本文選題：結(jié)核菌 + 耐多藥　； 參考：《北京協(xié)和醫(yī)學(xué)院》2010年博士論文

【摘要】：中國是全球22個(gè)結(jié)核病高負(fù)擔(dān)國家之一,患病人數(shù)僅次于印度居世界第二。我國的耐藥結(jié)核病流行狀況也很嚴(yán)重,耐多藥(MDR)結(jié)核的流行比例為9.3%,約是世界平均水平的2倍。耐多藥結(jié)核病治療周期長、藥物副作用大,而且治愈率低、死亡率高。因此,耐多藥結(jié)核病的控制對于中國當(dāng)前結(jié)核病的防治具有重要意義。 早期快速診斷結(jié)核分枝桿菌的耐藥性有利于耐多藥結(jié)核病的治療和控制。和基于培養(yǎng)的藥敏檢測方法相比,基于分子生物學(xué)的藥敏檢測方法具有快速、敏感和成本較低的優(yōu)勢。目前,已有商品化的產(chǎn)品INNO-LiPARif.TB (Innogenetics, Belgium)和GenoType MTBDR plus (Genotype MTBDR; Hain Lifescience, Nehren, Germany)被WHO認(rèn)為可以用于結(jié)核耐多藥性的快速診斷。但是由于不同地區(qū)流行菌株的耐藥相關(guān)基因突變頻率和類型不同,這些工具檢測耐藥性的靈敏度和特異度在不同地區(qū)也不同。因此,對于不同地區(qū)菌株的耐藥相關(guān)基因突變頻率和類型的全面了解,有利于這類新的診斷技術(shù)的開發(fā)和廣泛應(yīng)用。 在中國對耐藥結(jié)核菌,特別是耐多藥結(jié)核菌的耐藥相關(guān)基因的突變頻率和突變類型進(jìn)行的研究還很少。我們試圖通過改善的、全面的結(jié)核分支桿菌耐藥相關(guān)基因的研究,對河南省耐多藥結(jié)核菌4個(gè)一線抗結(jié)核藥耐藥相關(guān)基因的突變頻率和突變類型進(jìn)行詳細(xì)描述,并對各個(gè)耐藥相關(guān)基因的的變異規(guī)律進(jìn)行了深入的探討,為開發(fā)適合該地區(qū)耐多藥結(jié)核菌的高靈敏度和特異度的診斷方法提供更多的數(shù)據(jù)。 本研究中共收集來自河南省不同地區(qū)的115株耐多藥結(jié)核菌和22株對一線4個(gè)抗結(jié)核藥全敏感的菌株。使用WHO推薦的比例法檢測菌株對一線4個(gè)抗結(jié)核藥的藥物敏感性,獲得菌株的耐藥特征。 我們檢測了所有已知的一線4個(gè)抗結(jié)核藥物的9個(gè)耐藥相關(guān)基因的編碼區(qū)全長和部分啟動子區(qū),以期能獲得相關(guān)基因突變頻率和突變類型的更全面信息。通過對河南115株耐多藥結(jié)核菌4個(gè)一線抗結(jié)核藥的耐藥相關(guān)基因突變特征的全面分析,我們發(fā)現(xiàn)： (1)本研究利福平和異煙肼耐藥相關(guān)基因所有的已知突變的總突變頻率沒有明顯改變,如：本研究rpoB所有的已知突變的總突變頻率為97.3%,與多數(shù)研究報(bào)道的95%到98%一致。此外,MDR菌株中各耐藥相關(guān)突變位點(diǎn)的突變頻率的順位沒有改變。如：rpoB531rpoB526rpoB516,rpsL43rpsL88rrs513。 (2)同一地區(qū),在MDR菌株較多或全部為MDR菌株的研究中,多個(gè)耐藥相關(guān)基因的主要突變位點(diǎn)的突變頻率高于MDR菌株較少或單耐藥菌株的研究,這些突變位點(diǎn)有rpoV531、katG315、embB306、rpsL43。此外,有的耐藥相關(guān)基因的突變,如：embB306和rpsL 43,在MDR菌株中會出現(xiàn)在該突變對應(yīng)耐藥性的敏感菌株。 (3)共發(fā)現(xiàn)53個(gè)新突變位點(diǎn),16個(gè)新突變類型,這些新突變位點(diǎn)和類型在MDR結(jié)核菌的耐藥發(fā)生中的作用需要進(jìn)一步研究。 此外,我們還利用已獲得的耐多藥結(jié)核菌耐藥相關(guān)基因突變特征及其耐藥特征數(shù)據(jù),對embB基因突變(包括embB306突變)和MDR結(jié)核菌耐藥性間的關(guān)系進(jìn)行了分析,得到了如下結(jié)論： (1)在MDR結(jié)核菌中,embB基因上的突變,包括embB306突變,和乙胺丁醇耐藥統(tǒng)計(jì)學(xué)上相關(guān)。單獨(dú)檢測embB306突變預(yù)測乙胺丁醇耐藥靈敏度不高(62.7%),通過檢測embB基因所有突變,可以提高靈敏度至89.6%。這提示embB基因上多個(gè)位點(diǎn)均可能參與了乙胺丁醇的耐藥發(fā)生,大多數(shù)突變集中在300到500密碼子間。