結(jié)核分枝桿菌吡嗪酰胺耐藥診斷方法的建立及可靠性評(píng)估
發(fā)布時(shí)間：2018-07-25 13:52
【摘要】：吡嗪酰胺(Pyrazinamide,PZA)對(duì)細(xì)胞內(nèi)酸性環(huán)境下處于半休眠狀態(tài)的結(jié)核分枝桿菌具有獨(dú)特的殺菌活性,因此成為結(jié)核病短程化療的基石。由于價(jià)格便宜而療效好,PZA被廣泛用于結(jié)核病治療的強(qiáng)化期,并且也是耐多藥結(jié)核病治療的一種重要藥物。近年來的研究表明,PZA耐藥無論在耐多藥結(jié)核病患者中還是在非耐多藥結(jié)核病患者中都可能發(fā)生,因此常規(guī)開展PZA敏感性試驗(yàn)對(duì)于合理使用PZA至關(guān)重要。然而,由于PZA僅能在酸性環(huán)境下才能發(fā)揮作用,而酸性環(huán)境下的藥敏試驗(yàn)會(huì)影響細(xì)菌生長,因此PZA的藥敏試驗(yàn)不便于開展,并且可靠性也一直是個(gè)重要的問題。本研究建立并評(píng)估了多種PZA的敏感性試驗(yàn)方法,目的是為臨床尋求簡便、可靠、可操作的PZA藥敏試驗(yàn)方法。第一部分應(yīng)用煙酰胺微孔板法檢測結(jié)核分枝桿菌吡嗪酰胺耐藥性的方法的建立及評(píng)估目的:建立煙酰胺替代吡嗪酰胺的微孔板藥敏檢測方法并評(píng)估其可靠性。方法:選取125株結(jié)核分枝桿菌臨床分離株,另選取結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv、BCG標(biāo)準(zhǔn)株做為對(duì)照菌株。以倍比稀釋法設(shè)定9個(gè)煙酰胺藥物濃度(范圍從8到2000μg/ml),檢測菌株對(duì)煙酰胺的最低抑菌濃度,同時(shí)使用BACTEC MGIT960法檢測菌株對(duì)PZA敏感性,并對(duì)所有菌株進(jìn)行了pnc A基因及其啟動(dòng)子區(qū)的基因序列測定。結(jié)果:125株臨床分離株中有64株被BACTEC MGIT960法診斷為PZA耐藥。以BACTEC MGIT960法作為PZA耐藥性檢測的參考方法,以500μg/ml做為煙酰胺培養(yǎng)板法檢測PZA耐藥性的臨界值,則煙酰胺微孔板法的靈敏度為100%(61/61),特異度為95.3%(61/64),準(zhǔn)確度為97.6%(122/125)。對(duì)125臨床分離株進(jìn)行測序發(fā)現(xiàn)57株菌存在pnc A基因及其啟動(dòng)子區(qū)的突變,68株菌為野生株。以pnc A基因及其啟動(dòng)子區(qū)基因測序?yàn)閰⒖挤椒?煙酰胺微孔板法檢測PZA耐藥性的靈敏度為89.5%(51/57)、特異度為80.9%(55/68)、準(zhǔn)確度為84.8%(106/125)。結(jié)論:煙酰胺微孔板法檢測結(jié)核分枝桿菌吡嗪酰胺耐藥性方法具有操作簡易、時(shí)間快速、價(jià)格低廉、結(jié)果可靠和重復(fù)性較好的優(yōu)點(diǎn),具有較好的臨床推廣價(jià)值。第二部分:應(yīng)用Surveyor酶酶切法快速檢測吡嗪酰胺的耐藥性的方法的建立及評(píng)估目的:建立應(yīng)用Surveyor酶酶切法快速檢測MTB的pnc A基因及其啟動(dòng)子序列的基因突變的方法。方法:選取耐多藥(MDR)菌株91株作為研究對(duì)象。分別以上游引物:下游引物為2:5的比例擴(kuò)增所有菌株,同時(shí)以上游引物:下游引物為5:2的比例擴(kuò)增結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv,然后將PCR產(chǎn)物以1:1的比例混合雜交,應(yīng)用Surveyor酶酶切反應(yīng)產(chǎn)物后電泳。以雜交條帶能夠被Surveyor酶切割做為判定pnc A基因及其啟動(dòng)子序列存在基因突變的依據(jù)。以BACTEC MGIT960檢測PZA耐藥性方法和pnc A基因及其啟動(dòng)子區(qū)基因序列測定法作為對(duì)照,評(píng)價(jià)Surveyor酶酶切法的應(yīng)用價(jià)值。