结核分枝杆菌吡嗪酰胺耐药诊断方法的建立及可靠性评估
发布时间：2018-07-25 13:52
【摘要】：吡嗪酰胺(Pyrazinamide,PZA)对细胞内酸性环境下处于半休眠状态的结核分枝杆菌具有独特的杀菌活性,因此成为结核病短程化疗的基石。由于价格便宜而疗效好,PZA被广泛用于结核病治疗的强化期,并且也是耐多药结核病治疗的一种重要药物。近年来的研究表明,PZA耐药无论在耐多药结核病患者中还是在非耐多药结核病患者中都可能发生,因此常规开展PZA敏感性试验对于合理使用PZA至关重要。然而,由于PZA仅能在酸性环境下才能发挥作用,而酸性环境下的药敏试验会影响细菌生长,因此PZA的药敏试验不便于开展,并且可靠性也一直是个重要的问题。本研究建立并评估了多种PZA的敏感性试验方法,目的是为临床寻求简便、可靠、可操作的PZA药敏试验方法。第一部分应用烟酰胺微孔板法检测结核分枝杆菌吡嗪酰胺耐药性的方法的建立及评估目的:建立烟酰胺替代吡嗪酰胺的微孔板药敏检测方法并评估其可靠性。方法:选取125株结核分枝杆菌临床分离株,另选取结核分枝杆菌标准株H37Rv、BCG标准株做为对照菌株。以倍比稀释法设定9个烟酰胺药物浓度(范围从8到2000μg/ml),检测菌株对烟酰胺的最低抑菌浓度,同时使用BACTEC MGIT960法检测菌株对PZA敏感性,并对所有菌株进行了pnc A基因及其启动子区的基因序列测定。结果:125株临床分离株中有64株被BACTEC MGIT960法诊断为PZA耐药。以BACTEC MGIT960法作为PZA耐药性检测的参考方法,以500μg/ml做为烟酰胺培养板法检测PZA耐药性的临界值,则烟酰胺微孔板法的灵敏度为100%(61/61),特异度为95.3%(61/64),准确度为97.6%(122/125)。对125临床分离株进行测序发现57株菌存在pnc A基因及其启动子区的突变,68株菌为野生株。以pnc A基因及其启动子区基因测序为参考方法,烟酰胺微孔板法检测PZA耐药性的灵敏度为89.5%(51/57)、特异度为80.9%(55/68)、准确度为84.8%(106/125)。结论:烟酰胺微孔板法检测结核分枝杆菌吡嗪酰胺耐药性方法具有操作简易、时间快速、价格低廉、结果可靠和重复性较好的优点,具有较好的临床推广价值。第二部分:应用Surveyor酶酶切法快速检测吡嗪酰胺的耐药性的方法的建立及评估目的:建立应用Surveyor酶酶切法快速检测MTB的pnc A基因及其启动子序列的基因突变的方法。方法:选取耐多药(MDR)菌株91株作为研究对象。分别以上游引物:下游引物为2:5的比例扩增所有菌株,同时以上游引物:下游引物为5:2的比例扩增结核分枝杆菌标准株H37Rv,然后将PCR产物以1:1的比例混合杂交,应用Surveyor酶酶切反应产物后电泳。以杂交条带能够被Surveyor酶切割做为判定pnc A基因及其启动子序列存在基因突变的依据。以BACTEC MGIT960检测PZA耐药性方法和pnc A基因及其启动子区基因序列测定法作为对照,评价Surveyor酶酶切法的应用价值。结果:以BACTEC MGIT960法检测PZA耐药性作为对照方法,Surveyor酶酶切法诊断PZA耐药的灵敏度为100%(29/29),特异度为87.1%(54/62),准确度为91.2%(83/91)。以pnc A基因及其启动子区测序结果作为对照,Surveyor酶酶切法的灵敏度、特异度和准确度均为100%。结论:Surveyor酶酶切法检测pnc A基因及其启动子区突变的性能与基因测序高度一致。Surveyor酶切法具有快速、操作简便、价格低廉的特点,可以做为一个替代方法检测pnc A基因突变。第三部分评估Versa TREK Myco PZA kit法检测吡嗪酰胺耐药性的可靠性目的:评估Versa TREK Myco PZA kit法检测结核分枝杆菌吡嗪酰胺耐药性的可靠性及其临床应用价值。方法:选取192株结核分枝杆菌临床分离株。分别使用BACTEC MGIT960法和Versa TREK Myco PZA kit法检测待测菌株的PZA耐药性,并对所有纳入菌株的pnc A基因及其启动子区序列进行序列测定。结果:以BACTEC MGIT960法检测PZA耐药性作为对照方法,Versa TREK Myco PZA kit法的灵敏度为93.5%(58/62),特异度为91.5%(119/130),准确度为92.2%(177/192)。以pnc A基因测序法作为对照,Versa TREK Myco PZA kit法的灵敏度为88.4%(61/68)、特异度为82.9%(102/123)和准确度均为84.9%(163/192)。结论:Versa TREK Myco PZA kit法检测PZA耐药性与BACTEC MGIT960法具有很高的一致性;以pnc A基因直接测序法为标准,Versa TREK Myco PZA kit法具有较好的敏感性。第四部分探讨Wayne法检测吡嗪酰胺耐药性的可靠性目的:评估Wayne法检测吡嗪酰胺耐药性的可靠性及其临床应用价值。方法:选取91株结核分枝杆菌临床分离株,应用结核分枝杆菌标准株H37Rv、BCG标准株做为对照菌株。分别使用BACTEC MGIT960法和Wayne法检测纳入菌株的PZA耐药性,并对所有菌株的pnc A基因及其启动子区序列进行测序。结果:以BACTEC MGIT960法做为PZA耐药性检测的对照方法,Wayne法检测PZA耐药性的灵敏度、特异度、一致性分别为79.3%(23/29)、83.9%(52/62)、82.4%(75/91);以pnc A基因及其启动子区序列测定作为对照方法,Wayne法检测PZA耐药性的灵敏度、特异度、一致性结果分别为72.5%(29/40)、92.2%(47/51)、83.50%(76/91)。结论:Wayne法检测PZA耐药性的准确性较低,且要求的菌量比较大,结果需肉眼判断,操作相对繁琐,不宜在临床推广。
[Abstract]:Pyrazinamide (PZA) has unique bactericidal activity for Mycobacterium tuberculosis in the semi dormant state in the intracellular acidic environment. Therefore, it has become the cornerstone of the short range chemotherapy for tuberculosis. Because of its low price and good curative effect, PZA is widely used in the strengthening period of tuberculosis treatment and is also an important treatment for multidrug resistant tuberculosis. Drugs. Recent studies have shown that PZA resistance may occur both in patients with multidrug resistant tuberculosis or in non multidrug resistant TB patients, so routine PZA sensitivity tests are essential for the rational use of PZA. However, because PZA can only play a role in the acidic environment, the drug sensitivity test under the acidic environment The drug sensitivity test of PZA is not easy to carry out, and the reliability has always been an important problem. This study established and evaluated a variety of PZA sensitivity test methods. The purpose is to seek simple, reliable and operable PZA drug sensitivity test methods for clinical application. The first part uses nicotinamide microplate method to detect tuberculosis branch. The establishment and evaluation of the method of antimicrobial resistance of mycotoxin: to establish the microporous plate susceptibility testing method of nicotinamide instead of the methinamide and evaluate its reliability. Methods: 125 Mycobacterium tuberculosis clinical isolates were selected, the standard strain H37Rv of Mycobacterium tuberculosis was selected, and the BCG standard strain was used as the control strain. A double dilution method was used to set up 9. The nicotinamide concentration (range from 8 to 2000 g/ml) was used