【摘要】：背景 結(jié)核病是危害全球健康的重大公共衛(wèi)生問題，雖隨著短程督導(dǎo)化療方案的普遍實(shí)施，結(jié)核病疫情已經(jīng)有所控制，但當(dāng)前結(jié)核病控制仍面臨著耐多藥，合并HIV感染及流動(dòng)人口等問題帶來的巨大挑戰(zhàn)。非結(jié)核分枝桿菌是指結(jié)核與麻風(fēng)分枝桿菌以外的其他分枝桿菌，是一類在自然界中廣為分布的環(huán)境病原體。其可通過多種途徑感染易感人群引起局部或全身病灶，甚至導(dǎo)致死亡。國內(nèi)外越來越多的研究表明非結(jié)核分枝桿菌感染及其相關(guān)疾病的流行已呈現(xiàn)出上升的趨勢。 抗酸染色涂片鏡檢法簡單、快速，且是發(fā)現(xiàn)肺結(jié)核傳染源的有效方法。在結(jié)核病高發(fā)地區(qū)，其已成為結(jié)核病實(shí)驗(yàn)室診斷的主要指標(biāo)。但其只能證明抗酸桿菌的存在，不能區(qū)分結(jié)核與非結(jié)核分枝桿菌，在全球非結(jié)核分枝桿菌感染上升的背景下這使越來越多的涂陽病人有可能被誤診而接受不必要或不適當(dāng)?shù)目菇Y(jié)核治療。依靠培養(yǎng)的傳統(tǒng)菌種鑒定方法雖能達(dá)到區(qū)分兩者的目的，但在發(fā)展中國家的多數(shù)基層實(shí)驗(yàn)室尚無技術(shù)條件開展培養(yǎng)實(shí)驗(yàn)，上級(jí)分枝桿菌實(shí)驗(yàn)室雖能開展，但其繁瑣又費(fèi)時(shí)，遠(yuǎn)不能滿足臨床診療的需要。 目的 了解NTM的流行狀況和流行病學(xué)特征，初步評(píng)價(jià)IS6110熒光定量PCR法快速鑒定陽性痰涂片中的結(jié)核分枝桿菌的臨床應(yīng)用價(jià)值，同時(shí)對(duì)抗酸染色陽性痰涂片刮削物擴(kuò)增分枝桿菌利福平耐藥基因（rpoB基因）進(jìn)行初步探索。 第一部分廣州市越秀、海珠區(qū)非結(jié)核分枝桿菌流行狀況分析 一、研究對(duì)象及方法 選取2010年-2012年于越秀、海珠區(qū)兩個(gè)結(jié)核病防治所就診的可疑肺結(jié)核患者，收集其痰分枝桿菌培養(yǎng)等實(shí)驗(yàn)室資料。對(duì)于在研究期間有多次陽性培養(yǎng)和菌菌種鑒定的同一病例只按1例計(jì)算，且選擇在時(shí)間上靠近研究起點(diǎn)的實(shí)驗(yàn)室結(jié)果納入統(tǒng)計(jì)分析。 每個(gè)病例均在清晨留取深部痰標(biāo)本進(jìn)行分枝桿菌液體培養(yǎng)檢測，培養(yǎng)分離的菌株通過PNB生長抑制試驗(yàn)進(jìn)行MTC與NTM初步鑒別，NTM分離株按照中國防癆協(xié)會(huì)操作規(guī)程通過分枝桿菌的培養(yǎng)特征及一系列生化測試鑒定其菌種。 二、結(jié)果 1、可疑肺結(jié)核患者痰標(biāo)本中MTC與NTM的分離率分別為80.9%、19.1%。2010-2012各年度的NTM分離率分別為17.6%、17.1%、21.2%。 2、NTM分離者的男女比例為1.56：1，60歲及以上老年病例僅占26%，45-59歲、15-29歲年齡段為NTM分離高峰。 3、NTM菌種中快生長型占50.6%，慢生長型占49.4%。龜膿腫和鳥胞內(nèi)復(fù)合群為主要的分離菌種，分別占40.5%和24.1%。 第二部分痰涂片抗酸染色陽性患者分枝桿菌的分離情況分析 一、研究對(duì)象及方法 選取2012年~2013年于越秀、海珠區(qū)兩個(gè)結(jié)核病防治所診治的痰A(chǔ)FB涂片陽性且同期培養(yǎng)陽性的病例共331例。其中男性238例，女性93例(M/F=2.56)；年齡范圍15~87歲之間，平均39歲；初治225例，，復(fù)治106例。 所有病例均于清晨留取深部痰標(biāo)本用于涂片抗酸染色及分枝桿菌分離培養(yǎng)和鑒定。 二、結(jié)果 1、痰涂片AFB陽性患者中MTC和NTM的分離率分別為81%和19%。2012年NTM分離率為14.1%，2013年為25.2%。 2、初治和復(fù)治涂陽患者NTM分離率分別為12%和34%。 3、MTC組涂片陽性等級(jí)≤1+占55.97%，1+占44.03%。NTM組涂片陽性等級(jí)≤1+占82.54%，1+占17.46%。 第三部分IS6110-熒光定量PCR法快速鑒定抗酸染色陽性涂片中的結(jié)核分枝桿菌 一、研究對(duì)象及方法 331例同期培養(yǎng)陽性的抗酸染色陽性痰涂片來自2012年-2013年于越秀、海珠區(qū)結(jié)核病防治所就診的患者。其中陽性等級(jí)為1-9條/300視野的涂片25例，1+的涂片177例，2+涂片66例，3+涂片30例，4+涂片33例。根據(jù)其同期培養(yǎng)分群鑒定結(jié)果分為兩組，其中MTC組268例，NTM組63例。 應(yīng)用結(jié)核分枝桿菌(TB)核酸檢測試劑對(duì)所有痰涂片刮削物分枝桿菌DNA行熒光定量PCR檢測。 二、結(jié)果 1、熒光定量PCR法初步鑒定陽性涂片中分枝桿菌菌群的靈敏度和特異度分別為69.40%、76.19%。 2、熒光定量PCR法在鑒定抗酸染色涂片陽性標(biāo)本中的分枝桿菌菌群與培養(yǎng)法一致程度較差（kappa=0.324）。 3、MTC組各陽性等級(jí)涂片的陽性檢出率分別為26.67%、63.70%、68.97%、92.86%、93.75%。 4、FQ-PCR檢測的靶基因拷貝數(shù)和涂片陽性等級(jí)存在正相關(guān)（r=0.563）。第四部分抗酸染色陽性痰涂片刮削物擴(kuò)增分枝桿菌rpoB基因的初步探索一、研究對(duì)象及方法 18株臨床分離結(jié)核分枝桿菌來自廣州市胸科醫(yī)院“廣州地區(qū)分枝桿菌菌株庫”，80張抗酸染色陽性(3+～4+)痰涂片來自廣州市越秀區(qū)結(jié)核病防治所，涂片樣本來自經(jīng)分枝桿菌培養(yǎng)鑒定證實(shí)的肺結(jié)核患者。 設(shè)計(jì)兩對(duì)引物，以菌株、痰涂片刮削物中提取的基因組DNA為模板擴(kuò)增rpoB基因，PCR產(chǎn)物經(jīng)凝膠電泳檢測。 