儿童及老年人肺炎支原体感染的分子流行病学研究

发布时间：2018-06-27 22:58

本文选题：肺炎支原体 + P1基因　；参考：《东南大学》2017年硕士论文

【摘要】：研究背景与目的:肺炎支原体(MP)是引起呼吸道感染的主要病原体之一。人群普遍易感,每3-7年有一次流行高峰,春夏季是高发季节,各流行期MP亚型会发生一定变化。对MP基因的分型主要是通过PCR-RFLP分型方法和MLVA分型方法。MP基因型种类复杂、多样,且流行分布具有明显的时间、地点和人群分布差异。抗生素的滥用使得MP耐药问题日趋严重,研究显示MP耐药突变可能与基因型有关。随着分子技术的不断发展,MP分型方法的成熟,开展系统全面的MP基因型及其耐药突变的分子流行病学研究,从分子水平揭示MP亚型分布特征、流行趋势及耐药状况势在必行。本研究利用Meta分析,在系统全面了解MP基因型流行分布特征的基础上,开展以医院为基础的人群分子流行病学调查研究,了解目标医院患者人群MP基因型及耐药突变的流行分布情况,初步探讨MP两种分型方法分型结果之间可能存在的联系以及基因型与耐药突变之间的关系,旨在为当地医院MP感染的监测、预防和临床治疗提供科学依据。研究方法:1..Meta 分析:检索 PubMed、Web of Science、CNKI、CBM、万方等数据库,收集公开发表的与MP基因型有关的研究文献。按纳入与排除标准进行文献的筛选并进行数据提取工作,运用Freeman-Tukey双反正弦变换法对不同MP基因型的流行率进行合并计算,从研究时间、研究地点和研究人群三个方面进行亚组分析和Meta回归分析。Meta分析软件包括Stata19.0和SAS 9.3。2.MP基因分型方法:以南京市某综合性三甲医院儿科呼吸道感染患儿以及呼吸科老年呼吸道感染患者为研究对象。在知情同意的基础上采集咽拭子并进行流行病学个案调查,采用Nest-PCR扩增16S rRNA基因片段检测MP临床株,采用PCR-RFLP方法和mPCR-CE-MLVA方法进行MP基因分型。3.MP耐药突变分析方法:对MP临床株23S rRNA基因片段扩增并送生物公司测序。根据测序结果分析MP耐药基因位点突变。4.资料的统计分析方法:建立Excel数据库,使用SAS 9.3或SPSS19.0软件进行统计学分析。计数资料采用率或构成比描述,用χ2检验或Fisher确切概率法进行统计学分析,P0.05时差异具有统计学意义;计量资料采用中位数和四分位间距或均数±标准差(x±s)描述,采用Wilcoxon秩和检验或Kruskal-Wallis H检验进行统计学分析。研究结果:1..Meta分析共纳入42篇文献,其中与P1基因分型有关的文献40篇,成功分型的样本数为5027例,与MLVA分型有关的文献16篇。成功分型的样本数为1956例。P1-Ⅰ型流行率为 73.80%(95%CI:67.86-79.34);P1-Ⅱ 型流行率为 26.21%(95%CI:20.67-32.16);P1-Ⅱ 突变株流行率为 13.25%(95%CI:7.61-20.15);M4-5-7-2 型流行率为 68.97%(95%CI:59.55-77.70);M3-5-6-2 型流行率为 15.41%(95%CI:10.41-21.20);M3-6-6-2 型流行率为 5.49%(95%CI:2.34-9.90);其他型别的流行率为 6.71%(95%CI:4.72-9.02)。亚组分析和Meta回归分析发现,不同时间,地区和人群的MP基因型分布呈现明显差异。2.研究医院儿童呼吸道感染患者MP阳性率为27.6%。儿童患者中PCR-RFLP方法成功分型90例,P1-Ⅰ型流行率为21.1%(19/90),P1-Ⅱ型流行率78.9%(71/90),其中V2c突变株占50.7%(36/71)。MLVA分型方法成功分型102例,M3-6-6-2型流行率最高为 40.2%(41/102),M3-5-6-2 型流行率 37.3%(38/102),M4-5-7-2 型流行率为16.7%(17/102),其他基因型流行率为5.9%(6/102)。100%的M4-5-7-2型属于为P1-Ⅰ,81.2%的M3-5-6-2,M3-6-6-2型为P1-Ⅱ。老年患者P1基因成功分型24例,P1-Ⅰ 型 12.5%(3/24),P1-Ⅱ型 87.5%(21/24),V2c 突变株占 61.9%(13/21)。MLVA分型方法成功分型30例,M3-5-6-2、M3-6-6-2型型流行率均为43.3%(13/30),M4-5-7-2 型 10%(3/30),M3-6-7-2 型 1 例,流行率为 3.3%。100%的 M4-5-7-2 型为P1-Ⅰ,66.7%的 M3-5-6-2,M3-6-6-2 型为 P1-Ⅱ。3.儿童患者132例MP阳性标本中共发现21例存在耐药基因突变,耐药率为15.9%。其中90.5%(19/21)为A2063G突变,9.5%(2/21)为A2064G。老年MP感染者耐药率为 10%(3/30),A2063G 突变占 66.7%(2/3),T2611C 突变 33.3%(1/3)。成功分型的 141 例中,A2063G 突变中 M4-5-7-2 型 94.7%(18/19),M3-6-6-2 型 5.3%(1/19)。A2064G突变1例,属于M3-5-6-2型。T2611C突变1例,属于M3-6-6-2型。MP耐药可能与M4-5-7-2型有关。4.MP感染流行特征分析,MP感染可能与年龄、性别有关,与未感染MP的呼吸道感染患儿相比,MP感染易出现发热,消化道症状以及周身症状,而上呼吸道感染症状相对较少,N%和CRP值较高,L%值较低。儿童不同年龄间MP基因型分布存在统计学差异。小于3岁组患儿感染MP基因型以M3-6-6-2型为主,大于3岁的患儿以M3-5-6-2为主。研究结论:1.MP流行株P1基因型分布存在较大的时间差异性,2005年之前以P1-Ⅱ型为主,2005年之后P1-Ⅰ型占主导地位,并有向P1-Ⅱ型转换的趋势。MLVA分型型别一直以M4-5-7-2,M3-5-6-2,M3-6-6-2为主。在不同地区和人群中各基因型分布有很大的差别。2.研究医院儿童和老年患者MP基因型均以M3-6-6-2/P1-Ⅱ型,M3-5-6-2/P1-Ⅱ型为主。Mpn13位点可能与P1基因分型型别有一定的联系,但仍需更多的研究给予进一步的探讨。3.该地区MP耐药率与其他地区相比较低,耐药突变以A2063G为主。MP耐药突变可能与M4-5-7-2型有关。
