Bacterial Culture And Real-time PCR Assay For Detection Of Streptococcus Pneumoniae In Sputum Samples

Objective: Chronic obstructive pulmonary disease is one of the leading causes of mortality and morbidity in the world. Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) have significance on clinical progression. Several studies have shown that bacterial infection is the most frequent etiology of exacerbation. One of the most frequent pathogens is Streptococcus pneumoniae.Today the bacterial culture is the main methods for the detection of pathogens on the lower respiratory tract infection. Because some bacteria are fastidious bacteria and antibiotics abuse the positive rate of culture is lower. A large number of Clinical studies have shown that real-time polymerase chain reaction(real-time PCR) technology is helpful for diagnosis of lower respiratory tract infections. In this study, sputum samples of 51 AECOPD patients were analyzed by culture and real-time PCR. Then Streptococcus pneumoniae mixed with sputum samples from 30 patients with AECOPD and quantitative culture in blood agar. The purpose of the experiment is to explore the reasons of the lower positive rate of bacterial culture for Streptococcus pneumoniae from sputum samples of patients with chronic obstructive pulmonary disease and the role of Streptococcus pneumoniae for AECOPD.Methods:1. Sputum samples from 59 patients with AECOPD were analyzed by culture.2. Streptococcus pneumoniae mixed with the sputum of patients then quantitative cultured. The experimental group: the diluted 7 gradient of Streptococcus pneumoniae suspension mixed with a random sputum samples from 30 patients with AECOPD. Then quantitative culture in blood agar. The control group: the same concentration of pure strains of Streptococcus pneumoniae suspension was diluted to 7 gradient, then quantitative culture in blood agar. Compared the two Culture results.3. The results of real-time PCR: Streptococcus pneumoniae plasmid standard detected the self-made standard and 51 sputum samples of patients with AECOPD. Then compared the results.Results:1. A total of 59 sputum samples from patients with AECOPD were assessed. The culture findings revealed that 7 of the 59 sputum samples were positive. The pathogenic bacterias consist of Pseudomonas fluorescens(7.14%), Pseudomonas aeruginosa(7.14%), Proteus mirabilis(7.14%), Klebsiella pneumoniae(7.14%), Corynebacterium diphtheroides(7.14%), Candida albican(57.14%). All sputum samples were negative for streptococcus pneumoniae in culture.2. When the diluted suspensions of Streptococcus pneumoniae within the range 104-105 colony forming units (CFUs) per milliliter(CFU/ml) were cultured, the culture findings reveals that 27 of the 30 samples were positive in the control group while 4 of the 30 samples were positive in the experimental group(P<0.05). When the concentron more than 104CFU/ml, the positive culture results of the two sets were 100%(P>0.05).3. Using the real-time PCR assay, Streptococcus pneumoniae (≥104copies/ml) were found in 4/51 samples from the patients with AECOPD (7.8%) while Streptococcus pneumoniae(≥104cfu/ml) were found in 18/51 samples (35.3%).Conclusions:1. The culture of sputum samples from 59 AECOPD patients was negative for Streptococcus pneumoniae compared the the experimental group and the control group, we can conclude that certain factors in sputum can affect the growth of Streptococcus pneumoniae. But the specific factors have to be studied further.2. Real-time PCR assay appears to be more sensitive than culture in this study; We known that DNA can not be recycled completely in the process of DNA extraction as compared to the detection results of two standards. Further explained that some elements in the sputum may affect the growth of Streptococcus pneumoniae. But still could not explain what kind of factors affect the growth of Streptococcus pneumoniae.