| The incidence and infection of drug resistance tuberculosis (DR-TB), especially for the multi-drug resistance tuberculosis (MDR-TB) is one of the major cause to revive the epidemic of tuberculosis. WHO and IUATLD launched a global program for surveillance of drug resistance in 1994 to identify global DR-TB distribution, and conducted a resistance investigation on the first line drugs, including streptomycin(SM), isoniazid(INH) rifampicin (RFP) and ethambutol(EMB), among114 countries and area in the world from 1994 to 2003 to disclose the growing tendency of MDR-TB in developing countries. China is on the list of"Countries and Districts need more attention"proposed by WHO due to the severe situation of drug resistance. MDR-TB problem in China is also a"hot topic".The main task for TB control at present is to prevent and control MDT-TB, for which, the baseline information and epidemic characteristics of DT-TB shall be collected for reference to make preventive or administrative policies. However, both DR-TB information and epidemic characteristics show regional differences. Located in southwest, Chongqing is confronted with the pressure of large rural population and relatively underdeveloped economy. In the last decade, there is no local report on (M)DR-TB based on large sample size. In a word, the baseline information and epidemic characteristics of local (M)DR-TB is critical to DR-TB control. Section 1 in the present study disclosed smear positive samples sequentially collected by authorized local TB prevention organization (Tuberculosis Prevention and Control Institute, chest hospital etc.), total 1089 cases, and the analysis of drug resistant information and epidemic characteristics thereof.INH and EMB are the first line TB resistance drugs recommended by WHO and one of the indispensable compound drugs for TB treatment. According to the global surveillance report on drug resistance tuberculosis by WHO, the resistance rate of INH is rising yearly around the world. Of all the four TB drugs (INH, RFP, SM and EMB), the resistance rate of INH is the highest or the second highest, 5.6% on average. China's INH resistance rate is 17.6%, rather a high level. Generally speaking, the global EMB resistance rate is low, only 0.8% (0~24.8) on average, but rising rapidly every year. China's EMB resistance rate (1.5%) is higher than the global average value.To reduce death rate of DR-TB patients, infection sources and infection period, diagnosis and selection of sensitive drugs at the early stage is the most important method to help TB patients. However, the present drug sensitivity testing method based on culture is too time-consuming (1-3months) to be used in clinic timely. The developing molecular biological theories and techniques shed some light on the molecular mechanism of INH resistance and EMB resistance of mycobacterium tuberculosis. Up to now, researchers have proved that the mutation katG genes (especially katG315), an indispensible catalase- peroxide to INH activation by internal-coding of mycobacterium tuberculosis, is the major cause for INH resistance. Also the mutation rates of katG315 in INH-resistant mycobacterium tuberculosis shows regional differences (26%~95%). The mutation of embB (especially the 306th codon), the gene that is responsible for coding arabinose glycosyltransferase is the major cause to lead EMB resistance. embB 306 mutation was detected on 50%~70% EMB-resistant stains, so it is regarded as the molecular biological marker closely related to EMB resistance. katG315 mutation and embB306 mutation are used as the clinic testing methods for INH and EMB resistance respectively in some regions, by which the diagnosis and therapy of INF or EMB resistance are realized as well as showing high sensitivity and specificity. So it is safe to declare the feasibility of applying those rapid and economical molecular biological methods to clinic.Molecular biology is to estimate the phenotype of drug resistance through genotype, and the gene mutation of katG and embB shows significant regional differences. By now, there is no report on the mutation rate or mutation spectrum of those two genes in mycobacterium tuberculosis, whether katG315 and embB306 can be used as the marker to screen and test INH and EMB resistance of mycobacterium tuberculosis needs further exploration. Section 2 and Section 3 in this study used sequencing method to analyze the polymorphism of katG in 97 strains and embB in 101 strains of local patients by different compound drugs, and set a base to form a rapid molecular biological method to test INH resistance or EMB resistance in Chongqing. The main results and conclusions are as follows:1. The general DR-TB rate and MDR-TB rate is 27.6% (301/1089) and 7.3% (79/1089); the DR-TB rate and MDR-TB rate for new case is 22.7% and 4.1%; and the DR-TB rate and MDR-TB rate for re-treatment is 56.2% and 26.5%. In general, Chongqing's DR-TB level is in the middle level compared to the domestic or foreign surveillance results.2. The resistance rates for SM, INH, RFP and EMB are 16.3%, 14.0%, 10.7% and 4.7% respectively. In the recent three years, RFP resistance, EMB resistance and multi-drug resistance are constantly rising.3. Multi-drug resistance cases show no obvious gender of age difference, but they are closely related to chemotherapy history and living conditions (rural environment, annual income lower than average level etc.)4. The distribution of (M)DR-TB cases indicated that epidemic spreading of (M)DR-TB might exist within a certain scope to some extent in some area of Chongqing, so it is necessary to reinforce the surveillance of drug resistance, especially at the molecular level, in order to diagnose the expansion of mycobacterium tuberculosis at an early stage, which is also important to control the epidemic situation of (M)DR-TB.5. katG315 mutation spectra from INH resistant strains in Chongqing can be divided into three types: S315T, S315N and S315, with the general mutation 75.5% (37/49), including 81.1% (30/37) S315 mutation. katG315 mutation was not detected in INH sensitive strains.6. Compared to the traditional drug sensitivity test, , the method by using katG315 as the INH-resistant marker showed a high sensitivity 75.5%, specificity 100% and accuracy 87.6% (85/97). And according to other indicators, such method showed a fair clinic application value.7. katG315 mutation in MDR-TB strains was significantly higher than that in INH-resistant strains, and related to drug resistance quantity, so it is suitable to be used as the molecular marker for MDR-TB test.8. The analysis of katG mutation spectrum indicated that katG315 mutation is the major molecular mechanism to generate the local INH resistance. No katG315 mutation was detected in 24.5% INH resistant strains, indicating that there were some other mechanisms in the local area to generate INH resistance. The influence of mutation other rare point positions on the generating process of INH resistance needs further study.9. embB306 mutation rate in EMB-resistant strains (66.7%) was significantly higher than that in EMB-sensitive strains (60.0%). Compared to the traditional drug sensitivity test, the method by using embB306 as the EMB-resistant marker showed a high sensitivity 66.7%, specificity 90% and accuracy 78.2%, indicating that the rapid method by using embB306 mutation to test EMB resistant strains is applicable to the clinic.10. embB306 mutation rate in EMB-resistant MDR strains (84.0%) was significantly higher than that in non-MDR strains (60.0%), and ascending with the growth of drug resistance quantity, indicating that embB306 is more suitable for testing EMB-resistant MDR strains as a marker.11. embB mutation spectrum is in broad distribution. In the present study, embB306 mutation covered 66.7% EMB-resistant strains, indicating that embB306 mutation is the major molecular mechanism to generate EMB resistance in Chongqing, and that other molecular mechanisms are probably related to a number of rare mutations and the common results of all those rare mutations.In conclusion, the present study outlined the baseline information and the major epidemic characteristics of (M)DR-TB in Chongqing, and identified that katG315 and embB306 are the major molecular mechanisms for INH resistance and EMB resistance respectively, so the method by using INH or EMB as the marker for rapid test of drug resistance shows a fair clinic application value. Besides, the results of this study also provides reference for prevention and control of TB, and (M)DR-TB in Chongqing...
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