| Backgrouds and objectives:Community acquired pneumonia (CAP) has been one of major threats to human health. Datas from the United States revealed that CAP is the sixth cause of death. In British, CAP ranks No.4 cause of death. In China, millions of people suffer from CAP each year. Empirical treatment of CAP is still the main therapy method, but the failure rate has been increasing because of the increased drug resistant strains. The sensitivity and specificity of existing diagnostic tools themselves are limited in determining the pathogens of CAP, so we need to build a rapid diagnosis method to guide the accurate medical treatment. Streptococcus pneumoniae (S. pneumoniae) is the maining pathogen of CAP. The traditional method of sputum culture is time-consuming, and often leads to false negative results after antibiotics treated. Although the sensitivity of immunochromatography is reliable, its specificity is still uncertain. In recent years, polymerase chain reaction (PCR)-based was progressively applied to detect a variety of respiratory trect infection pathogens. Currently, more advanced quantitative PCR based on development of the traditional PCR technology. For example, fluorescence quantitative PCR (FQ-PCR), which is integrated high sensitivity of PCR, high specificity of DNA hybridization probe and exact quantitation of spectrum technical. Detection of target gene can appear in real time and the complete process can be accomplished in 1-2h. Therefore, the purpose of this study is to evaluate the clinical value of FQ-PCR to detect S. pneumoniae in CAP patients'sputum samples. Targets and Methods:Experimental group sequentialiy selected 49 patients who were conformed to the diagnosis stantard of CAP of the first affiliated hospital of Dalian medical university from September 2008 to Juanary 2010. At corresponding time, we selected healthy adult volunteers as control group contained 42 cases. There was no significant diference between the control group and experimental group on age and sex. Collected consecutive morning sputum samples of experimental group patients for three times and control group only one time respectively. A part of experimental group's sputum samples for sputum cultured, and another 1ml of samples retained in sterile plastic tubes, stored in-80℃, whose gene LytA (S. pneumoniae) to be detected by FQ-PCR,. The morning sputum samples (1ml) of control group in sterile plastic tubes were stored in-80℃for detecting. The positive control was obtained from quantitative cultures of S. pneumoniae. And the concentration gradient of bacteria liquid is 107,106,105,104 CFU per milliliter. And the standard curve was made according to the results of the positive control from FQ-PCR.Results:In experimental group,44 of 49 patients'qualified morning sputum samples were acquired. Results of sputum culture showed that 1/44 (2.3%) cases had infection with pneumoniae S of erythromycin-resistance, 1/44 (2.3%) cases had infection with Staphylococcus aureus of methicillin-sensitive, candida albicans were detected in 3/44 (6.8%) cases' sputum samples. According to the results of FQ-PCR, we defined initial template content of S. pneumoniae-DNA≥104 CFU/ml was positive. The results showed that 10/44(22.7%) cases had infection with S. pneumoniae. Among these cases,2/10(20%) cases'S. pneumoniae-DNA≥105 CFU/ml, 8/10 (80%) cases'S. pneumoniae-DNA were bewteen 105-104 CFU/ml, 16/44 (36.4%) cases'DNA were bewteen 104-103 CFU/ml,10/44 (22.7%) cases'DNA<103 CFU/ml,and 8/44 (18.1%) cases were not detected S. pneumoniae. In the positive group, there is no difference (p>0.05) compared the results of FQ-PCR with the symptoms of iconography, white blood cell count and treatment response.9/10 cases (90%) were positive of FQ-PCR but negetive of sputum culture. Therefore, there was significant difference between the two methods in detecting S. pneumoniae (p<0.05). In the control group, S. pneumoniae infection was detected in 3/42 (7.1%) cases' sputum samples S. pneumoniae-DNA≥104 CFU/ml,8/42 cases'S. pneumo-niae-DNA were bewteen 104-103(cfu/ml),18/42 (42.9%)cases'S. pneumo-niae-DNA<103 (cfu/ml), and 13/42 (30.0%) cases were not detected S. pneumoniae. There was significant difference between experimental group and control group in detecting S. pneumoniae (p<0.05). In experimental group,27 patients'sputum samples were collected more than one times. Among the 27 cases, S. pneumoniae were detected in 21 cases'sputum samples, and 15/21 (71.4%) cases'content of S. pneumoniae-DNA were decreasing day by day.Conclusions:FQ-PCR, as the supplement of traditional diagnosis methods, which has higher sensitivity and specificity in detection of S. pneumoniae. |
PDF Full Text Request