二、結(jié)果 1、兩對(duì)引物對(duì)18例臨床分離株基因組DNA進(jìn)行擴(kuò)增，均能見到明亮目的條帶，擴(kuò)增產(chǎn)物經(jīng)測序證實(shí)為rpoB基因。 2、痰涂片刮削物DNA經(jīng)不同模板量、不同退火溫度，兩組引物分別行普通PCR、Touchdown PCR、巢式PCR、二次PCR擴(kuò)增rpoB基因均未有目的條帶。熒光定量PCR檢測兩種不同方法提取的痰涂片DNA拷貝數(shù)分別為102-103copies/ul和104-105copies/ul。 3、菌株基因組DNA經(jīng)100-107一系列倍數(shù)稀釋后，兩對(duì)引物擴(kuò)增rpoB基因分別在104、103以下的稀釋倍數(shù)能觀察到明亮目的條帶，對(duì)應(yīng)的熒光定量PCR檢測拷貝數(shù)分別為106copies/ul、107copies/ul。 結(jié)論 1、可疑肺結(jié)核患者痰標(biāo)本中NTM分離率19.1%。 2、NTM感染人群以中青年為主。 3、NTM以快速生長分枝桿菌較多見。 4、涂陽患者NTM分離率為19%,2013年涂陽患者NTM分離率明顯高于2012年。 5、復(fù)治涂陽病人比初治者更易分離到NTM。 6、非結(jié)核分枝桿菌痰涂片陽性標(biāo)本以1+以下多見。 7、IS6110-熒光定量PCR法檢測到的結(jié)核分枝桿菌基因拷貝數(shù)與涂片陽性等級(jí)成正相關(guān)。 8、IS6110-熒光定量PCR法可作為陽性等級(jí)3+以上痰涂片中分枝桿菌快速初步鑒定的較好方法。 9、陽性痰涂片刮取物中提取的DNA量不足是rpoB基因未能成功擴(kuò)增的主要原因。
[Abstract]:background Tuberculosis is a major public health problem that is harmful to the global health, although with the general implementation of the short-range supervision and chemotherapy scheme, the tuberculosis epidemic has been controlled, but the current tuberculosis control is still facing the problems of multi-drug resistance, the combination of HIV infection and the floating population. The non-tuberculous mycobacteria refer to the other mycobacteria other than the Mycobacterium tuberculosis and the M. jatropha. It is a kind of environmental pathogen that is widely distributed in nature. a body that can infect a susceptible population by a variety of ways to cause local or systemic lesions, or even death The more and more studies at home and abroad have shown that the prevalence of non-tuberculous mycobacteria infection and its related diseases has been increasing. The anti-acid staining smear microscopy method is simple and rapid, and is the source of the infection of the pulmonary tuberculosis. Effective method. It has become the principal of the tuberculosis laboratory diagnosis in a high-incidence area of tuberculosis. However, it can only prove that the presence of antacid can not distinguish between the tuberculosis and the non-tuberculous mycobacteria, and in the background of the rise of the infection of the non-mycobacterium tuberculosis in the world, the more and more smear-positive patients are likely to be misdiagnosed with unnecessary or inappropriate antiknot Nuclear therapy. Although the traditional identification method of the traditional culture can achieve the purpose of distinguishing the two, in most basic-level laboratories in developing countries, there are no technical conditions to carry out the culture experiment, but the superior mycobacteria laboratory can be carried out, but it is tedious and time-consuming and can not meet the clinical diagnosis