Patients
This study was conducted as a part of CAP studies that investigated the prevalence and clinical features of atypical pneumonia and evaluated the usefulness of diagnostic methods for the diagnosis of this condition. All patients with CAP who visited 12 institutions participating in the Atypical Pathogen Study Group from January 2008 to December 2011 were enrolled in this study. The diagnosis was based on clinical signs and symptoms of lower respiratory tract infection (cough, fever, productive sputum, dyspnea, chest pain, or abnormal breath sounds) and the presence of new infiltrates on chest radiographs that were at least segmental and were not caused by preexisting or other known causes. Informed consent was obtained from all patients; the study protocol was approved by the Ethics Committee at Kawasaki Medical School.
Microbiological laboratory tests
Microbiological tests, such as Gram stain, cultures, real-time polymerase chain reaction (PCR), urinary antigen tests and serological tests, were performed as described previously [
19]. Nasopharyngeal swab specimens were obtained from all patients and, if pleural fluid and sputum were available, a Gram stain test and a quantitative culture were obtained. Blood cultures were obtained from all adolescent and adult patients. Sputum data were only evaluated when the Gram stain test revealed numerous leukocytes (>25 in a × 100 microscopic field) and few squamous epithelial cells (<10 in a × 100 microscopic field). Invasive methods, such as bronchoscopic examination, were employed to obtain specimens in some patients after full explanation of the procedures. A bronchoscopic examination was undertaken for clinical indications. These specimens were also used for culturing and PCR. Cultures for
M. pneumoniae and
Legionella species were performed on pleuropneumonia-like organism broth (Difco, Detroit, MI, USA) and buffered charcoal-yeast extract alpha agar, respectively. Cultures for
Chlamydophila pneumoniae and
C. psittaci were performed using cycloheximide-treated HEp-2 cells grown in a 24-well cell culture plate. All specimens were examined twice. Culture confirmation was done by fluorescent-antibody staining with
C. pneumoniae and
C. psittaci species-specific and genus-specific monoclonal antibodies. The target DNA sequences for PCR were a region of the 53-kDa gene for
C. pneumoniae, the major outer membrane gene for
Chlamydia, the P1 cytadhesin gene for
M. pneumoniae, and the nucleotide sequence of the 5S-ribosomal DNA for
Legionella. DNA was extracted from respiratory samples using a QIAamp DNA Mini Kit (QIAGEN K. K., Tokyo, Japan) in accordance with the manufacturer’s instructions. The assays were performed as described previously [
19]. Nasopharyngeal swab specimens were also tested for influenza A and B viruses by a direct enzyme immunoassay.
Paired serum samples were collected at intervals of at least 4 weeks after onset. Complement fixation (CF) tests were done in all patients for antibodies to influenza A and B viruses, adenovirus, respiratory syncytial virus, cytomegalovirus, and parainfluenza virus types 1, 2, and 3. Antibodies against M. pneumoniae were measured with the use of a particle agglutination (PA) test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan), Legionella species by a microagglutination test (detection of L. pneumophila serogroups 1 ~ 6, L. bozemanii, L. dumoffii, L. gormanii, and L. micdadei), and Coxiella burnetii by an indirect immunofluorescence test. A microimmunofluorescence test was used for the titration of IgG and IgM antibodies against chlamydial species using formalinized elementary bodies of C. pneumoniae KKpn-15, C. trachomatis L2/434/Bu, and C. psittaci Budgerigar-1 strains as antigens. Rheumatoid factors were absorbed with GullSORB (Meridian Bioscience Inc., OH, USA) before IgM titration. In addition to serology, culturing, and/or PCR, urinary antigen tests (Binax NOW, Binax Inc. Portland, ME, USA) for S. pneumoniae and L. pneumophila were performed in adolescent and adult patients.
Criteria for the determination of microbial etiology
The microbial etiology was classified as "definitive", "presumptive", or "unknown" as reported previously [
19]. A definitive etiology was defined if one of the following conditions was present: (1) blood or pleural fluid cultures yielding the presence of bacterial or fungal pathogen; (2) urinary antigen test results positive for
L. pneumophila or
S. pneumoniae; (3) respiratory specimen culture or PCR results positive for
M. pneumoniae, C. psittaci or
Legionella species; (4) nasopharyngeal antigen test results positive for influenza A and B viruses; (5) a fourfold increase in the antibody titer for viruses,
M. pneumoniae (to ≥1:160),
Legionella species (to ≥1:128),
C. burnetii, or
Chlamydia species (IgM or IgG); or (6) a single increase in IgM titer for
Chlamydia species ≥1:32. A presumptive etiology was considered if any of the following conditions were present: (1) an organism showing heavy (≥10
7 cfu/mL) or moderate (10
6 cfu/mL) growth of a predominant bacterium on a sputum culture in combination with Gram stain findings; (2) any microorganism isolated from bronchoscopic specimens when its concentration reached ≥10
5 cfu/mL in quantitative culture; (3) an antibody titer of ≥1:320 for
M. pneumoniae in either an acute-phase or convalescent-phase serum sample; (4) an antibody titer of ≥1:256 for
Legionella species in either an acute-phase or convalescent-phase serum sample; or (5) respiratory specimens culture of PCR results positive for
C. pneumoniae. An unknown etiology was considered if any of the following conditions were present: (1) respiratory specimens culture results were “normal flora”; (2) an organism showing light growth on a sputum culture; or (3) cases not fulfilling any of the above conditions.
Detection of point mutations associated with resistance in domain V of 23S rRNA
A search for mutations at sites 2063, 2064, and 2617 in the
M. pneumoniae 23S rRNA domain V gene region was performed using a direct sequencing method in samples with a positive PCR result, as reported previously [
12,
15,
18]. Specifically, nested PCR was performed using a thermal cycler (PCR Thermal Cycler Dice Gradient, Takara Bio, Inc., Shiga, Japan) with primers (Sigma-Aldrich, Japan), Taq polymerase (Takara Ex
Taq Version; Takara Bio, Inc.), and extracted DNA. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN). The purified products were electrophoresed in a 3% Nusieve 3:1 agarose gel (Lonza) and, after the single band was confirmed, labeled using a BigDye Terminator V3.1 cycle sequencing kit (Applied Biosystems) and applied to an ABI Prism 3130x1 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer’s instructions. The presence or absence of gene mutations at each site was determined by reading using a sequence scanner (Applied Biosystems).
Minimum inhibitory concentrations
The minimum inhibitory concentrations (MICs) of three agents for
M. pneumoniae isolates were determined using microdilution methods with PPLO broth, as reported previously [
12,
15]. These agents were erythromycin, minocycline, and levofloxacin.
M. pneumoniae strain M129 was used as a control. Serial twofold dilutions of antibiotics prepared in PPLO broth containing 10
4 to 10
5 CFU/mL of
M. pneumoniae were placed in 96-well microplates [
12,
15]. The microplates were sealed with adhesive sheets and incubated at 37°C. The MIC was determined as the lowest concentration of antimicrobial agent at which the color of the control medium changed.
Statistical analysis
Statistical analysis was performed using Stat View version 5.0. (SAS Institute Inc, Cary, NC, USA). The incidence of clinical findings was analyzed using Fisher's Exact test, and laboratory data were compared using Student's t test.