The study was retrospective and included adult patients with CAP who underwent HRCT. In all patients, CT images were obtained before treatment with antibiotics. Patients were seen at Kurashiki Daiichi Hospital, Kawasaki Medical School Kawasaki Hospital, and Kawasaki Medical School Hospital, Okayama, Japan, between April 2000 and December 2008. None of the patients were immunocompromised; namely patients with HIV infection, neutropenia secondary to chemotherapy, or patients on immunosuppressants; or patients with history of chronic lung diseases, from nursing homes, or patients with recent (<30 days) admission to hospital. The diagnosis was based on clinical signs and symptoms (cough, fever, productive sputum, dyspnea, chest pain, or abnormal breath sounds), and radiographic pulmonary abnormalities that were at least segmental and were not due to pre-existing or other known causes. All cases of pneumonia occurring more than three days after hospitalization were considered nosocomial and were excluded. The study protocol was approved by the Ethics Committee at Kawasaki Medical School.
Microbiological laboratory tests
Microbiological tests such as Gram stain, cultures, urinary antigen tests, and serological tests were performed as described previously [3, 16]. Blood cultures and nasopharyngeal swab specimens were obtained from all patients on admission and, if pleural fluid and sputum were available, a Gram stain test and a quantitative culture were obtained. 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). Certain invasive methods such as bronchoscopic examination were employed to obtain specimens in some patients after full explanation of the procedures. These specimens were also used for culturing of M. pneumoniae and Legionella species on pleuropneumonia-like organism broth and agar (70% pleuropneumonia-like organism broth [Difco, Inc., Detroit, MI, USA] supplemented with 20% heat-inactivated horse serum, 10% fresh yeast extract [25%], thallium acetate [final concentration 0.5 mg/mL], and sterile penicillin G [final concentration 1,000 U/mL]) 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 passed twice. Culture confirmation was done by fluorescent-antibody staining with C. pneumoniae and C. psittaci species-specific and genus-specific monoclonal antibodies. Bronchoscopic specimens were also used for polymerase chain reaction (PCR) of M. pneumoniae and chlamydial species. The M. pneumoniae-specific primers used for the PCR were from the DNA base sequence within the P1 cytadhesin gene, and amplification was performed as reported by Ramirez et al. . The C. pneumoniae-specific and genus-specific primers used for PCR were from the DNA base sequence within the 53-kDa protein gene and major outer membrane protein gene, respectively, established in our laboratory. These assays were performed as described previously [3, 18].
Paired serum samples were collected at intervals of at least four weeks after onset. Complement fixation 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 to M. pneumoniae were measured by a passive agglutination test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan), Legionella species by a microagglutination test (detection of L. pneumophila serogroups 1~6, L. bozoemanii, 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 titrations. In addition to serology, culturing, and/or PCR, a urinary antigen test (Binax NOW, Binax Inc. Portland, ME, USA) was used for the detection of S. pneumoniae and L. pneumophila.
Criteria for the determination of microbial etiology
Microbial etiology was classified as "definitive", "presumptive", or "unknown" as reported previously . Bacteria were considered to be definitive causative agents when isolated from blood or pleural fluid cultures. The results of sputum cultures were considered in combination with Gram stain findings. An organism showing heavy (≥ 107 cfu/mL) or moderate (106 cfu/mL) growth of a predominant bacterium on a sputum culture was considered to be a presumptive pathogen. Any microorganism isolated from bronchoscopic specimens was considered to be a presumptive pathogen when its concentration reached >105 cfu/mL in quantitative culture. If M. pneumoniae or Legionella species were isolated from a specimen, that specimen was considered to be a definitive pathogen even if the culture showed little growth. L. pneumophila and S. pneumoniae were considered to be presumptive agents when the urinary antigen test was positive. For serological tests, a four-fold rise in the antibody titer level between paired sera was considered definitive. Acute C. pneumoniae or C. psittaci infection was defined as IgM ≥ 1:32 or a four-fold rise in IgG or IgM titer between acute and convalescent serum samples.
Chest CT findings
CT examinations were performed using a Hi-Speed Advantage scanner (General Electric Medical Systems, Milwaukee, WI, USA), Hi-Speed LX/i Advantage (General Electric Medical Systems), or a Light Speed Ultra (General Electric Medical Systems). All scans were obtained during suspended end inspiration with the patient in a spine position. HRCT was performed with 1-mm collimation at 10-mm intervals. Images were obtained at lung (level -700 HU; width, 1,500 HU) and mediastinal (level 20–40 HU; width, 400 HU) levels. The time between clinical onset of pneumonia (fever and/or respiratory symptoms) and CT ranged from 1 to 10 days (mean, 5.1 days) for M. pneumoniae pneumonia and from 1 to 12 days (mean, 5.2 days) for S. pneumoniae pneumonia. Contrast material was used in 12 patients with M. pneumoniae or S. pneumoniae pneumonia.
Three chest radiologists (16, 12 and 10 years experience, respectively), who were blinded to the diagnoses, retrospectively and independently assessed the presence of consolidation, ground-glass attenuation, centrilobular nodules, thickening of the bronchial wall, reticular or linear opacity, pleural effusion, and lymphadenopathy. Readers also evaluated if the pneumonia was unilateral or bilateral and identified the opacity pattern of pneumonia. In addition, it was recorded which lobe of the lung was involved.
Consolidation was defined as air-space opacification with obscuration of the underlying vasculature. Ground-glass attenuation was defined as mildly increased attenuation without obscuration of the underlying vasculature. A centrilobular nodule was defined as a nodule identified around the peripheral pulmonary arterial branches or 3 to 5 mm away from the pleura, interlobular septa, or pulmonary veins. Bronchial wall thickening was defined as the thickening identified widespread areas not close to areas of ground-glass attenuation and/or consolidation. Interlobular septa thickening, intralobular interstitial thickening, and areas of irregular linear opacity were all classified as reticular or linear opacity. The reticular framework in ground-glass attenuation that is described as crazy-paving appearance was not classified as an area of reticular or linear opacity. Mediastinal lymphadenopathy was judged to be present when the minimal diameter of a lymph node was larger than 10 mm. Hilar lymphadenopathy was judged to be present only if the maximum diameter of the ipsilateral hilum exceeded that of the contralateral hilum by 1.5-fold or more. The final decisions for the presence of each finding and the opacity pattern for each case were reached by the consensus of the three radiologists.
Differential diagnosis in JRS guidelines
The JRS guidelines include six parameters and criteria based on underlying conditions, clinical symptoms, and laboratory findings by multiple regression analysis . These parameters are; 1) under 60 years of age, 2) no or minor co-morbid illness, 3) the patient has stubborn cough, 4) the patient has poor chest auscultatory findings, 5) no sputum or identified etiological agent by rapid diagnostic tests, and 6) a peripheral white blood cell (WBC) count below 10,000/mm3. When there is a correlation of more than four parameters out of six, then the guidelines recommend the use of macrolides or tetracyclines for suspected M. pneumoniae pneumonia . If these criteria are not met, the guidelines recommend the use of β-lactams for suspected S. pneumoniae pneumonia .
Statistical analysis was performed using Stat View version 5.0. (SAS Institute Inc, Cary, NC, USA). The incidence of underlying conditions, clinical findings, and radiographic findings were analyzed using Fisher's Exact test. Mean age of patients and laboratory data were compared using Student's t test. For each radiological finding, kappa values were calculated between the readers.