吡嗪酰胺酶活性对肺结核临床疗效的影响探讨

发布时间：2018-09-11 06:43

【摘要】：背景吡嗪酰胺(pyrazinamide,PZA)是非常重要的抗结核一线药物,它能够杀灭处于酸性环境中或巨噬细胞内半休眠状态的结核分枝杆菌(简称结核菌),有着其他药物无法替代的抗结核作用。它的使用能够有效地缩短结核病的疗程,并降低远期复发率。PZA的作用机制比较特别,它在体内能表现出很强的杀菌作用,而体外仅在酸性培养基中才具备杀菌活性。同时,其本身并无杀菌作用,它需在结核菌细胞内吡嗪酰胺酶(pyrazinamidase,PZase)的催化下才能转化为真正具有杀菌活性的吡嗪酸(pyrazinoic acid,POA),PZase的高效活性是其发挥抗菌作用的必要条件,其酶活性的降低或消失将使得PZA无法转化为POA而发挥抗菌作用。随着PZA的广泛应用,其耐药情况亦日渐严重。目前的研究发现,pnc A基因突变造成其所编码的PZase活性失活是PZA耐药的主要原因。目的本研究旨在探讨pnc A基因突变及PZase活性对肺结核临床疗效的影响。第一部分吡嗪酰胺耐药对初治肺结核疗效影响的队列研究一、对象与方法1.对象:从广州地区分枝杆菌菌株库中保存有菌株且已行PZA药敏试验的患者中选择2012年7月至2016年1月在我院完成诊治的75例PZA耐药并采用PZA治疗的初治肺结核患者作为PZA耐药组。采用1:1配对原则,选择同期PZA敏感并采用PZA治疗、年龄相差在10岁以内的初治肺结核患者75例作为PZA敏感组。2.方法:采用回顾性队列研究的方法,收集两组患者的一般资料及治疗前后的痰菌和影像学变化情况。比较PZA耐药组和PZA敏感组治疗2月末及6月末的疗效差异,包括痰菌阴转、病灶吸收和空洞缩小情况。二、结果1.治疗2月末,PZA耐药组和敏感组的痰菌阴转率分别为76.00%、85.33%(c2=2.095,P=0.148);病灶吸收率分别为81.33%、88.00%(c2=1.284,P=0.257);空洞缩小率分别为43.14%、60.38%(c2=3.085,P=0.079)。2.治疗6月末,PZA耐药组和敏感组的痰菌阴转率分别为85.33%、93.33%(c2=2.519,P=0.113);病灶吸收率分别为89.33%、94.67%(c2=1.714,P=0.190);空洞缩小率分别为54.90%、71.70%(c2=3.162,P=0.067)。第二部分pnc A基因突变对肺结核临床疗效影响的研究一、对象与方法1.对象:第一部分入选的PZA耐药者和PZA敏感者冻存在广州地区分枝杆菌菌株库中的结核分枝杆菌菌株,共计150株,其中PZA耐药株和敏感株各75株。2.方法:对上述冻存的150株结核菌进行复苏培养,采用PCR-DNA测序技术对复苏成功的菌株进行pnc A基因扩增及序列分析。同时,以有pnc A基因突变的耐药患者为突变组,选择已匹配的无pnc A基因突变的敏感患者为未突变组,比较两组患者治疗2月末及6月末的疗效差异。二、结果1.冻存的150株结核菌共成功复苏141株,其中PZA耐药菌株成功复苏67株,PZA敏感菌株成功复苏74株。2.67株PZA耐药菌株中有16株发生pnc A基因突变,74株PZA敏感菌株中有1株发生pnc A基因突变,耐药菌株和敏感菌株的pnc A基因突变率分别为23.88%、1.35%(c2=16.832,P=0.000),提示PZA耐药与pnc A基因突变有关(r=0.327)。3.突变组和未突变组患者各16例,治疗2月末,突变组和未突变组的痰菌阴转率分别为68.75%、87.50%(P=0.394);病灶吸收率分别为75.00%、93.75%(P=0.333);空洞缩小率分别为41.67%、69.23%(P=0.238);两组治疗有效率相似。4.突变组和未突变组患者各16例,治疗6月末,突变组和未突变组的痰菌阴转率分别为81.25%、100.0%(P=0.226);病灶吸收率分别为87.50%、100.0%(P=0.484);空洞缩小率分别为50.00%、84.62%(P=0.097);两组治疗有效率相似。第三部分吡嗪酰胺酶活性对肺结核临床疗效的影响探讨一、对象与方法1.对象:第二部分成功复苏的141株结核分枝杆菌,其中PZA耐药者67株,PZA敏感者74株。2.方法:采用Wayne法对上述菌株进行PZase活性的测定,分析PZase活性与PZA表型药敏及pnc A基因之间的关系。同时,以PZase阴性的耐药患者为阴性组,选择已匹配的PZase阳性的敏感患者为阳性组,回顾性分析两组患者治疗2月末及6月末的疗效差异。二、结果1.67株PZA耐药菌株中,28株PZase活性为阴性,39株PZase活性为阳性。74株PZA敏感菌株中,4株PZase活性为阴性,70株PZase活性为阳性。耐药株和敏感株的PZase活性的阴性率分别为41.79%、5.41%(c2=26.534,P=0.000),提示PZA耐药与PZase活性丧失有关(r=0.398)。2.PZase阴性的32株菌株中,有17株发生pnc A基因突变,PZase阳性的109株菌株中,pnc A基因均无突变。PZase阴性菌株和阳性菌株pnc A基因突变率分别为53.13%、0.00%(c2=60.930,P=0.000),提示pnc A基因突变与PZase活性丧失有关(r=0.564)。3.PZase阴性组和PZase阳性组患者各27例,治疗2月末,阴性组和阳性组的痰菌阴转率分别为77.78%、85.19%(c2=0.491,P=0.484);病灶吸收率分别为77.78%、88.89%(c2=0.533,P=0.465);空洞缩小率分别为42.86%、68.18%(c2=2.794,P=0.095);两组治疗有效率相似。4.PZase阴性组和PZase阳性组患者各27例,治疗6月末,阴性组和阳性组的痰菌阴转率分别为85.19%、100.00%(c2=2.430,P=0.119);病灶吸收率分别为88.89%、100.00%(c2=1.412,P=0.235);空洞缩小率分别为57.14%、81.82%(c2=3.101,P=0.078);两组治疗有效率相似。结论1.PZA表型耐药的初治肺结核患者使用含PZA方案治疗,其疗效与对PZA敏感患者相似。2.PZA表型耐药与pnc A基因突变有关。3.pnc A基因突变的初治肺结核患者使用含PZA方案治疗,其疗效与pnc A未突变PZA表型敏感患者相似。4.PZA表型耐药与PZase活性丧失有关。5.pnc A基因突变与PZase活性丧失有关。6.PZase阴性的初治肺结核患者使用含PZA方案治疗,其疗效与PZase阳性PZA表型敏感的患者相似。
