初治涂阳肺结核患者治疗前后结核分枝杆菌毒力变化的探讨

Analysis of Mycobacterium tuberculosis virulence before and after anti-tuberculosis treatment for smear positive pul- monary tuberculosis patients

目的分析初治涂阳肺结核患者经抗结核药物治疗前后结核分枝杆菌（MTB）的毒力变化情况。方法收集2014--2015年入选“武汉市初治涂阳肺结核免费隔离治疗项目”的患者中初治涂阳肺结核且经抗结核治疗2周后痰涂片仍未转阴者（未转阴组）治疗前、治疗1周后及治疗2周后分离的MTB（15株）；初治涂阳肺结核经抗结核药物治疗后2周内痰涂片转阴患者（转阴组）治疗前分离的MTB（4株）；耐多药结核病患者（对照组）治疗前分离的MTB（1株）。将上述不同类别患者不同时间点痰培养分离的MTB菌株于体外分别感染巨噬细胞3h和96h后，应用流式细胞术检测巨噬细胞早期凋亡水平，同时计数菌落数量，分析其变化情况。结果未转阴组治疗前、治疗1周后、治疗2周后分离的MTB菌株感染巨噬细胞3h后，诱导巨噬细胞早期凋亡率分别为（2．95±1．13）％、（4．21±1．92）％、（3．73±1．36）％，差异无统计学意义（F=3．38，P=0．086）；感染巨噬细胞96h后，诱导巨噬细胞早期凋亡率分别为（4．25±0．48）oA、（5．69±2．03）％、（5．06±1．25）％，差异无统计学意义（F=1．69，P=0．245）。未转阴组、转阴组及对照组患者治疗前痰培养分离的MTB菌株感染巨噬细胞3h后诱导巨噬细胞早期凋亡率分别为（2．95±1．13）％、（4．05±1．87）％、（4．38±0．29）％，差异无统计学意义（F=1．94，P=0．380）；感染巨噬细胞96h后，诱导巨噬细胞早期凋亡率分别为（4．25±0．48）％、（4．11±0．26）oA、（5．39±0．24）%，差异有统计学意义（F=11．97，P=0．003）。各组不同治疗时间所分离的MTB菌株感染巨噬细胞后菌落数量差异无统计学意义。结论初治涂阳肺结核经抗结核治疗后2周痰涂片仍未转阴患者所携带的MTB菌株毒力在治疗前后没有变化。

Objective Evaluation of virulence of Mycobacterium tuberculosis （MTB） isolated before and after treatment for newly diagnosed smear positive pulmonary tuberculosis （TB） patients. Methods Patients were re- cruited from Project of Free Quarantined Hospital Treatment for Newly Diagnosed Smear Positive Pulmonary TB Patients in Wuhan during 2014--2015. Fifteen strains of MTB isolated from patients before the initiation of anti-TB treatment, after one week or two weeks of anti-TB treatment from smear positive pulmonary TB patients who are still sputum smear positive after 2 weeks of anti-TB treatment, four MTB strains from pulmonary TB patients who are sputum smear negative during 2 weeks of anti-TB treatment and one multidrug-resistant MTB strain before the initiation of anti-TB treatment. Macrophages were infected with the different MTB strains cultured from different time points of sputum isolated from the different types of patients mentioned above in vitro for 3 hours or 96 hours. Flow cytometry was conducted to detect the early apoptotic level of macrophages. At the same time, the number of MTB colonies was counted. Results The percentage of early apoptosis of macrophages infected with cultured MTB from sputum isolated before, after one week or two weeks of anti-TB treatment from smear positive pulmonary TB patients who were still sputum smear positive after 2 weeks of anti-TB treatment were （2.95±1.13）%, （4. 21±1.92）% and （3.73±1.36）% repectively, after infection for 3 h,displaying no significant difference （F= 3.38, P=0. 086）% （4.25±0. 48） %, （5.69±2.03） %, （5.06±1.25）% after infection for 96 h, displaying no significant difference （F=1. 69,P= 0. 245）. The percentage of early apoptosis of macrophages infected with cultured MTB from sputum isolated before treatment from smear positive pulmonary TB patients who were still sputum smear positive after treatment were （2.95 ±1.13）%, （4. 05±1.87） % for who were negative after treatment and （4. 38±0. 29） % for MDR MTB after infection for 3 h, displaying no significant difference （F=1. 94,P=0. 380）. The percentage were （4. 25±0. 48）%, （4. 11±0.26） % and （5.39±0. 24） % after infection for 96 h respectively, displaying no significant difference （F=11.97, P=0. 003）. No significant difference was observed as to the count of MTB colonies. Conclusion The virulence of MTB isolated is not significantly changed from MTB patients who are still sputum smear positive after 2 weeks of anti-TB treatment.