某些對乙胺丁醇敏感的MDR菌株發(fā)生embB基因突變(包括embB306突變),這與多數(shù)研究結(jié)論一致,表明embB基因突變不僅和乙胺丁醇耐藥相關(guān),還可能與MDR相關(guān)。 (2)總體上embB306位點(diǎn)突變菌株比例隨耐藥數(shù)目增加而增加,趨勢卡方檢驗(yàn)顯示相關(guān)性有統(tǒng)計(jì)學(xué)顯著性。但耐3藥組embB306位點(diǎn)突變菌株比例低于耐2藥組,這可能是因?yàn)楸狙芯恐心玩溍顾氐腗DR菌株發(fā)生突變的比例(17.9%)較低；也可能是因?yàn)槟?藥(MDR)組樣本量較少而影響了統(tǒng)計(jì)分析的穩(wěn)定性。同樣,embB基因各位點(diǎn)總和突變與耐藥數(shù)目增加的相關(guān)性有統(tǒng)計(jì)學(xué)顯著性,其中發(fā)生embB497突變的MDR菌株在耐4藥組明顯多于耐3藥組,提示embB基因其他位點(diǎn)突變可能也有隨耐藥數(shù)目增加而增加的趨勢。 (3)耐4藥組菌株中有90.2%發(fā)生embB基因突變。通過檢測embB基因4個(gè)位點(diǎn)的突變(embB306、embB354、embB406和embB497)可以發(fā)現(xiàn)83.6%的一線4重耐藥菌株,靈敏度較高,用于一線4重耐藥性初步篩選有很好的潛在應(yīng)用價(jià)值,特別適用于像河南這種耐一線4個(gè)抗結(jié)核藥在MDR結(jié)核菌中比例(53%)較高的地區(qū)。
[Abstract]:China is one of the 22 countries with high TB burden in the world, the number of patients in the world is second only to the world in India. The prevalence of drug-resistant tuberculosis in China is also very serious. The prevalence rate of multi drug resistant (MDR) tuberculosis is 9.3%, about 2 times the world average. Therefore, the control of multidrug-resistant tuberculosis is of great significance for the prevention and treatment of tuberculosis in China.
Early rapid diagnosis of Mycobacterium tuberculosis resistance is beneficial to the treatment and control of multidrug resistant tuberculosis. Compared with the culture based drug sensitivity detection methods, molecular biology based drug sensitivity detection method has the advantages of rapid, sensitive and low cost. Currently, the commercial product INNO-LiPARif.TB (Innogenetics, Belgium) and Gen have been developed. OType MTBDR plus (Genotype MTBDR; Hain Lifescience, Nehren, Germany) is considered to be used for the rapid diagnosis of multidrug resistance in tuberculosis. However, the sensitivity and specificity of these tools are also different in different regions due to the different frequency and type of mutation of resistance related genes in different regions. A comprehensive understanding of the frequency and type of drug resistance related genes in different regions is conducive to the development and wide application of these new diagnostic techniques.
