydia pneumoniae in patients with COPD remains unclear. C. pneumoniae infection, diagnosed serologically, caused 5 to 10% of acute exacerbations in patients with chronic bronchitis [4, 5]. The seroprevalence and geometric mean titres of C. pneumoniae IgG [5, 6]. and IgA [7] antibodies were higher in COPD patients than controls, irrespective of exacerbation status. Von Hertzen et al measured serum IgG and IgA, salivary secretory IgA, circulating immune complexes, and nucleic acid, and found evidence of "chronic" C. pneumoniae infection in 65% of COPD patients [8], and recently Lieberman et al showed an association between C. pneumoniae serology, determined by a commercial immunoassay, and COPD exacerbation [9]. However, the reproducibility and validity of serologic testing remain uncertain [10].

Given these limitations of serology, the direct detection of C. pneumoniae nucleic acids may facilitate studies of its role in chronic disease etiology. The presence of C. pneumoniae has been directly demonstrated in lung tissue [11], and correlated with detection of C. pneumoniae DNA in peripheral blood mononuclear cells (PBMC) by PCR (Blasi et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000). However, there are limited data regarding the prevalence of C. pneumoniae DNA in PBMC of COPD patients.

In a previous study, we found that PBMC C. pneumoniae DNA detection among cardiac patients [12] was associated with current smoking and winter/spring season. The primary objective of this study was to examine the prevalence and clinical determinants of C. pneumoniae DNA in COPD patients, and in particular to validate the observed association with current smoking and season. Secondary objectives were to explore whether C. pneumoniae detection was associated with unique symptoms, pulmonary function, or sputum cell counts, and to contrast the prevalence among respirology and cardiology patients.

C. pneumoniae DNA detection in PBMC or sputum by categorical clinical characteristics is tabulated in Table 1, with unadjusted odds ratios, and by continuous clinical and laboratory characteristics in Table 2.

Current smoking (OR = 3.5, 95% CI: 1.2, 9.9, P = 0.02) and season (November to April, OR = 3.9, 95% CI: 1.4, 10.9, P = 0.008) were associated with DNA detection, independent of age and gender, whereas male gender was not (OR = 2.2, 95% CI: 0.8, 6.3, P = 0.14). A month-by-month breakdown for C. pneumoniae DNA positive PBMC is illustrated in Figure 1. The composite of current smoking or season identified 25 of 27 (92.6%) C. pneumoniae positives, and was strongly associated with DNA detection (age and gender adjusted OR = 11.1, 95% CI: 2.4, 50.5, P = 0.002). The composite of smoking or season was also associated with DNA detection in PBMC alone (adjusted OR = 9.0, 95% CI: 2.0, 41.4, P = 0.005).

To further explore the relationship between current smoking status and the detection of C. pneumoniae DNA, we measured plasma nicotine metabolites in 73 subjects. Levels = 25 μg/L indicate current smoking status. At this threshold, seven former smokers were reclassified as current smokers, and one current smoker as a former smoker. Nicotine metabolites = 25 μg/L were associated with C. pneumoniae DNA with OR of 2.1 (95% CI: 0.8, 5.7, P = 0.13). There was no relationship between higher plasma nicotine levels and the probability of detecting Chlamydia pneumoniae DNA (see Figure 2.)

Self-reported chronic sputum production was associated with C. pneumoniae DNA detection (age, gender, smoking and season-adjusted OR = 4.2, 95% CI: 1.1, 16.0, P = 0.04), whereas self-reported chronic cough, use of corticosteroids, or a history of coronary artery disease or diabetes mellitus were not (see Table 1). Chronic sputum production identified 24 of 27 (88.9%) of patients with C. pneumoniae DNA detected. In a forward selection model including sputum production, smoking, season, gender and age, only sputum production (OR = 5.3, 95% CI: 1.4, 19.6, P = 0.01) and season (OR = 2.8, 95% CI: 1.1, 7.5, P = 0.04) were independent predictors. Two-way interactions were not statistically significant.

We examined the association between C. pneumoniae DNA and measures of pulmonary function (Table 2), and sputum cell counts. Pulmonary function was moderately to severely impaired, and no relationship was found with C. pneumoniae DNA detection. Sputum neutrophil, macrophage, and eosinophil counts were not associated with C. pneumoniae detection.

We also examined the association between C. pneumoniae DNA and spirometry by linear regression modeling of FEV₁. Age, gender, current smoking, C. pneumoniae DNA status, and season were included in the model (Table 3). In the entire study group, age and male gender were predictors of FEV₁. However, if we used the per cent predicted FEV₁ as the outcome variable, no variable was significant at the P < 0.05 level (data not shown), and only smoking approached this level of significance (P = 0.09). We then examined subjects aged 65 years or younger, and those older than 65 years, separately. In the younger age group, age predicted FEV₁, whereas current smoking, male gender and C. pneumoniae DNA status were associated at P < 0.10. In the older age group, male gender was associated with FEV₁, but age, current smoking, season or DNA status were not.