[Abstract]:Research background and objective: Mycoplasma pneumoniae (MP) is one of the main pathogens causing respiratory infection. The population is generally susceptible to a peak epidemic peak every 3-7 years, a high onset season in spring and summer, and a certain change in the MP subtypes in each epidemic period. The classification of MP genes is mainly through the PCR-RFLP typing and the MLVA typing of.MP genotypes. The complexity, diversity, and epidemic distribution have obvious time, location and population distribution. The abuse of antibiotics makes the problem of MP resistance become increasingly serious. The research shows that the MP resistance mutation may be related to the genotype. With the development of the molecular technology, the maturation of the MP typing method, the opening of the systematic and comprehensive MP genotypes and their resistant mutants Epidemiological studies, from molecular level to reveal the distribution characteristics of MP subtype, epidemic trend and drug resistance are imperative. In this study, based on the systematic understanding of the characteristics of the epidemic distribution of MP genotypes, Meta analysis was used to investigate the molecular epidemiology of the population based on the hospital, and to understand the MP genotype of the patients in the target hospital and to understand the genotype of the population in the target hospital. The epidemic distribution of drug-resistant mutations, the possible relationship between the results of the MP two typing methods and the relationship between genotyping and drug resistance mutations is discussed. The aim is to provide scientific basis for the monitoring, prevention and clinical treatment of MP infection in local hospitals. Research methods: 1..Meta analysis: PubMed, Web of Science, CNKI, CBM, and 10 million. The published literature related to the MP genotypes was collected. According to the inclusion and exclusion criteria, the literature was screened and the data was extracted. The Freeman-Tukey double sine transformation method was used to combine the prevalence rates of different MP genotypes. From the study time, the research site and the research population were carried out in three aspects. Subgroup analysis and Meta regression analysis of.Meta analysis software included Stata19.0 and SAS 9.3.2.MP genotyping methods: children with respiratory tract infection in a comprehensive three a hospital in Nanjing and patients with respiratory infection in the Department of respiration. The 16S rRNA gene fragment was amplified by Nest-PCR to detect the MP clinical strain, and the PCR-RFLP method and mPCR-CE-MLVA method were used to analyze the.3.MP resistance mutation of the MP genotyping: the 23S rRNA gene fragment of the MP clinical strain was amplified and the biological company was sequenced. Xcel database, using SAS 9.3 or SPSS19.0 software for statistical analysis. Enumeration data rate or composition ratio description, x 2 test or Fisher exact probability method for statistical analysis, P0.05 difference has statistical significance; measurement data use the median and four division spacing or mean number + standard deviation (x + s) description, using Wilcoxon rank sum Test or Kruskal-Wallis H test for statistical analysis. Results: 1..Meta analysis included a total of 42 documents, including 40 literature related to P1 genotyping, the number of successful typing samples was 5027, and 16 documents related to MLVA typing. The number of successful typing samples was 73.80% (95%CI:67.86-79.34) and P1- in 1956 cases (95%CI:67.86-79.34); P1- The prevalence rate of type II