| Objective: 1.Bacterial infection accounted for a large proportion in chronic obstructive pulmonary disease with acute exacerbation (AECOPD) . With the traditional method of sputum culture ,the positive rate was only 10%-30% or less.According to more reports in foreign literature, streptococcus pneumoniae (SP) was one of the common pathogen in AECOPD, while the results were quite different from different regions of China. This study was designed to establish a fast testing method- fluorescence quantitative PCR(FQ-PCR,RT-PCR), to detect the SP in sputum of AECOPD patients. Compared the results with the related researches of recent years, this method showing itself more advantages. 2. Traditional method of sputum culture was influenced by many factors,but clinical studies were more concentrated on the process of collecting specimens and the operation of culture ,which can cause a series of sad effects .But there was no report about the effect of sputum itself on the growth of bacteria .This study aimed to comparing the culure of SP with sputum itself and sputum mixed with streptococcus pneumoniae, observing if there is any effect on the growth of streptococcus pneumoniae in sputum.Methods:From Jan 2010 to Mar 2011, there were 59 patients with AECOPD diagnosed in respiratory department of our hospital, 45 males and 14 females, aged from 51 to 89, forty five patients were heavy smokers,6 patients were injected the hormone in latter times. The diagnosis criterion referenced Chinese Medical Association 2007 Institute of Respiratory Diseases, diagnosis and treatment of chronic obstructive pulmonary disease guidelines; The sputum samples from patients elected the first 3 days'samples admitting to hospital .If there was no sputum or who had difficulties in expectoration, using the method of aerosol inhalation to get or using sputum aspirator to help .Cultured all sputum samples and observed the culture results.Selected the autolysin lytA as a specific gene to design standard substance, primers and probes. Used the technique of fluorescence quantitative PCR to detect the SP in the samples, and according to the result of pure fluid of SP, estimated the infection rate of SP; 2.From the same conditions, diluted the fluid SP into 7 concentration ,from10-1 to 10-7,chose 10μl from 4 gradients which were 10-1,10-2,10-3,10-4 to culture respectively. Made the same concentration sputum mixed with streptococcus pneumoniae,and cultured them . Compared the two culture results .Results:The positive rate of sputum culture in fifty nine patients was 22%,Candida albicans(7cases, 11.8%), this might be due to contamination. Pseudomonas aeruginosa(2 cases,3.3%), Klebsiella pneumoniae(1 case,1.7%), diphtheria bacilli(1 case,1.7%), Proteus bacteria(1 case,1.7%),and 1 case of Pseudomonas fluorescens (1.7%),but there was no Streptococcus pneumoniae growing on the petri dish.The total samples were a hundred and four in the first 3 days, of which 70 were positive used fluorescence quantitative PCR, the positive rate was 67.3%, higher than culture Significantly (22%).According to the result,we can roughly estimate the infection of SP in AECOPD patients.We concluded that there were 18 patients infected with SP.Used Fisher's test andχ2 test to compare SP infection with drug use and other clinical factors, we saw that all of the tests were no significant differences (P>0.05).Cultured a certain concentration of SP and the same concentration of pure broth mixed with sputum, the quantity of colonies were significant differences.There were more than a thousand colonies from the culture results which concentration was 1.15×105cfu /ml, while the other class had only a few. The concentration which was 1.15×103 cfu /ml still had SP and the other class had none. Conclusions:1.Fluorescence quantitative PCR,as a quick testing technology,its positive rate of bacterial detection is significant higher than traditional methods;2.Sputum itself has effect on the growth of streptococcus pneumoniae. |
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