【摘要】：背景吡嗪酰胺(pyrazinamide,PZA)是非常重要的抗結核一線藥物,它能夠殺滅處于酸性環(huán)境中或巨噬細胞內半休眠狀態(tài)的結核分枝桿菌(簡稱結核菌),有著其他藥物無法替代的抗結核作用。它的使用能夠有效地縮短結核病的療程,并降低遠期復發(fā)率。PZA的作用機制比較特別,它在體內能表現(xiàn)出很強的殺菌作用,而體外僅在酸性培養(yǎng)基中才具備殺菌活性。同時,其本身并無殺菌作用,它需在結核菌細胞內吡嗪酰胺酶(pyrazinamidase,PZase)的催化下才能轉化為真正具有殺菌活性的吡嗪酸(pyrazinoic acid,POA),PZase的高效活性是其發(fā)揮抗菌作用的必要條件,其酶活性的降低或消失將使得PZA無法轉化為POA而發(fā)揮抗菌作用。隨著PZA的廣泛應用,其耐藥情況亦日漸嚴重。目前的研究發(fā)現(xiàn),pnc A基因突變造成其所編碼的PZase活性失活是PZA耐藥的主要原因。目的本研究旨在探討pnc A基因突變及PZase活性對肺結核臨床療效的影響。第一部分吡嗪酰胺耐藥對初治肺結核療效影響的隊列研究一、對象與方法1.對象:從廣州地區(qū)分枝桿菌菌株庫中保存有菌株且已行PZA藥敏試驗的患者中選擇2012年7月至2016年1月在我院完成診治的75例PZA耐藥并采用PZA治療的初治肺結核患者作為PZA耐藥組。采用1:1配對原則,選擇同期PZA敏感并采用PZA治療、年齡相差在10歲以內的初治肺結核患者75例作為PZA敏感組。2.方法:采用回顧性隊列研究的方法,收集兩組患者的一般資料及治療前后的痰菌和影像學變化情況。比較PZA耐藥組和PZA敏感組治療2月末及6月末的療效差異,包括痰菌陰轉、病灶吸收和空洞縮小情況。二、結果1.治療2月末,PZA耐藥組和敏感組的痰菌陰轉率分別為76.00%、85.33%(c2=2.095,P=0.148);病灶吸收率分別為81.33%、88.00%(c2=1.284,P=0.257);空洞縮小率分別為43.14%、60.38%(c2=3.085,P=0.079)。2.治療6月末,PZA耐藥組和敏感組的痰菌陰轉率分別為85.33%、93.33%(c2=2.519,P=0.113);病灶吸收率分別為89.33%、94.67%(c2=1.714,P=0.190);空洞縮小率分別為54.90%、71.70%(c2=3.162,P=0.067)。第二部分pnc A基因突變對肺結核臨床療效影響的研究一、對象與方法1.對象:第一部分入選的PZA耐藥者和PZA敏感者凍存在廣州地區(qū)分枝桿菌菌株庫中的結核分枝桿菌菌株,共計150株,其中PZA耐藥株和敏感株各75株。2.方法:對上述凍存的150株結核菌進行復蘇培養(yǎng),采用PCR-DNA測序技術對復蘇成功的菌株進行pnc A基因擴增及序列分析。同時,以有pnc A基因突變的耐藥患者為突變組,選擇已匹配的無pnc A基因突變的敏感患者為未突變組,比較兩組患者治療2月末及6月末的療效差異。二、結果1.凍存的150株結核菌共成功復蘇141株,其中PZA耐藥菌株成功復蘇67株,PZA敏感菌株成功復蘇74株。2.67株PZA耐藥菌株中有16株發(fā)生pnc A基因突變,74株PZA敏感菌株中有1株發(fā)生pnc A基因突變,耐藥菌株和敏感菌株的pnc A基因突變率分別為23.88%、1.35%(c2=16.832,P=0.000),提示PZA耐藥與pnc A基因突變有關(r=0.327)。3.突變組和未突變組患者各16例,治療2月末,突變組和未突變組的痰菌陰轉率分別為68.75%、87.50%(P=0.394);病灶吸收率分別為75.00%、93.75%(P=0.333);空洞縮小率分別為41.67%、69.23%(P=0.238);兩組治療有效率相似。4.突變組和未突變組患者各16例,治療6月末,突變組和未突變組的痰菌陰轉率分別為81.25%、100.0%(P=0.226);病灶吸收率分別為87.50%、100.0%(P=0.484);空洞縮小率分別為50.00%、84.62%(P=0.097);兩組治療有效率相似。第三部分吡嗪酰胺酶活性對肺結核臨床療效的影響探討一、對象與方法1.對象:第二部分成功復蘇的141株結核分枝桿菌,其中PZA耐藥者67株,PZA敏感者74株。2.方法:采用Wayne法對上述菌株進行PZase活性的測定,分析PZase活性與PZA表型藥敏及pnc A基因之間的關系。同時,以PZase陰性的耐藥患者為陰性組,選擇已匹配的PZase陽性的敏感患者為陽性組,回顧性分析兩組患者治療2月末及6月末的療效差異。二、結果1.67株PZA耐藥菌株中,28株PZase活性為陰性,39株PZase活性為陽性。74株PZA敏感菌株中,4株PZase活性為陰性,70株PZase活性為陽性。耐藥株和敏感株的PZase活性的陰性率分別為41.79%、5.41%(c2=26.534,P=0.000),提示PZA耐藥與PZase活性喪失有關(r=0.398)。2.PZase陰性的32株菌株中,有17株發(fā)生pnc A基因突變,PZase陽性的109株菌株中,pnc A基因均無突變。PZase陰性菌株和陽性菌株pnc A基因突變率分別為53.13%、0.00%(c2=60.930,P=0.000),提示pnc A基因突變與PZase活性喪失有關(r=0.564)。3.PZase陰性組和PZase陽性組患者各27例,治療2月末,陰性組和陽性組的痰菌陰轉率分別為77.78%、85.19%(c2=0.491,P=0.484);病灶吸收率分別為77.78%、88.89%(c2=0.533,P=0.465);空洞縮小率分別為42.86%、68.18%(c2=2.794,P=0.095);兩組治療有效率相似。4.PZase陰性組和PZase陽性組患者各27例,治療6月末,陰性組和陽性組的痰菌陰轉率分別為85.19%、100.00%(c2=2.430,P=0.119);病灶吸收率分別為88.89%、100.00%(c2=1.412,P=0.235);空洞縮小率分別為57.14%、81.82%(c2=3.101,P=0.078);兩組治療有效率相似。結論1.PZA表型耐藥的初治肺結核患者使用含PZA方案治療,其療效與對PZA敏感患者相似。2.PZA表型耐藥與pnc A基因突變有關。3.pnc A基因突變的初治肺結核患者使用含PZA方案治療,其療效與pnc A未突變PZA表型敏感患者相似。4.PZA表型耐藥與PZase活性喪失有關。5.pnc A基因突變與PZase活性喪失有關。6.PZase陰性的初治肺結核患者使用含PZA方案治療,其療效與PZase陽性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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R521

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