結(jié)果:以BACTEC MGIT960法檢測PZA耐藥性作為對(duì)照方法,Surveyor酶酶切法診斷PZA耐藥的靈敏度為100%(29/29),特異度為87.1%(54/62),準(zhǔn)確度為91.2%(83/91)。以pnc A基因及其啟動(dòng)子區(qū)測序結(jié)果作為對(duì)照,Surveyor酶酶切法的靈敏度、特異度和準(zhǔn)確度均為100%。結(jié)論:Surveyor酶酶切法檢測pnc A基因及其啟動(dòng)子區(qū)突變的性能與基因測序高度一致。Surveyor酶切法具有快速、操作簡便、價(jià)格低廉的特點(diǎn),可以做為一個(gè)替代方法檢測pnc A基因突變。第三部分評(píng)估Versa TREK Myco PZA kit法檢測吡嗪酰胺耐藥性的可靠性目的:評(píng)估Versa TREK Myco PZA kit法檢測結(jié)核分枝桿菌吡嗪酰胺耐藥性的可靠性及其臨床應(yīng)用價(jià)值。方法:選取192株結(jié)核分枝桿菌臨床分離株。分別使用BACTEC MGIT960法和Versa TREK Myco PZA kit法檢測待測菌株的PZA耐藥性,并對(duì)所有納入菌株的pnc A基因及其啟動(dòng)子區(qū)序列進(jìn)行序列測定。結(jié)果:以BACTEC MGIT960法檢測PZA耐藥性作為對(duì)照方法,Versa TREK Myco PZA kit法的靈敏度為93.5%(58/62),特異度為91.5%(119/130),準(zhǔn)確度為92.2%(177/192)。以pnc A基因測序法作為對(duì)照,Versa TREK Myco PZA kit法的靈敏度為88.4%(61/68)、特異度為82.9%(102/123)和準(zhǔn)確度均為84.9%(163/192)。結(jié)論:Versa TREK Myco PZA kit法檢測PZA耐藥性與BACTEC MGIT960法具有很高的一致性;以pnc A基因直接測序法為標(biāo)準(zhǔn),Versa TREK Myco PZA kit法具有較好的敏感性。第四部分探討Wayne法檢測吡嗪酰胺耐藥性的可靠性目的:評(píng)估Wayne法檢測吡嗪酰胺耐藥性的可靠性及其臨床應(yīng)用價(jià)值。方法:選取91株結(jié)核分枝桿菌臨床分離株,應(yīng)用結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv、BCG標(biāo)準(zhǔn)株做為對(duì)照菌株。分別使用BACTEC MGIT960法和Wayne法檢測納入菌株的PZA耐藥性,并對(duì)所有菌株的pnc A基因及其啟動(dòng)子區(qū)序列進(jìn)行測序。結(jié)果:以BACTEC MGIT960法做為PZA耐藥性檢測的對(duì)照方法,Wayne法檢測PZA耐藥性的靈敏度、特異度、一致性分別為79.3%(23/29)、83.9%(52/62)、82.4%(75/91);以pnc A基因及其啟動(dòng)子區(qū)序列測定作為對(duì)照方法,Wayne法檢測PZA耐藥性的靈敏度、特異度、一致性結(jié)果分別為72.5%(29/40)、92.2%(47/51)、83.50%(76/91)。結(jié)論:Wayne法檢測PZA耐藥性的準(zhǔn)確性較低,且要求的菌量比較大,結(jié)果需肉眼判斷,操作相對(duì)繁瑣,不宜在臨床推廣。
[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.
【學(xué)位授予單位】：北京市結(jié)核病胸部腫瘤研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：北京市結(jié)核病胸部腫瘤研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R52;R446.5
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