PBMC results from the COPD patients in this study were compared with patients from the coronary angiography study reported previously [12]. COPD patients were more likely to have C. pneumoniae DNA in PBMC than patients undergoing coronary angiography (24 of 100 versus 24 of 208, adjusted OR = 2.9, 95% CI: 2.4, 9.9, P < 0.001), independent of current smoking status, gender, or season. The highest monthly prevalence was found in March and April, irrespective of year or study group. Among cardiology patients, the C. pneumoniae prevalence in March and April 1999 was 16% (20 of 122), compared with a prevalence of 5% (3 of 60) in the months of May to October. Similarly, in COPD patients studied in 2000 and 2001, March and April prevalence was 50% (9 of 18), compared with 15% (6 of 41) in the months of May to October. In the combined dataset, season (November to April OR = 4.3, 95% CI: 2.0, 9.2, P < 0.001) and smoking (OR = 2.7, 95% CI: 1.3, 5.4, P = 0.005) predicted C. pneumoniae DNA detection in PBMC, whereas male gender did not (OR = 1.5, 95% CI: 0.7, 3.2, P = 0.28). The combined measure of current smoking or season was associated with DNA detection (OR = 10.1, 95% CI: 3.0, 33.8, P < 0.001).

Among clinically stable patients with smoking-related COPD and moderate to severe pulmonary function impairment, we detected a relatively high prevalence of C. pneumoniae DNA. Peripheral blood (PBMC) specimens were superior to induced sputa, and an association between C. pneumoniae DNA detection, current smoking and winter/spring months was demonstrated. Chronic sputum production was associated with C. pneumoniae, but we found no clear association with other clinical symptoms, measures of lung function, or sputum cell counts.

Data on the prevalence of C. pneumoniae DNA in PMBC of COPD patients have been published solely in abstract form. Black et al found a prevalence of 14 of 20 (70.0%, 95% CI: 48.1, 85.5) Australian patients, using whole blood or PBMC (Black et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000). Blasi et al found a prevalence of 10 among 21 (47.6%, 95% CI: 28.3, 67.6) Italian patients, compared with 11 of 21 (52.4%) who had C. pneumoniae DNA in trans-bronchial lung biopsy specimens (Blasi et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000). Our prevalence of 24.0% in PBMC of Canadian COPD patients is slightly lower than these previous estimates, although within the 95% confidence interval for Blasi et al's estimate. Although this estimate was higher for patients with chronic respiratory disease compared with patients undergoing coronary angiography, we have recently found a high prevalence (31/103, 29.8%) among family practice controls without acute cardiorespiratory symptoms (Smieja et al, unpublished data).

We found a low prevalence (11%) of C. pneumoniae DNA in induced sputum among patients with chronic stable COPD. By contrast, Von Hertzen et al detected DNA in spontaneous sputum of 59% of patients with severe COPD [8], and Black et al detected DNA in 94% of patients hospitalized for COPD exacerbation (Black et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000). We obtained induced sputum to measure sputum cell counts by a validated methodology, but spontaneous sputum may be superior for routine C. pneumoniae detection. Conversely, sputum positivity may be higher during acute exacerbations, and our study intentionally excluded such patients. Our data support the utility of PBMC in preference to sputum for C. pneumoniae DNA detection.

A clearer relationship between C. pneumoniae and smoking is emerging. Hahn first proposed that smoking was a potential confounder of the association between C. pneumoniae antibody levels and cardiovascular disease [16]. Von Hertzen et al showed that smoking was associated with higher C. pneumoniae IgA levels in smoking-discordant twins [17], and Karvonen et al showed that C. pneumoniae IgG seropositivity to be higher in smokers in a population-based study [18]. This study has validated our previous observation that current smoking was associated with a higher prevalence of C. pneumoniae nucleic acid detection [12]. Whether C. pneumoniae associations with chronic diseases such as COPD or coronary artery disease are confounded by inaccurate ascertainment of smoking status, or whether infection is directly involved in the causal pathway for smoking-associated diseases, will require careful investigation.

We identified no clear relationship between FEV₁ and C. pneumoniae DNA detection. In the sub-group of patients aged 65 years or younger, current smoking and C. pneumoniae DNA status were each associated with a reduced of FEV₁ approximately 400 mL, although neither was nominally significant at a level of P < 0.05. A larger sample size may allow more precise estimation of this relationship, although only a prospective study design will adequately determine whether DNA detection is associated with greater decline in FEV₁. Our data suggest that a study enrolling subjects 65 years or younger may have greater power for examining whether C. pneumoniae is associated with lung function decline independently of smoking status.

We identified self-reported chronic sputum production as a strong risk factor for C. pneumoniae DNA detection. However, this association requires validation, and was not significant at P < 0.01 as we pre-specified for secondary analyses. Nevertheless, C. pneumoniae DNA detection in stable COPD patients may be associated with respiratory symptoms.

In conclusion, C. pneumoniae was prevalent in patients with COPD and associated with current smoking and winter/spring season, and may be associated with chronic sputum production. C. pneumoniae DNA detection in PBMC may facilitate the undertaking of large prospective epidemiologic studies examining the role of C. pneumoniae in COPD etiology and progression.