Identification of isolates of Streptococcus pneumoniae recovered from community acquired pneumonia with molecular methods

Streptococcus pneumoniae is the leading microorganism in the etiology of community acquired pneumonia and continues to be an important cause of morbidity and mortality despite vaccination and antimicrobial therapy. Community acquired pneumonia is a frequent and potentially fatal infectious disease with a high cost of therapy. In spite of the sophisticated diagnostic procedures today, the etiologic microorganisms can not be determined in more than half of the patients with community acquired pneumonia. Traditional methods used for the identification of pneumococci are optochin susceptibility test, bile solubility test, and serologic tests. Recently, molecular diagnostic methods, which are based on the replication of nucleic acids in vitro conditions, are being used for the diagnosis of etiologic agents in community acquired pneumonia. Real-time polymerase chain reaction (real-time PCR), a method quantifying the amount of DNA/RNA in the specimen, is being increasingly used for the identification of S. pneumoniae in addition to conventional polymerase chain reaction (PCR). The aim of this study was to identify the S. pneumoniae with PCR and real time PCR from isolates of samples from lower respiratory tracts of patients with community acquired pneumonia and diagnosed to be S. pneumoniae with classical diagnostic methods, and also to compare these methods from the aspect of sensitivity and specificity. In this study, samples of sputum, tracheal aspiration, bronchoalveolar lavage (BAL), and pleural effusion that were obtained from adult patients with probable lower respiratory tract infection between January 2004 and January 2005, were assessed. Specimens that had typical appearance of S. pneumoniae in Gram staining XIwere cultivated onto 5% sheep blood agar. Optochin susceptibility test and bile solubility test were applied to the colonies causing alpha hemolysis on blood agar. Those isolates that were thought to be pneumococci with classical methods were analyzed again with PCR and real-time PCR, and the results were compared. A total of 180 lower respiratory tract samples were assessed, of which 139 (77.2%) were sputum, 16 (8.9%) were BAL, 16 (8.9%) were tracheal aspirates and 9 (5%) were pleural fluid. Of the patients, 46.6% were men, and 53.3% were women. Ninety one (65.5%) sputum samples, 3 (18.7%) BAL samples, 6 (37.5%) tracheal aspiration samples, 4 (44.5%) pleural effusion samples were alpha hemolytic specimens, and showed typical morphologic appearance of pneumococci. These samples were accepted as culture positive, and optochin susceptibility test and bile solubility test were applied. Sixty five (62.5%) of 104 culture positive isolates were optochin sensitive, 12 (11.5%) had low sensitivity, and 27 (26%) were optochin resistant. Of the 104 isolates, 73 (70.2%) isolates were found to be positive with bile solubility test, and 31 (29.8%) were negative. Bile solubility test was found to be positive in all of the 65 optochin sensitive isolates. Eight (66.7%) of the 12 isolates with low sensitivity to optochin were positive with bile solubility test, and 4 (33.3%) of them were negative. All of the 27 optochin resistant isolates were negative in bile solubility test. Of the culture positive isolates, 55 (60.4%) sputum samples were positive with conventional PCR, and 36 (39.6%) were negative. Three (75%) pleural fluid samples were positive, and 1 (25%) sample was negative with conventional PCR. All of the samples of BAL and tracheal aspiration were found to be positive with conventional PCR. Sixty one (67%) of sputum samples were positive, and 30 (33%) were negative with real-time PCR. Of the pleural fluid samples, 3 (75%) were positive, and 1 (25%) was negative with real-time PCR. All of the BAL samples and tracheal aspiration samples were positive in real-time PCR too. While 67 (64.4%) of 104 culture positive isolates were found to be positive with both PCR and real-time PCR, 31 (29.8%) isolates were found to be negative with both tests. Six (5.8%) isolates that were negative with PCR, were found to be positive with real-time PCR.In conclusion, conventional PCR and real-time PCR were observed to be as sensitive as other classical methods (microscopy, cultivation, optochin susceptibility test, bile solubility test) in the diagnosis of S. pneumoniae. Real-time PCR method is more sensitive and specific than conventional PCR in the diagnosis of pneumococci, and can be used safely in daily laboratory practice. xm