| Background and Objective:Mycoplasma pneumoniae (MP)is a major cause of respiratory tract infection and is transmitted mainly through aerosol or direct contact. There are not any useful methods to detect the MP rapidly. Recently, a rapid identification of MP medium was developed in China; this culture medium contained growth factors and biological inhibitors, which only allowed MP to grow quickly. The aim of this study was to evaluate the effectiveness of this commercial rapid MP medium method, and had epidemiological investigation of MP infection in the inpatients of Department of Respiratory Medicine, Military General Hospital of Beijing PLA, 2009.Influenza is a kind of acute respiratory infection, caused by influenza virus; it is characterized by strong infectivity, rapid and wide spread. The influenza virus A H1N1 in 2009 had a serious impact on people’s lives and work. The number of outpatients with influenza-like symptoms has dramatically increased. We detected outpatients in fever clinic by gold imunochrochromatographic assay (GICA) test for screening influenza. We summarized and analyzed clinical data and results of GICA test in that time for better understanding of the epidemiological characteristics of influenza. To improve the sensitivity and specificity of GICA test for influenza virus, we cloned and expressed the M1 protein of influenza virus A, laid a good foundation for future research.Methods:1. Patients (132 cases, 64 male and 68 female) admitted to the respiratory department of Beijing General Hospital from December 1st, 2008 to March 1st, 2009 were enrolled in this study.A throat swab was taken to do the rapid culture by rapid identification MP medium the morning after admission to the respiratory department. MP PCR test and routine bacterial and fungal microorganism culture test were used to identify the organisms following MP culture. In addition, serum specimens were also collected on the day after admission, and tested for MP antibody(IgG+IgM+IgA)using the passive particle agglutination test.2. From January 2009 to December 2009, there were 500 patients enrolled in this epidemiological investigation, including 300 male, 200 female. We used the passive particle agglutination test to detect the MP (IgG+IgM+IgA)antibody, meanwhile summarized and analyzed clinical data.3. All patients who visited the outpatient department with influenza-like symptoms (fever and/or at least one of the following symptoms: sore throat, cough, rhinorrhea, or nasal congestion) and screened by GICA test were enrolled in this study. Between May 2009 to January 2010, 7804 patients with influenza-like symptoms were screened for influenza virus A and B by GICA test. We summarized and analyzed clinical data and results of GICA test.4. Design primer of the M1 of influenza virus A according the M1 gene in NCBI, amplified the M1 gene by RT-PCR, The amplified product was cloned into the expression vector PET32a, recombinant plasmid was transformed into E.coli BL21(DE3) competent cells and induced with 1 mmol/L IPTG. Finally, use the western blot to identify the fusion protein.Results:1. The rapid identification culture showed that 41 of 132 patients were MP positive. We did routine PCR for all the medium after culture, there was only one medium PCR show MP gene positive, which is also shown positive by rapid culture. Compared to routine PCR, the negative rate is 100%, positive rate is 2.5%.We did real time-PCR test on 50 samples of the rapid identification medium after 24 hours of culture (25 were positive based on rapid identification culture and 25 were deemed negative). The PCR test showed 6 samples positive, accounting for 24 % of culture positive specimens. The consistency test showed kappa=0.24, indicating that rapid M. pneumoniae identification culture had poor concordance with the MP PCR test.For the microorganism culture and identification, we chose 10 specimens that were deemed MP positive based on the rapid identification culture. The result showed no bacterial contamination but all had at least one fungus.Compared with the passive particle agglutination test, consistency test showed kappa=0.34. The rapid MP identification culture method had no good concordance with the passive particle agglutination test. 2. If the passive particle agglutination test showing antibody titers≥1:80 is considered positive, from January 2009 to December 2009, 176 of 500 patients were MP positive, the infection rate is 35.2%, the first quarter and fourth quarter had higher infection rate, but the differences of infection rate among months and quarters were not significant.We divided patients into 4 groups according to age. The patients younger than 40 years old had higher infection rate of MP, while the patients older than 80 years old had lower infection rate of MP, and both had statistical significance. The difference of infection rate between 40-60 years old and 60-80 years old had no significance.If MP antibody≥1:160 were considered as recent acute infection, recent acute infection patients were younger than other group, and the difference was significant. Among the recent acute infection patients, 29.5% patients were acute exacerbation of chronic lung disease (including chronic bronchitis, asthma, bronchiectasis, and interstitial lung diseases), 36.2% pneumonia, and 14.3% bronchitis.3.From May 2009 to January 2010, 7804 patients with influenza-like symptoms were screened for influenza virus A and B by GICA test. 202 patients were influenza virus-positive; accounting for 2.59% of all cases detected. Among the 202 influenza virus-positive patients, 171 patients were influenza virus A-positive, 24 were influenza virus B-positive, and 7 were co-infected with influenza virus A and B. The positive influenza patients were identified mainly in the summer and autumn of 2009. The prevalence of influenza showed no difference between male and female. The symptoms such as fever, sore throat, nasal congestion, sneezing, runny nose, and joint pain were more frequently observed in influenza virus positive patients.4. Amplified M1 gene of influenza virus A by RT-PCR, successfully cloned it into vector PET32a, and expressed the fusion protein in E.coli BL21(DE3).Conclusion:1. The rapid identification of MP culture test is easy to perform and provides results fast, but has poor concordance with the MP PCR and serum MP antibody test due to the high fungus contamination. This commercial rapid MP medium needs further improvement.2. MP infection can occur in any time of the year, the first quarter and fourth quarter had higher infection rate, but the difference of infection rate among every months and every quarter was not significant. MP infection mainly occurs in young adults, manifesting as pneumonia or bronchitis. MP infection is a major cause for acute exacerbation of chronic lung disease.3. Symptoms such as fever, sore throat, nasal congestion, sneezing, runny nose, and joint pain were more frequently observed in influenza virus positive patients. But these symptoms are not specific for influenza patients. The GICA kit is very useful for screening large number of patients with influenza-like symptoms.4. Successfully cloned and expressed the M1 of influenza virus A, which laid a foundation of M1 for further researching and using. |
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