was 26.21% (95%CI:20.67-32.16); the prevalence rate of P1- II mutant was 13.25% (95%CI:7.61-20.15); M4-5-7-2 prevalence was 68.97% (95%CI:59.55-77.70); M3-5-6-2 prevalence was 15.41% (95%CI:10.41-21.20); M3-6-6-2 prevalence rate was 5.49% (95%CI: 2.34-9.90); the prevalence rate of other types was 6.71% (95%CI:4.72-9.02) Subgroup analysis and Meta regression analysis found that the distribution of MP genotypes in different times, regions and people showed significant differences in the MP positive rate of children with respiratory infection in.2. research hospitals 90 cases of PCR-RFLP, P1- I prevalence rate was 21.1% (19/ 90), P1- II prevalence rate 78.9% (71/90), and V2c mutant strain 102 cases were divided into 50.7% (36/71).MLVA typing, the highest prevalence rate of M3-6-6-2 type was 40.2% (41/102), the prevalence rate of M3-5-6-2 type was 37.3% (38/102), the prevalence rate of M4-5-7-2 type was 16.7% (17/102), and the M4-5-7-2 genotype of other genotypes was 5.9% (6/102).100%. 24 cases of successful typing, P1- type I 12.5% (3/24), P1- type II 87.5% (21/24), V2c mutant 61.9% (13/21).MLVA typing, 30 cases were successfully classified, M3-5-6-2, M3-6-6-2 type prevalence rates were 43.3% (13/30), M4-5-7-2 type 10% (3/30), 1 cases, 66.7% 132 MP positive specimens of P1- II.3. children were found to have resistance gene mutations in 21 cases, the drug resistance rate was 90.5% (19/21) as A2063G mutation, 9.5% (2/21) was 10% (3/30), A2063G mutation accounted for 66.7% (2/3) and 33.3% mutation 33.3% (2/21). M4-5-7-2 type 94.7% (18/19), M3-6-6-2 type 5.3% (1/19).A2064G mutation in 1 cases, belonging to 1 cases of M3-5-6-2 type.T2611C mutation, belongs to M3-6-6-2 type.MP resistance may be associated with the M4-5-7-2 type.4.MP infection epidemic characteristics analysis, MP infection may be related to age, sex, compared with children with respiratory tract infection without infection, the infection is prone to fever, digestive tract Symptoms and body symptoms, while upper respiratory tract infection symptoms are relatively less, N% and CRP values are higher, L% values are lower. The distribution of MP genotypes in children at different ages is statistically different. The MP genotype of children younger than 3 years old is mainly M3-6-6-2 type, and the children older than 3 years with M3-5-6-2 as the main. Research conclusion: P1 genotype distribution of 1.MP epidemic strains exist. The major time difference was P1- type II before 2005, and P1- I was dominant after 2005, and there was a trend towards P1- type II transformation, and the.MLVA typed type had been dominated by M4-5-7-2, M3-5-6-2 and M3-6-6-2. The distribution of genotypes in different regions and populations had a great difference in the MP genotype of children and elderly patients in.2. research hospitals. M3-6-6-2/P1- type II and M3-5-6-2/P1- II type.Mpn13 loci may be associated with P1 genotyping, but more research is needed to further explore the lower MP resistance rate of.3. in this region. The mutation of the drug resistant mutation with A2063G as the dominant.MP resistance may be related to the M4-5-7-2 type.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.1;R725.6

【参考文献】