and treatment. to be required Objective To study the prevalence and epidemiological characteristics of NTM, and to evaluate the rapid identification of Mycobacterium tuberculosis in the positive sputum smear by using the IS6110 fluorescence quantitative PCR method. Clinical application value of mycobacterium tuberculosis rifampin-resistant gene (rpoB gene) was amplified by anti-acid staining positive sputum smear. Preliminary exploration. The first part of Guangzhou Yuexiu, non-tuberculosis in Haizhu District M. M. prevalence I. The study object and method selected the suspected pulmonary tuberculosis patients treated by the two tuberculosis prevention and control centers in Yuexiu and Haizhu District in 2010-2012, and collected it. For laboratory data, such as the culture of Mycobacterium tuberculosis, only one case was calculated for the same case with multiple positive culture and bacterial strain identification during the study, and the selection was close to the study in time The laboratory results of the point were included in the statistical analysis. In each case, the deep sputum samples were collected in the morning for detection of mycobacteria liquid culture, and the isolated strains were inhibited by the growth of the PNB The MTC and NTM were initially identified by the test, and the NTM isolates were cultured in accordance with the operation procedures of the Chinese Anti-Japanese Association through the culture of Mycobacteria. features and one The isolation rates of MTC and NTM in sputum specimens of patients with suspected pulmonary tuberculosis were 80.9% and 19.1%, respectively. The ratio of the male to female was 1.56:1, the age of 60 and older was only 26%, and 45- 59-year-old,15-29-year-old was the peak of NTM separation. The fast-growing type (50.6%) and the slow-growing type (49.4%) in the species (49.4%). The main separation strains were 40.5% and 24.1%, respectively. The second part of the sputum smear is acid-fast Analysis of the isolation of M. positive patients with positive staining. The study object and method were selected from 2012 to 2013 in Yuexiu and Haizhu District. There were 331 cases of sputum AFB smear positive and positive in the same period, of which 238 cases were male and 93 cases were female (M/ F = 2.56). Between the ages of 15 and 87, the average age was 39; in the first treatment,225 cases were treated and 106 cases were retreated. All cases All in the morning The separation rate of MTC and NTM in sputum smear AFB positive patients was 81. % and 19%. The NTM separation rate in 2012 was 14.1%,2013 The rate of NTM was 12% and 34%, respectively. The positive grade of the smear positive of the MTC group was 55.97%. + accounted for 