to detect the minimum inhibitory concentration of nicotinamide, and the sensitivity of the strain to PZA was detected by the BACTEC MGIT960 method, and the gene sequence of PNC A gene and the promoter region of all strains was measured. Results: 64 of the 125 strains of clinical isolates were diagnosed as PZA by BACTEC MGIT960 method. Resistance. The BACTEC MGIT960 method was used as the reference method for the detection of PZA resistance. The critical value of PZA resistance was detected by 500 mu g/ml as a nicotinamide culture plate method. The sensitivity of the nicotinamide microplate method was 100% (61/61), the specificity was 95.3% (61/64), and the accuracy was 97.6% (122/ 125). The sequence of 57 strains of 125 clinical isolates was found to exist PNC A. The mutation of the gene and its promoter region, 68 strains of the wild strain. The sensitivity of the PNC A gene and its promoter region gene was analyzed as the reference method. The sensitivity of the nicotinamide microplate method to detect the resistance of PZA was 89.5% (51/57), the specificity was 80.9% (55/68), and the accuracy was 84.8% (106/125). Conclusion: the nicotinamide microplate method was used to detect the Mycobacterium tuberculosis of the Mycobacterium tuberculosis. The drug resistance method has the advantages of simple operation, quick time, low price, reliable results and good reproducibility, and has good clinical value. The second part: the establishment and evaluation of the method of rapid detection of the drug resistance of the drug by Surveyor enzyme digestion method: the establishment of PNC A for rapid detection of MTB by using Surveyor enzyme digestion method The method of gene mutation of the gene and its promoter sequence. Methods: 91 strains of MDR strain were selected as the research object. The above primers were amplified by the downstream primers for the proportion of 2:5, and the above primers were amplified by the downstream primers for 5:2, and then the PCR product was proportional to the proportion of 1:1. Hybrid hybridization, using Surveyor enzyme digestion after the reaction product electrophoresis. The hybrid strip can be cut by Surveyor enzyme as the basis for determining the existence of gene mutation in the PNC A gene and its promoter sequence. The BACTEC MGIT960 detection of PZA resistance method and the PNC A gene and the promoter region gene sequencing method are used as control to evaluate the Surveyor enzyme enzyme. Results: the BACTEC MGIT960 method was used to detect the resistance of PZA as a control method. The sensitivity of Surveyor enzyme digestion was 100% (29/29), the specificity was 87.1% (54/62), and the accuracy was 91.2% (83/91). The sensitivity and specificity of the PNC A gene and the sequencing result of the promoter region were taken as the control, the sensitivity and specificity of the Surveyor enzyme digestion method. And accuracy is 100%. conclusion: Surveyor enzyme digestion method for detection of PNC A gene and its promoter region mutation performance and gene sequencing highly consistent.Surveyor enzyme cutting method is rapid, easy to operate, low price characteristics, can be used as an alternative to detect the PNC A gene mutation. The third part evaluates Versa TREK Myco PZA Kit Method Detection Objective: To evaluate the reliability and clinical value of Versa TREK Myco PZA kit for the detection of drug-resistant Mycobacterium tuberculosis and its clinical application. Methods: 192 strains of Mycobacterium tuberculosis were selected as clinical isolates. BACTEC MGIT960 method and Versa TREK Myco PZA kit method were used to detect the drug resistance of the strains. PNC A gene and its promoter region sequence were sequenced. Results: PZA resistance was detected by BACTEC MGIT960 as a control method, and the sensitivity of Versa TREK Myco PZA kit method was 93.5% (58/62), specificity was 91.5% (119/130), and the accuracy was 92.2%. The sensitivity of the Myco PZA kit method is 88.4% (61/68), the specificity is 82.9% (102/123) and the accuracy is 84.9% (163/192). Conclusion: the Versa TREK Myco PZA kit method has a high consistency with the method of detecting the resistance of PZA with the method of direct sequencing. Fourth parts have good sensitivity. Objective: