Asthma is a chronic lung disease characterized by airway obstruction, inflammation, and bronchial hyperresponsiveness to a variety of stimuli, including infections. Infections with viruses such as respiratory syncytial virus, parainfluenza virus, influenza A and B virus, coronavirus, adenovirus, and rhinovirus have been identified in 10% to 60% of children and adults with acute exacerbations of asthma. [1] [2] [3] [4] [5] Infection with other organisms, such as Mycoplasma pneumoniae, have also been associated with acute exacerbations of asthma. 6, 7 Another atypical respiratory pathogen, Chlamydia pneumoniae, has been established as an important cause of acute respiratory illness, including pharyngitis, bronchitis, and pneumonia. 8, 9 In a previous prospective study designed to assess the role of acute C. pneumoniae infection in bronchitis and atypical pneumonia, Hahn et al 10, 11 reported an association between serologic evidence of acute C. pneumoniae infection and adult-onset asthma and asthmatic bronchitis. Emre et al 12 also examined the prevalence of C. pneumoniae infection in children with acute exacerbations of reactive airway disease. C. pneumoniae was isolated from 11% of children in their study.
To study the role of C. pneumoniae in acute exacerbation of adult asthma, we assessed the frequency of C. pneumoniae infection in adult patients with exacerbations of asthma by isolation and polymerase chain reaction (PCR) from nasopharyngeal swabs and by serology.
Patients studied were 168 adults (16 to 80 years of age, mean 49.2 years; 91 males and 77 females) with asthma having recurrent episodes of reversible wheezing and breathlessness 13 who were seen at the Kawasaki Medical School Hospital between January 1995 and December 1996 for acute wheezing attacks, often with status asthmaticus. The initial mean percent of predicted FEV 1 was 70.3 (range 38 to 124 and SD 16.2). The mean duration of wheezing was 18.2 years (range 1 to 52 years); 124 (74%) patients were on regular treatment with bronchodilators. Twenty eight percent were current smokers, 42% were ex-smokers and 30% had never smoked. Non-asthmatic controls matched for age, sex, and smoking status were selected from patients attending the same hospital. The criteria for inclusion were no signs and symptoms of acute respiratory illness and normal pulmonary function test with a percent of predicted FEV 1 of 90 or greater (mean 101.6, range 90 -134, and SD 9.2). Informed consent was obtained from all subjects. The mean duration of follow up was 3.2 months (range 2 to 12 months). Culture and PCR Nasopharyngeal swab specimens were obtained from all patients and controls for isolation in cell culture and PCR. The swab specimens were placed in sucrose-phosphate-glutamate (SPG) transport medium. Culture for C. pneu-moniae was performed in cycloheximide-treated HEp-2 cells grown in a 24well cell culture plate as reported previously. 14 All specimens were passed once. Culture confirmation was done by fluorescent-antibody staining with C. pneumoniae species-specific and genus-specific monoclonal antibodies. 14, 15 The C. pneumoniae-specific primers used for PCR were from the DNA base sequence within the 53-kD protein gene as reported previously. 16 Cell culture grown C. pneumoniae strain KKp-15 17 was used as positive controls, and chlamydia transport medium was used as negative controls in every run. After electrophoresis of amplification products on a 1.5% agarose gel at 100 V, the band was visualized by staining with ethidium bromide. The appearance of a 499 base pair amplification product was taken as positive. Serology Paired serum samples were collected at intervals of 4 weeks for controls. Also serum samples of asthma patients were collected several times for at least 4 weeks after onset. All sera were stored at Ϫ70°C until testing. The microimmunofluorescence (MIF) test was used for titration of IgG, IgA, and IgM antibodies against chlamydial species, 18 using formalinized elementary bodies of C. pneumoniae KK-pn15, C. trachomatis L2/434/Bu and C. psittaci 6BC strains as antigens. Rheumatoid factors were absorbed with Gullsorb (Gull Laboratories, Salt Lake City, USA) before IgA and IgM titrations. 19 Acute or current infection was defined by IgM Ն 1: 16 
Demographic characteristics of study populations are shown in Table 1 . There were no significant differences between cases and controls in age, sex, or smoking status.
