肺炎支原体及流感病毒快速检测方法的评估应用及探索研究
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摘要
目的:
肺炎支原体(mycoplasma pneumoniae,MP)是一种常见的呼吸道感染病原,快速诊断困难。目前临床常用的检查手段为ELISA、被动凝胶颗粒检测抗体、PCR检测抗原等,但均不理想。近年来国内专家研发了一种MP快速鉴定培养基,本研究对这种快速鉴定培养方法进行了应用评估,同时对2009年全年收住北京军区总医院呼吸内科的住院患者进行了MP感染情况的流行病学调查。
流行性感冒(流感)是由流感病毒引起的急性呼吸道传染病,具有传染性强,蔓延快及波及面广的特点。2009年甲型H1N1流感病毒变异株给人们的生活、生产带了严重影响,发热门诊的流感样病例患者激增,为了快速筛查流感患者,我们对就诊的流感样病例患者进行了胶体金免疫层析检测(gold imunochrochromatographic assay, GICA)法的流感快速检测,为了更好的了解流感患者的临床特征,我们对进行GICA法快速筛查患者的基本资料、临床症状等进行了总结分析。同时,克隆和表达了甲型流感病毒的M1蛋白,为下一步改进GICA法检测流感病毒的敏感性和特异性进行了初步探索性的研究。
方法:
1.2008年12月1日到2009年3月1日收住北京军区总医院呼吸科的患者,共132例,(男64例,女68例)。采集患者入院后次日晨咽拭子即刻放入快速鉴定培养基进行MP培养,同时抽取静脉血,离心取血清,应用被动凝集检测试剂盒检测患者血清MP抗体。最后收集培养后咽拭子快速培养基进行MP基因片段PCR扩增,并行常规细菌、真菌培养鉴定。
2.2009年1月到2009年12月收住北京军区总院呼吸科的各种急性呼吸道疾病患者500例(男300例,女200例),采集患者入院后次日晨静脉血,分离血清,应用被动凝集检测试剂盒检测患者血清MP抗体,同时结合患者的临床资料,分析MP感染人群、季节及各疾病种类的感染情况。
3.从2009年5月到2010年1月北京军区总医院发热门诊共接诊成人患者约8000余例,其中对7804例流感样症状患者进行了GICA法甲流快速检测。本研究对其临床资料进行了总结、整理、分析。
4.从NCBI中查找甲流病毒的M1基因,并设计引物,提取H5N1的病毒RNA,反转录后,扩增全长,并连接到PET32a原核表达载体中,转化入大肠杆菌BL21(DE23),应用适当的条件对其进行诱导表达,并对诱导表达的融合蛋白进行western blot验证。
结果:
1.132例患者咽拭子MP快速培养,41例结果阳性。对快速培养MP后的培养基进行MP基因片段的普通PCR扩增检测,结果显示:41个MP培养阳性的培养基,仅有1个培养基中MP基因PCR扩增阳性。培养结果阴性的培养基,MP基因扩增全部为阴性。故两种方法阳性符合率仅为2.5%,阴性符合率为100%。
对MP快速培养后的50个培养基进行了MP基因片段real-time PCR扩增,(其中包括25个MP培养阳性,25个培养阴性的培养基),结果仅有6个MP培养阳性的培养基PCR扩增MP基因片段为阳性,且含量极低,两种方法相比,阳性符合率为24%,阴性符合率100%。两种方法的一致性检验kappa值仅为0.24,即MP快速鉴定培养法与PCR检测方法一致性不高。
快速培养MP阳性的培养基,经纯化培养、鉴定均未发现有细菌菌落,但存在至少两种真菌生长。
MP快速培养法与MP抗体被动颗粒凝集检测法相比,两种检测方法kappa值仅为0.34,一致性不高。
2.以MP抗体滴度≥1:80作为患者感染MP的标准,2009年1月至2009年12月,共检测500例患者,其中176例患者检测MP阳性,感染率为35.2%。第一季度和第四季度的MP感染率较其它季度稍高,但各季度、各月份之间差异无显著性意义。将500例患者分为4个年龄阶段,对各年龄段MP感染率进行分析,小于40岁年龄组MP感染率明显高于其它三组患者,大于80岁年龄组的MP感染率明显低于其它三组,差异有显著性意义。40至60岁和60至80岁组患者的MP感染率差异无显著性意义。