To evaluate the reliability of the Wayne method for the detection of the drug resistance of the drug: the reliability and clinical value of the Wayne method for the detection of the drug resistance of the drug. Methods: 91 clinical isolates of Mycobacterium tuberculosis were selected, the standard strain H37Rv of Mycobacterium tuberculosis was used and the BCG standard strain was used as the control strain. The BACTEC MGIT960 method and the Wayn were used respectively. The e method was used to detect the PZA resistance of the strains and the sequence of PNC A gene and its promoter region of all strains. Results: the sensitivity, specificity and consistency of Wayne method were 79.3% (23/29), 83.9% (52/62) and 82.4% (75/91), respectively, by BACTEC MGIT960 as the control method of PZA resistance detection. The sensitivity, specificity and consistency of Wayne method were 72.5% (29/40), 92.2% (47/51) and 83.50% (76/91) respectively. Conclusion: the accuracy of Wayne method for detecting PZA resistance is low, and the amount of bacteria required is relatively large. The results need to be judged by naked eyes, and it is not suitable for clinical push. Wide.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52;R446.5
[Abstract]:Pyrazinamide (PZA) has unique bactericidal activity for Mycobacterium tuberculosis in the semi dormant state in the intracellular acidic environment. Therefore, it has become the cornerstone of the short range chemotherapy for tuberculosis. Because of its low price and good curative effect, PZA is widely used in the strengthening period of tuberculosis treatment and is also an important treatment for multidrug resistant tuberculosis. Drugs. Recent studies have shown that PZA resistance may occur both in patients with multidrug resistant tuberculosis or in non multidrug resistant TB patients, so routine PZA sensitivity tests are essential for the rational use of PZA. However, because PZA can only play a role in the acidic environment, the drug sensitivity test under the acidic environment The drug sensitivity test of PZA is not easy to carry out, and the reliability has always been an important problem. This study established and evaluated a variety of PZA sensitivity test methods. The purpose is to seek simple, reliable and operable PZA drug sensitivity test methods for clinical application. The first part uses nicotinamide microplate method to detect tuberculosis branch. The establishment and evaluation of the method of antimicrobial resistance of mycotoxin: to establish the microporous plate susceptibility testing method of nicotinamide instead of the methinamide and evaluate its reliability. Methods: 125 Mycobacterium tuberculosis clinical isolates were selected, the standard strain H37Rv of Mycobacterium tuberculosis was selected, and the BCG standard strain was used as the control strain. A double dilution method was used to set up 9. The nicotinamide concentration (range from 8 to 2000 g/ml) was used to detect the minimum inhibitory concentration of nicotinamide, and the sensitivity of the strain to PZA was detected by the BACTEC MGIT960 method, and the gene sequence of PNC A gene and the promoter region of all strains was measured. Results: 64 of the 125 strains of clinical isolates were diagnosed as PZA by BACTEC MGIT960 method. Resistance. The BACTEC MGIT960 method was used as the reference method for the detection of PZA resistance. The critical value of PZA resistance was detected by 500 mu g/ml as a nicotinamide culture plate method. The sensitivity of the nicotinamide microplate method was 100% (61/61), the specificity was 95.3% (61/64), and the accuracy was 97.6% (122/ 125). The sequence of 57 strains of 125 clinical isolates was found to exist PNC A. The mutation of the gene and its promoter region, 68 strains of the wild strain. The sensitivity of the PNC A gene and its promoter region gene was analyzed as the reference method. The sensitivity of the nicotinamide microplate method to detect the resistance of PZA was 89.5% (51/57), the specificity was 80.9% (55/68), and the accuracy was 84.8% (106/125). Conclusion: the nicotinamide microplate method was used to detect the Mycobacterium tuberculosis of the Mycobacterium tuberculosis. The drug resistance method