C. pneumoniae was isolated from the nasopharynx from 2 (1.2%) asthma patients and none from controls. C. pneumoniae was detected by PCR from 9 (5.4%) cases and one (0.9%) control. Culture positive specimens were all positive by PCR.
Antibodies against C. pneumoniae are shown in Table 2 Other respiratory tract pathogen including viruses and M. pneumoniae were identified in 21 (12.5%) patients by serologic test. Fourteen (8.3%) were found to have infections with viruses (seven influenza A, three parainfluenza 3, three respiratory syncytial virus, one dual infection with influenza A, and adenovirus) and seven (4.2%) with M. pneumoniae. Dual infections of C. pneumoniae and viruses or M. pneumoniae were not observed.
The results of laboratory tests and treatment of nine culture or PCR-positive patients are summarized in Table  3 . Five of nine patients who were culture or PCR positive had serologic evidence of acute infection. Three patients had stable IgG antibody titer, all of them had IgA. One had no IgG and IgA antibody. These four patients, however, had clinical and laboratory evidence of infection. All had symptoms of acute respiratory tract infection and elevation of inflammatory signs, CRP, and ESR. C. pneumoniae was successfully eradicated from the nasopharynx of all infected patients following antibiotic therapy. Five patients were treated with new macrolide antibiotics (clarithromycin in four and roxithromycin in one) and four patients with levofloxacin for 14 to 21 days (Table 3 ). All nine patients were treated with combined therapy with intravenous theophylline (in all nine patients) and 5 to 7-day courses of oral prednisone (in six patients) and antibiotics because of severe asthma attacks and achieved remission. moniae respiratory infection and acute exacerbation of asthma, and antibody titers and wheeze. In our study, exacerbations of asthma associated with C. pneumoniae were seen more often in older age groups, while exacerbations of asthma not associated with C. pneumoniae antibody were seen more often in young adults. Seroepidemiologic studies showed older patients were more likely to be having reinfections with C. pneumoniae, while younger patients were more likely to be having a primary infection. 20 In our study, most patients with acute C. pneumoniae infection had no IgM antibody titers suggesting a reinfection in these patients. The sample size in our study is too small to confirm the epidemiologic observations.
It has been suggested that presence of serum IgA antibodies is a useful marker for diagnosis of some chronic bacterial infections because the half life of serum IgA is less than 1 week 21 ; therefore, its continuous presence may indicate persistent antigenic stimulation to the immune system. In support of IgA antibodies as a marker for chronic infection, microbe-specific serum IgA has been shown to associate with a variety of chronic illnesses by bacterial pathogens, including C. trachomatis. 22 In particular, C. pneumoniae-specific IgA has been associated with C. pneumoniae reinfection, 23 chronic obstructive pulmonary disease, 24 and symptomatic asthma. 25 Our data also showed a significant association of C. pneumoniae-specific serum IgA antibodies and symptomatic reversible airway obstruction in adults. These findings indicate that chronic infection or reinfection with C. pneumoniae is common in asthma patients. Asthma exacerbations in patients with evidence of C. pneumoniae infection occurred in older ages when infection with C. pneumoniae is often a reinfection. It has been proposed that chronic C. trachomatis infection induces immunopathologic disease. 9 Reinfection with C. pneumoniae may therefore trigger an immunopathologic process in the lungs, perhaps involving epithe-lial damage and mediator release, 26, 27 or induce delayed hypersensitivity to chlamydial protein antigens 28 resulting in chronic airway inflammation characteristic of asthma.
The rates of infections with viruses and M. pneumoniae in our study are similar to some reports, 3, 4 but is remarkably lower in comparison to some other reports. 5, 6 The low prevalence of viral and mycoplasmal infection found in our study is probably due to the use of the CF test which has a low sensitivity and specificity.
In conclusion, we have shown an association of C. pneumoniae with acute exacerbations of asthma in some asthmatic adults. We were not able to assess whether eradication of C. pneumoniae after antichlamydial therapy produced improvement of asthma symptoms because bronchodilators or corticosteroids were also used.