以抗体滴度≥1:160作为患者MP近期急性感染的指标,则检测的500例患者中有105例为近期感染。近期感染组的年龄与其他抗体滴度组相比,年龄相对较小,差异有显著性意义。MP近期感染患者中,约29.5%为慢性肺部疾病加重(包括慢性支气管炎、哮喘、支气管扩张、间质性肺疾病),36.2%为肺炎,14.3%为支气管炎。
3.2009年5月到2010年1月在甲流大流行期间,应用GICA法对北京军区总医院发热门诊7804例流感样症状患者进行了流感筛查,其中202例患者GICA法检测流感阳性,占检测人群的2.59%。其中检出甲型流感患者171例,乙型流感患者24例,甲、乙流感混合感染患者7例。流感的高发季节主要为夏季和秋季,感染人群多为青壮年,主要临床症状为发热、咽痛、鼻塞、打喷嚏、流鼻涕、关节酸痛。
4.成功构建了PET32a/M1原核表达质粒,并用western blot对融合蛋白的表达进行了验证。
结论:
1.MP快速鉴定培养基的配方在抗真菌方面还有待进一步改进,否则会对结果造成影响。
2.呼吸内科住院患者全年均有MP感染,第一季度和第四季度感染率稍高,但各季度MP感染率差异无显著意义;MP感染人群主要为青壮年,多为肺炎和支气管炎。在慢性肺部疾病的患者中,MP感染可能是引起患者病情急性加重住院的因素之一。
3.从2009年5月到2010年1月,流感病毒感染主要在夏季和秋季,临床表现为发热、咽痛、鼻塞、流涕,但仅根据临床症状无法判断是否为流感病毒感染。GICA法快速检测流感病毒抗原操作简便,快捷,在流感流行时可用于大量流感样病例筛查。
4.成功构建了甲流病毒M1蛋白的原核表达系统,为利用M1蛋白抗体建立甲流的GICA诊断方法,进一步提高其敏感性和特异性进行了探索性研究。
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.
肺炎支原体(mycoplasma pneumoniae,MP)是一种常见的呼吸道感染病原,快速诊断困难。目前临床常用的检查手段为ELISA、被动凝胶颗粒检测抗体、PCR检测抗原等,但均不理想。近年来国内专家研发了一种MP快速鉴定培养基,本研究对这种快速鉴定培养方法进行了应用评估,同时对2009年全年收住北京军区总医院呼吸内科的住院患者进行了MP感染情况的流行病学调查。
流行性感冒(流感)是由流感病毒引起的急性呼吸道传染病,具有传染性强,蔓延快及波及面广的特点。2009年甲型H1N1流感病毒变异株给人们的生活、生产带了严重影响,发热门诊的流感样病例患者激增,为了快速筛查流感患者,我们对就诊的流感样病例患者进行了胶体金免疫层析检测(gold imunochrochromatographic assay, GICA)法的流感快速检测,为了更好的了解流感患者的临床特征,我们对进行GICA法快速筛查患者的基本资料、临床症状等进行了总结分析。同时,克隆和表达了甲型流感病毒的M1蛋白,为下一步改进GICA法检测流感病毒的敏感性和特异性进行了初步探索性的研究。
方法:
1.2008年12月1日到2009年3月1日收住北京军区总医院呼吸科的患者,共132例,(男64例,女68例)。采集患者入院后次日晨咽拭子即刻放入快速鉴定培养基进行MP培养,同时抽取静脉血,离心取血清,应用被动凝集检测试剂盒检测患者血清MP抗体。最后收集培养后咽拭子快速培养基进行MP基因片段PCR扩增,并行常规细菌、真菌培养鉴定。
2.2009年1月到2009年12月收住北京军区总院呼吸科的各种急性呼吸道疾病患者500例(男300例,女200例),采集患者入院后次日晨静脉血,分离血清,应用被动凝集检测试剂盒检测患者血清MP抗体,同时结合患者的临床资料,分析MP感染人群、季节及各疾病种类的感染情况。