has the advantages of simple operation, quick time, low price, reliable results and good reproducibility, and has good clinical value. The second part: the establishment and evaluation of the method of rapid detection of the drug resistance of the drug by Surveyor enzyme digestion method: the establishment of PNC A for rapid detection of MTB by using Surveyor enzyme digestion method The method of gene mutation of the gene and its promoter sequence. Methods: 91 strains of MDR strain were selected as the research object. The above primers were amplified by the downstream primers for the proportion of 2:5, and the above primers were amplified by the downstream primers for 5:2, and then the PCR product was proportional to the proportion of 1:1. Hybrid hybridization, using Surveyor enzyme digestion after the reaction product electrophoresis. The hybrid strip can be cut by Surveyor enzyme as the basis for determining the existence of gene mutation in the PNC A gene and its promoter sequence. The BACTEC MGIT960 detection of PZA resistance method and the PNC A gene and the promoter region gene sequencing method are used as control to evaluate the Surveyor enzyme enzyme. Results: the BACTEC MGIT960 method was used to detect the resistance of PZA as a control method. The sensitivity of Surveyor enzyme digestion was 100% (29/29), the specificity was 87.1% (54/62), and the accuracy was 91.2% (83/91). The sensitivity and specificity of the PNC A gene and the sequencing result of the promoter region were taken as the control, the sensitivity and specificity of the Surveyor enzyme digestion method. And accuracy is 100%. conclusion: Surveyor enzyme digestion method for detection of PNC A gene and its promoter region mutation performance and gene sequencing highly consistent.Surveyor enzyme cutting method is rapid, easy to operate, low price characteristics, can be used as an alternative to detect the PNC A gene mutation. The third part evaluates Versa TREK Myco PZA Kit Method Detection Objective: To evaluate the reliability and clinical value of Versa TREK Myco PZA kit for the detection of drug-resistant Mycobacterium tuberculosis and its clinical application. Methods: 192 strains of Mycobacterium tuberculosis were selected as clinical isolates. BACTEC MGIT960 method and Versa TREK Myco PZA kit method were used to detect the drug resistance of the strains. PNC A gene and its promoter region sequence were sequenced. Results: PZA resistance was detected by BACTEC MGIT960 as a control method, and the sensitivity of Versa TREK Myco PZA kit method was 93.5% (58/62), specificity was 91.5% (119/130), and the accuracy was 92.2%. The sensitivity of the Myco PZA kit method is 88.4% (61/68), the specificity is 82.9% (102/123) and the accuracy is 84.9% (163/192). Conclusion: the Versa TREK Myco PZA kit method has a high consistency with the method of detecting the resistance of PZA with the method of direct sequencing. Fourth parts have good sensitivity. Objective: To evaluate the reliability of the Wayne method for the detection of the drug resistance of the drug: the reliability and clinical value of the Wayne method for the detection of the drug resistance of the drug. Methods: 91 clinical isolates of Mycobacterium tuberculosis were selected, the standard strain H37Rv of Mycobacterium tuberculosis was used and the BCG standard strain was used as the control strain. The BACTEC MGIT960 method and the Wayn were used respectively. The e method was used to detect the PZA resistance of the strains and the sequence of PNC A gene and its promoter region of all strains. Results: the sensitivity, specificity and consistency of Wayne method were 79.3% (23/29), 83.9% (52/62) and 82.4% (75/91), respectively, by BACTEC MGIT960 as the control method of PZA resistance detection. The sensitivity, specificity and consistency of Wayne method were 72.5% (29/40), 92.2% (47/51) and 83.50% (76/91) respectively. Conclusion: the accuracy of Wayne method for detecting PZA resistance is low, and the amount of bacteria required is relatively large. The results need to be judged by naked eyes, and it is not suitable for clinical push. Wide.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52;R446.5
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