[Abstract]:Background Prazinamide (PZA) is a very important first-line anti-tuberculosis drug. It can kill Mycobacterium tuberculosis (M. tuberculosis) in acidic environment or semi-dormant macrophages. It has an irreplaceable anti-tuberculosis effect. Its use can effectively shorten the course of treatment of tuberculosis and reduce the long-term effect. Phase recurrence rate. PZA has a special mechanism of action, it can show strong bactericidal effect in vivo, but it has bactericidal activity only in acidic medium in vitro. At the same time, it has no bactericidal effect itself. It needs to be catalyzed by pyrazinamidase (PZase) in Mycobacterium tuberculosis cells to be converted into truly bactericidal activity. The high activity of pyrazinoic acid (POA) and PZase is a necessary condition for them to exert their antimicrobial effect. The decrease or disappearance of their enzyme activity will make PZA unable to convert into POA and play an antimicrobial role. Sexual inactivation is the main cause of PZA resistance. Objective To investigate the effect of PNC A gene mutation and PZase activity on the clinical efficacy of pulmonary tuberculosis. Seventy-five patients with PZA-resistant pulmonary tuberculosis who had been treated in our hospital from July 2012 to January 2016 were selected as the PZA-resistant group. A retrospective cohort study was conducted to collect the general data and the changes of sputum bacteria and imaging in the two groups before and after treatment. The negative conversion rates of sputum bacteria were 76.00% and 85.33% (c2 = 2.095, P = 0.148), 81.33% and 88.00% (c2 = 1.284, P = 0.257), 43.14% and 60.38% (c2 = 3.085, P = 0.079) respectively, and 93.33% (c2 = 2.519, P = 0.113), respectively. At the end of June, the negative conversion rates of sputum bacteria were 85.33% and 93.33% (c2 = 2.519, P = 0.113) in the PZA resistant and sensitive groups. 4.67% (c2 = 1.714, P = 0.190) and 54.90% and 71.70% (c2 = 3.162, P = 0.067) respectively. Methods: 150 strains of tuberculosis were resuscitated and cultured. PCR-DNA sequencing was used to amplify and sequence the PNC A gene of the resuscitated strains. At the same time, the matched non-pnc A gene mutation was selected from the drug-resistant patients with PNC A gene mutation as the mutation group. Results 1. A total of 141 strains of cryopreserved 150 strains of tuberculosis were successfully resuscitated, 67 strains of which were resistant to PZA, 16 strains of which were resistant to PZA, and 74 strains of which were sensitive to PZA. PNC A gene mutation occurred in one strain. The mutation rates of PNC A gene in resistant strains and susceptible strains were 23.88% and 1.35% (c2 = 16.832, P = 0.000), suggesting that pZA resistance was associated with PNC A gene mutation (r = 0.327). 3. The sputum negative conversion rates in mutant group and non-mutant group were 68.75% and 87.50% respectively at the end of February (P = 0.39). 