44.03%. The positive grade of smear positive in NTM group was 82.54%, and 1 + was 17.46%. . Part III IS Rapid identification of Mycobacterium tuberculosis one, study object and method in acid-fast stain positive smear by 6110-fluorescence quantitative PCR The positive anti-acid stained positive sputum smear from 2012 to 2013 in Yuexiu, Haizhu District, for the prevention and treatment of tuberculosis, in which the positive grade is 1-9/ There were 25 smear of 300 eyes,177 cases of 1 + smear,66 cases of 2 + smear and 30 cases of 3 + smear. And 33 cases of 4 + smears were divided into two groups according to the group identification results of the same period, including 268 cases of MTC group and 63 cases of NTM group. .. Detection of the fluorescence quantitative PCR of Mycobacterium tuberculosis DNA by using Mycobacterium tuberculosis (TB) nucleic acid detection reagent. 1. The sensitivity and specificity of the bacterial flora in the positive smear were 69.40% and 76.19%, respectively. The fluorescence quantitative PCR method has a poor degree of consistency with the culture method (kappa = 0.324) in the identification of the positive specimen of the acid-fast stain smear. The positive rate of positive grade smear in MTC group was 26.67%, 63.70% and 68.9 respectively. 7%, 92.86%, 93.75%.4, FQ-PCR-detected target gene copy Number and smear positive, etc. There was a positive correlation (r = 0.563) in the stage. The fourth part of the acid-fast staining positive sputum smear was used to amplify the rpoB gene of the Mycobacterium tuberculosis. >,80 anti-acid staining positive (3 + ~ 4 +) sputum smear from the tuberculosis prevention and control department in Yuexiu District, Guangzhou, and the smear samples from the culture and identification of the mycobacterium tuberculosis solid lung junction designing two pairs of primers to amplify the rpoB gene by using the genomic DNA extracted from the bacterial strain and the sputum smear scraper as a template, 2. Results 1 and 2 pairs of primers were used to amplify the genomic DNA of 18 clinical isolates. A. The rpoB gene was amplified by ordinary PCR, Touchdown PCR, nested PCR and secondary PCR with different template and different annealing temperatures. the DNA copy number of the sputum smear extracted by the two different methods is 102-103 copies/ ul and 104-105 copies/ ul.3, and the two pairs of primers amplify the rpoB gene respectively after the bacterial strain genomic DNA is diluted by a series of multiple times of 100-107, at 1 the dilution factor below 04,103 can be observed for a bright target strip, for example, The copy number of the fluorescence quantitative PCR should be 涓

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