河南省耐多药结核菌耐药相关基因的分子流行病学研究

发布时间：2018-05-04 21:37

本文选题：结核菌 + 耐多药　；参考：《北京协和医学院》2010年博士论文

【摘要】：中国是全球22个结核病高负担国家之一,患病人数仅次于印度居世界第二。我国的耐药结核病流行状况也很严重,耐多药(MDR)结核的流行比例为9.3%,约是世界平均水平的2倍。耐多药结核病治疗周期长、药物副作用大,而且治愈率低、死亡率高。因此,耐多药结核病的控制对于中国当前结核病的防治具有重要意义。早期快速诊断结核分枝杆菌的耐药性有利于耐多药结核病的治疗和控制。和基于培养的药敏检测方法相比,基于分子生物学的药敏检测方法具有快速、敏感和成本较低的优势。目前,已有商品化的产品INNO-LiPARif.TB (Innogenetics, Belgium)和GenoType MTBDR plus (Genotype MTBDR; Hain Lifescience, Nehren, Germany)被WHO认为可以用于结核耐多药性的快速诊断。但是由于不同地区流行菌株的耐药相关基因突变频率和类型不同,这些工具检测耐药性的灵敏度和特异度在不同地区也不同。因此,对于不同地区菌株的耐药相关基因突变频率和类型的全面了解,有利于这类新的诊断技术的开发和广泛应用。在中国对耐药结核菌,特别是耐多药结核菌的耐药相关基因的突变频率和突变类型进行的研究还很少。我们试图通过改善的、全面的结核分支杆菌耐药相关基因的研究,对河南省耐多药结核菌4个一线抗结核药耐药相关基因的突变频率和突变类型进行详细描述,并对各个耐药相关基因的的变异规律进行了深入的探讨,为开发适合该地区耐多药结核菌的高灵敏度和特异度的诊断方法提供更多的数据。本研究中共收集来自河南省不同地区的115株耐多药结核菌和22株对一线4个抗结核药全敏感的菌株。使用WHO推荐的比例法检测菌株对一线4个抗结核药的药物敏感性,获得菌株的耐药特征。我们检测了所有已知的一线4个抗结核药物的9个耐药相关基因的编码区全长和部分启动子区,以期能获得相关基因突变频率和突变类型的更全面信息。通过对河南115株耐多药结核菌4个一线抗结核药的耐药相关基因突变特征的全面分析,我们发现： (1)本研究利福平和异烟肼耐药相关基因所有的已知突变的总突变频率没有明显改变,如：本研究rpoB所有的已知突变的总突变频率为97.3%,与多数研究报道的95%到98%一致。此外,MDR菌株中各耐药相关突变位点的突变频率的顺位没有改变。如：rpoB531rpoB526rpoB516,rpsL43rpsL88rrs513。 (2)同一地区,在MDR菌株较多或全部为MDR菌株的研究中,多个耐药相关基因的主要突变位点的突变频率高于MDR菌株较少或单耐药菌株的研究,这些突变位点有rpoV531、katG315、embB306、rpsL43。此外,有的耐药相关基因的突变,如：embB306和rpsL 43,在MDR菌株中会出现在该突变对应耐药性的敏感菌株。 (3)共发现53个新突变位点,16个新突变类型,这些新突变位点和类型在MDR结核菌的耐药发生中的作用需要进一步研究。此外,我们还利用已获得的耐多药结核菌耐药相关基因突变特征及其耐药特征数据,对embB基因突变(包括embB306突变)和MDR结核菌耐药性间的关系进行了分析,得到了如下结论： (1)在MDR结核菌中,embB基因上的突变,包括embB306突变,和乙胺丁醇耐药统计学上相关。单独检测embB306突变预测乙胺丁醇耐药灵敏度不高(62.7%),通过检测embB基因所有突变,可以提高灵敏度至89.6%。这提示embB基因上多个位点均可能参与了乙胺丁醇的耐药发生,大多数突变集中在300到500密码子间。某些对乙胺丁醇敏感的MDR菌株发生embB基因突变(包括embB306突变),这与多数研究结论一致,表明embB基因突变不仅和乙胺丁醇耐药相关,还可能与MDR相关。 (2)总体上embB306位点突变菌株比例随耐药数目增加而增加,趋势卡方检验显示相关性有统计学显著性。但耐3药组embB306位点突变菌株比例低于耐2药组,这可能是因为本研究中耐链霉素的MDR菌株发生突变的比例(17.9%)较低；也可能是因为耐2药(MDR)组样本量较少而影响了统计分析的稳定性。同样,embB基因各位点总和突变与耐药数目增加的相关性有统计学显著性,其中发生embB497突变的MDR菌株在耐4药组明显多于耐3药组,提示embB基因其他位点突变可能也有随耐药数目增加而增加的趋势。 (3)耐4药组菌株中有90.2%发生embB基因突变。通过检测embB基因4个位点的突变(embB306、embB354、embB406和embB497)可以发现83.6%的一线4重耐药菌株,灵敏度较高,用于一线4重耐药性初步筛选有很好的潜在应用价值,特别适用于像河南这种耐一线4个抗结核药在MDR结核菌中比例(53%)较高的地区。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R52;R181.3

【参考文献】