4) The absorption rate was 75.00% and 93.75% (P = 0.333), the cavity reduction rate was 41.67% and 69.23% (P = 0.238), and the effective rate was similar between the two groups. In the third part, the effect of pyrazinamidase activity on the clinical efficacy of tuberculosis was discussed. Objectives and methods 1. Objectives: In the second part, 141 strains of Mycobacterium tuberculosis were successfully resuscitated, of which 67 strains were resistant to PZA and 74 strains were sensitive to PZA. Methods: Wayne method was used. The PZase activity of the above strains was determined to analyze the relationship between the PZase activity and the PZA phenotypic susceptibility and PNC A gene. At the same time, the matched PZase-positive susceptible patients were selected as the positive group and the negative PZase-negative drug-resistant patients as the negative group. The therapeutic effects of the two groups at the end of February and the end of June were analyzed retrospectively. 2. Results 1.67 strains of PZA were resistant. Among the drug-resistant strains, 28 were negative for PZase activity, 39 were positive for PZase activity, 4 were negative for PZase activity and 70 were positive for PZase activity. The negative rates of PZase activity of drug-resistant strains and sensitive strains were 41.79% and 5.41% respectively (c2 = 26.534, P = 0.000), suggesting that PZA resistance was related to the loss of PZase activity (r = 0.398).2.PZase negative 32. The mutation rates of PNC A gene in PZase negative and positive strains were 53.13% and 0.00% (c2 = 60.930, P = 0.000), respectively, suggesting that the mutation of PNC A gene was related to the loss of PZase activity (r = 0.564). 3. PZase negative and PZase positive groups were 27 cases, respectively. At the end of 2 months, the sputum negative conversion rates of negative group and positive group were 77.78% and 85.19% (c2 = 0.491, P = 0.484), 77.78% and 88.89% respectively (c2 = 0.533, P = 0.465), 42.86% and 68.18% respectively (c2 = 2.794, P = 0.095), and the effective rates of the two groups were similar.4.PZase negative group and 27 PZase positive group, respectively. The negative conversion rates of sputum bacteria in sex group and positive group were 85.19% and 100.00% (c2 = 2.430, P = 0.119), 88.89% and 100.00% respectively (c2 = 1.412, P = 0.235), 57.14% and 81.82% respectively (c2 = 3.101, P = 0.078), and the effective rates were similar between the two groups. PZA phenotypic resistance is associated with mutation of PNC A gene. 3. Primary pulmonary tuberculosis patients with mutation of PNC A gene are treated with PZA containing regimen. The efficacy is similar to that of PNC A non-mutation sensitive patients with PZA phenotypic sensitivity. 4. PZA phenotypic resistance is associated with loss of PZase activity. 5. PNC A gene mutation is associated with loss of PZase activity. 6. PZase negative. The efficacy of PZA-containing regimen was similar to that of PZA-sensitive patients with PZase-positive pulmonary tuberculosis.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R521

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