3.从2009年5月到2010年1月北京军区总医院发热门诊共接诊成人患者约8000余例,其中对7804例流感样症状患者进行了GICA法甲流快速检测。本研究对其临床资料进行了总结、整理、分析。
4.从NCBI中查找甲流病毒的M1基因,并设计引物,提取H5N1的病毒RNA,反转录后,扩增全长,并连接到PET32a原核表达载体中,转化入大肠杆菌BL21(DE23),应用适当的条件对其进行诱导表达,并对诱导表达的融合蛋白进行western blot验证。
结果:
1.132例患者咽拭子MP快速培养,41例结果阳性。对快速培养MP后的培养基进行MP基因片段的普通PCR扩增检测,结果显示:41个MP培养阳性的培养基,仅有1个培养基中MP基因PCR扩增阳性。培养结果阴性的培养基,MP基因扩增全部为阴性。故两种方法阳性符合率仅为2.5%,阴性符合率为100%。
对MP快速培养后的50个培养基进行了MP基因片段real-time PCR扩增,(其中包括25个MP培养阳性,25个培养阴性的培养基),结果仅有6个MP培养阳性的培养基PCR扩增MP基因片段为阳性,且含量极低,两种方法相比,阳性符合率为24%,阴性符合率100%。两种方法的一致性检验kappa值仅为0.24,即MP快速鉴定培养法与PCR检测方法一致性不高。
快速培养MP阳性的培养基,经纯化培养、鉴定均未发现有细菌菌落,但存在至少两种真菌生长。
MP快速培养法与MP抗体被动颗粒凝集检测法相比,两种检测方法kappa值仅为0.34,一致性不高。
2.以MP抗体滴度≥1:80作为患者感染MP的标准,2009年1月至2009年12月,共检测500例患者,其中176例患者检测MP阳性,感染率为35.2%。第一季度和第四季度的MP感染率较其它季度稍高,但各季度、各月份之间差异无显著性意义。将500例患者分为4个年龄阶段,对各年龄段MP感染率进行分析,小于40岁年龄组MP感染率明显高于其它三组患者,大于80岁年龄组的MP感染率明显低于其它三组,差异有显著性意义。40至60岁和60至80岁组患者的MP感染率差异无显著性意义。
以抗体滴度≥1:160作为患者MP近期急性感染的指标,则检测的500例患者中有105例为近期感染。近期感染组的年龄与其他抗体滴度组相比,年龄相对较小,差异有显著性意义。MP近期感染患者中,约29.5%为慢性肺部疾病加重(包括慢性支气管炎、哮喘、支气管扩张、间质性肺疾病),36.2%为肺炎,14.3%为支气管炎。
3.2009年5月到2010年1月在甲流大流行期间,应用GICA法对北京军区总医院发热门诊7804例流感样症状患者进行了流感筛查,其中202例患者GICA法检测流感阳性,占检测人群的2.59%。其中检出甲型流感患者171例,乙型流感患者24例,甲、乙流感混合感染患者7例。流感的高发季节主要为夏季和秋季,感染人群多为青壮年,主要临床症状为发热、咽痛、鼻塞、打喷嚏、流鼻涕、关节酸痛。
4.成功构建了PET32a/M1原核表达质粒,并用western blot对融合蛋白的表达进行了验证。
结论:
1.MP快速鉴定培养基的配方在抗真菌方面还有待进一步改进,否则会对结果造成影响。
2.呼吸内科住院患者全年均有MP感染,第一季度和第四季度感染率稍高,但各季度MP感染率差异无显著意义;MP感染人群主要为青壮年,多为肺炎和支气管炎。在慢性肺部疾病的患者中,MP感染可能是引起患者病情急性加重住院的因素之一。
3.从2009年5月到2010年1月,流感病毒感染主要在夏季和秋季,临床表现为发热、咽痛、鼻塞、流涕,但仅根据临床症状无法判断是否为流感病毒感染。GICA法快速检测流感病毒抗原操作简便,快捷,在流感流行时可用于大量流感样病例筛查。
4.成功构建了甲流病毒M1蛋白的原核表达系统,为利用M1蛋白抗体建立甲流的GICA诊断方法,进一步提高其敏感性和特异性进行了探索性研究。
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.
引文
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