There are few studies in China on the mutation frequency and mutation types of drug-resistant Mycobacterium tuberculosis, especially MDR resistant genes. We attempt to improve the mutation frequency of 4 first line anti tuberculosis drug resistance genes in Henan province by improving the comprehensive drug resistance related genes of Mycobacterium tuberculosis. The mutation types are described in detail, and the variation rules of various resistance related genes are discussed in depth to provide more data for the development of the diagnostic methods suitable for the high sensitivity and specificity of multi drug resistant Mycobacterium tuberculosis in this area.
In this study, 115 strains of multidrug resistant Mycobacterium tuberculosis from different regions of Henan province and 22 strains of 4 all sensitive anti tuberculosis drugs were collected. The sensitivity of strains to 4 anti tuberculosis drugs was detected by the proportion method recommended by WHO, and the resistance characteristics of the strains were obtained.
We examined the full length and the partial promoter region of the coding region of all 9 drug resistance related genes of 4 known antituberculous drugs, in order to obtain more comprehensive information about the frequency of mutation and the type of mutation of the related genes. Through the comprehensive classification of the mutation characteristics of the resistance related gene of 4 first-line anti tuberculosis drugs in Henan. Analysis, we found:
(1) the total mutation frequency of all known mutations in rifampicin and isoniazid resistance related genes did not change significantly, for example, the total mutation frequency of all known mutations of rpoB in this study was 97.3%, consistent with the 95% to 98% reported in the study. In addition, the mutation frequency of each resistance related mutation site in the MDR strain did not change. Such as: rpoB531rpoB526rpoB516, rpsL43rpsL88rrs513.
(2) in the same area, in the study of more or all MDR strains of MDR strain, the mutation frequency of the main mutation loci of multiple resistance related genes is higher than that of MDR strains or single resistant strains. These mutation sites are rpoV531, katG315, embB306, rpsL43., and some mutations of resistance related genes, such as embB306 and rpsL 43, in MD. The mutant strain of R will be sensitive to the drug resistance.
(3) 53 new mutation sites, 16 new mutation types, and the role of these new mutation sites and types in the occurrence of drug-resistance in MDR TB need to be further studied.
In addition, we also analyzed the relationship between the embB gene mutation (including embB306 mutation) and the drug resistance of MDR tuberculosis by using the acquired resistance related gene mutation characteristics and drug resistance characteristic data. The following conclusions were obtained:
(1) in MDR tuberculosis, mutations in embB gene, including embB306 mutation, and ethambutol resistance are statistically related. The sensitivity of embB306 mutation to predict ethambutol resistance is not high (62.7%), and the sensitivity to 89.6%. can be increased by detecting all mutations of embB gene, which suggests that multiple sites in the embB gene may be involved in B The resistance to amethbutanol occurred, most of the mutations were concentrated between 300 and 500 ciphers. Some of the MDR strain sensitive to ethambutol occurred embB gene mutation (including embB306 mutation), which was in accordance with most of the findings, indicating that the mutation of the embB gene was not only associated with ethambutol resistance, but also associated with MDR.
(2) on the whole, the proportion of embB306 mutant strains increased with the increase of the number of drug resistance, and the trend chi square test showed that the correlation was statistically significant. However, the proportion of embB306 mutant strains in the 3 drug resistant group was lower than that of the 2 drug resistant group, which may be due to the lower proportion of the mutation of streptomycin resistant strains of streptomycin in this study (17.9%); it may also be because of the ratio of the mutation of streptomycin resistant strains (17.9%) in this study. The less sample size of the 2 drug resistant (MDR) group affected the stability of the statistical analysis. Similarly, the correlation between the total mutation of the embB gene points and the increase of the number of drug resistance was statistically significant, of which the MDR strain of the embB497 mutation was significantly more than the 3 drug resistant group in the 4 drug resistant group, suggesting that the mutation of the other loci of the embB gene may also increase with the number of resistance. Add and increase the trend.
(3) 90.2% of the 4 drug resistant strains had embB mutation. By detecting 4 loci mutations of the embB gene (embB306, embB354, embB406 and embB497), 83.6% front-line 4 heavy resistant strains were found to be highly sensitive. It was useful for preliminary screening of first line 4 heavy resistance, especially for 4 resistant lines like Henan. The proportion of antitubercular drugs in MDR TB (53%) was higher.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R52;R181.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Mokrousov Igor;Narvskaya Olga;;Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing,China[J];Chinese Medical Journal;2007年09期

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本文編號：1844748