本文選題：肺炎支原體 + P1基因�。� 參考：《東南大學》2017年碩士論文

【摘要】：研究背景與目的:肺炎支原體(MP)是引起呼吸道感染的主要病原體之一。人群普遍易感,每3-7年有一次流行高峰,春夏季是高發(fā)季節(jié),各流行期MP亞型會發(fā)生一定變化。對MP基因的分型主要是通過PCR-RFLP分型方法和MLVA分型方法。MP基因型種類復雜、多樣,且流行分布具有明顯的時間、地點和人群分布差異�？股氐臑E用使得MP耐藥問題日趨嚴重,研究顯示MP耐藥突變可能與基因型有關。隨著分子技術的不斷發(fā)展,MP分型方法的成熟,開展系統(tǒng)全面的MP基因型及其耐藥突變的分子流行病學研究,從分子水平揭示MP亞型分布特征、流行趨勢及耐藥狀況勢在必行。本研究利用Meta分析,在系統(tǒng)全面了解MP基因型流行分布特征的基礎上,開展以醫(yī)院為基礎的人群分子流行病學調查研究,了解目標醫(yī)院患者人群MP基因型及耐藥突變的流行分布情況,初步探討MP兩種分型方法分型結果之間可能存在的聯(lián)系以及基因型與耐藥突變之間的關系,旨在為當?shù)蒯t(yī)院MP感染的監(jiān)測、預防和臨床治療提供科學依據(jù)。研究方法:1..Meta 分析:檢索 PubMed、Web of Science、CNKI、CBM、萬方等數(shù)據(jù)庫,收集公開發(fā)表的與MP基因型有關的研究文獻。按納入與排除標準進行文獻的篩選并進行數(shù)據(jù)提取工作,運用Freeman-Tukey雙反正弦變換法對不同MP基因型的流行率進行合并計算,從研究時間、研究地點和研究人群三個方面進行亞組分析和Meta回歸分析。Meta分析軟件包括Stata19.0和SAS 9.3。2.MP基因分型方法:以南京市某綜合性三甲醫(yī)院兒科呼吸道感染患兒以及呼吸科老年呼吸道感染患者為研究對象。在知情同意的基礎上采集咽拭子并進行流行病學個案調查,采用Nest-PCR擴增16S rRNA基因片段檢測MP臨床株,采用PCR-RFLP方法和mPCR-CE-MLVA方法進行MP基因分型。3.MP耐藥突變分析方法:對MP臨床株23S rRNA基因片段擴增并送生物公司測序。根據(jù)測序結果分析MP耐藥基因位點突變。4.資料的統(tǒng)計分析方法:建立Excel數(shù)據(jù)庫,使用SAS 9.3或SPSS19.0軟件進行統(tǒng)計學分析。計數(shù)資料采用率或構成比描述,用χ2檢驗或Fisher確切概率法進行統(tǒng)計學分析,P0.05時差異具有統(tǒng)計學意義;計量資料采用中位數(shù)和四分位間距或均數(shù)±標準差(x±s)描述,采用Wilcoxon秩和檢驗或Kruskal-Wallis H檢驗進行統(tǒng)計學分析。研究結果:1..Meta分析共納入42篇文獻,其中與P1基因分型有關的文獻40篇,成功分型的樣本數(shù)為5027例,與MLVA分型有關的文獻16篇。成功分型的樣本數(shù)為1956例。P1-Ⅰ型流行率為 73.80%(95%CI:67.86-79.34);P1-Ⅱ 型流行率為 26.21%(95%CI:20.67-32.16);P1-Ⅱ 突變株流行率為 13.25%(95%CI:7.61-20.15);M4-5-7-2 型流行率為 68.97%(95%CI:59.55-77.70);M3-5-6-2 型流行率為 15.41%(95%CI:10.41-21.20);M3-6-6-2 型流行率為 5.49%(95%CI:2.34-9.90);其他型別的流行率為 6.71%(95%CI:4.72-9.02)。亞組分析和Meta回歸分析發(fā)現(xiàn),不同時間,地區(qū)和人群的MP基因型分布呈現(xiàn)明顯差異。2.研究醫(yī)院兒童呼吸道感染患者MP陽性率為27.6%。兒童患者中PCR-RFLP方法成功分型90例,P1-Ⅰ型流行率為21.1%(19/90),P1-Ⅱ型流行率78.9%(71/90),其中V2c突變株占50.7%(36/71)。MLVA分型方法成功分型102例,M3-6-6-2型流行率最高為 40.2%(41/102),M3-5-6-2 型流行率 37.3%(38/102),M4-5-7-2 型流行率為16.7%(17/102),其他基因型流行率為5.9%(6/102)。100%的M4-5-7-2型屬于為P1-Ⅰ,81.2%的M3-5-6-2,M3-6-6-2型為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 突變株占 61.9%(13/21)。MLVA分型方法成功分型30例,M3-5-6-2、M3-6-6-2型型流行率均為43.3%(13/30),M4-5-7-2 型 10%(3/30),M3-6-7-2 型 1 例,流行率為 3.3%。100%的 M4-5-7-2 型為P1-Ⅰ,66.7%的 M3-5-6-2,M3-6-6-2 型為 P1-Ⅱ。3.兒童患者132例MP陽性標本中共發(fā)現(xiàn)21例存在耐藥基因突變,耐藥率為15.9%。其中90.5%(19/21)為A2063G突變,9.5%(2/21)為A2064G。老年MP感染者耐藥率為 10%(3/30),A2063G 突變占 66.7%(2/3),T2611C 突變 33.3%(1/3)。成功分型的 141 例中,A2063G 突變中 M4-5-7-2 型 94.7%(18/19),M3-6-6-2 型 5.3%(1/19)。A2064G突變1例,屬于M3-5-6-2型。T2611C突變1例,屬于M3-6-6-2型。MP耐藥可能與M4-5-7-2型有關。4.MP感染流行特征分析,MP感染可能與年齡、性別有關,與未感染MP的呼吸道感染患兒相比,MP感染易出現(xiàn)發(fā)熱,消化道癥狀以及周身癥狀,而上呼吸道感染癥狀相對較少,N%和CRP值較高,L%值較低。兒童不同年齡間MP基因型分布存在統(tǒng)計學差異。小于3歲組患兒感染MP基因型以M3-6-6-2型為主,大于3歲的患兒以M3-5-6-2為主。研究結論:1.MP流行株P1基因型分布存在較大的時間差異性,2005年之前以P1-Ⅱ型為主,2005年之后P1-Ⅰ型占主導地位,并有向P1-Ⅱ型轉換的趨勢。MLVA分型型別一直以M4-5-7-2,M3-5-6-2,M3-6-6-2為主。在不同地區(qū)和人群中各基因型分布有很大的差別。2.研究醫(yī)院兒童和老年患者MP基因型均以M3-6-6-2/P1-Ⅱ型,M3-5-6-2/P1-Ⅱ型為主。Mpn13位點可能與P1基因分型型別有一定的聯(lián)系,但仍需更多的研究給予進一步的探討。3.該地區(qū)MP耐藥率與其他地區(qū)相比較低,耐藥突變以A2063G為主。MP耐藥突變可能與M4-5-7-2型有關。
[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.
【學位授予單位】：東南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R563.1;R725.6

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