Study population
Our study included 42 consecutive cases of Legionella pneumonia diagnosed in our hospital. Further, 33 cases with other bacterial pneumonia, 19 cases with active pulmonary tuberculosis, and 29 age-adjusted control subjects were also included in this study. The pathogens isolated from the other bacterial pneumonia cases included Streptococcus pneumoniae (12 cases), Hemophilus influenzae (10 cases), Klebsiella pneumoniae (7 cases), Mycoplasma pneumoniae (4 cases), and others (3 cases). Control subjects were healthy without any infections, lung diseases, or liver diseases.
This study was approved by the University of the Ryukyus Institutional Review Board. The need for informed consent from each patient for inclusion in the study was waived because of the retrospective approach of this study, which caused no additional adverse events in any of the subjects. However, prior informed consent had been obtained from each patient before performing any procedure or obtaining any sample.
Blood samples were obtained for conventional clinical diagnosis from each patient. Bronchoalveolar fluids were obtained when required for the diagnosis of Legionella pneumonia. These samples were stored at -80°C until further use. Medical chart reviews were used to obtain information regarding the laboratory findings and clinical outcome of each patient.
Diagnosis of pneumonia and tuberculosis
The diagnosis of pneumonia was based on the clinical presentation (symptoms and physical examination), chest X-ray findings, and laboratory data. The diagnosis of Legionella pneumonia was confirmed by the detection of Legionella by culture, elevation of antibody titers in paired sera, and/or detection of its specific antigen in the urine. The other pneumonia cases were diagnosed based upon the bacteriological investigations (blood culture and culture of the expectorated sputa with satisfactory quality for examination), pulmonary tuberculosis was diagnosed following a positive smear test for acid-fast bacilli as well as positive culture.
HGF determination
Sera were prepared conventionally and stocked at -80°C until further investigation. The stock period was up to years without freeze-thaw cycle. The HGF level for each serum sample was determined by a sandwich ELISA (R&D Systems, Minneapolis, MN) using recombinant human HGF as a standard. The lowest detection limit for HGF was 40 pg/mL. This kit detects both active form and pro-form of HGF. Inter- and intra-assay reproducibilities are reported as 7.0% and 5.6%, respectively.
Statistical methods
Data are reported as mean ± standard deviation (SD). The logarithmic transformation of several data values (HGF, white blood cell counts (WBC), and lactate dehydrogenase (LDH)) allowed Gaussian approximation (demonstrated by the Kolmogorov -Smirnov and Shapiro-Wilk tests). Differences in the logarithmically transformed values for HGF levels between multiple groups were examined by using analysis of variance (ANOVA) and Bonferroni's multiple comparison test. Differences between two groups were examined using unpaired t-test with Welch's correction and Mann-Whitney test. The relationship between two parameters was determined by Pearson's correlation coefficient test. These tests were performed using statistical software programs (Prism 4, Graphpad Software Inc., California; and SPSS version 15.0J, SPSS Japan Inc., Tokyo). P < 0.05 was considered statistically significant.