采用罗氏培养基进行MTB药物敏感性试验的影响因素分析

Analysis on Influencing Factors of Mycobacterium tuberculosis Drug Susceptibility Test by Using L^wenstein Jensen Medium

目的评价基于罗氏培养基绝对浓度法的结核分枝杆菌药物敏感性试验（简称“药敏试验”）检测异烟肼、利福平、乙胺丁醇的药物浓度标准，以及不同接种菌量、接种方式对耐药性检测结果的影响。方法按照世界卫生组织推荐方法从可能敏感患者和可能耐药患者痰液样本中的分离菌株，分别测定两类菌株对异烟肼、利福平、乙胺丁醇的最低抑菌浓度（MIC）并计算累计百分比以两类菌株累计百分比差值最大的MIC确定耐药界限；分别将菌量为10-3mg和10-4mg的结核分枝杆菌，接种于比例法耐药界限的3种药物含药培养基，经4周孵育后比较耐药结果；比较接种量均为10-6mg菌量的两种接种方法（接种环和滴管接种）孵育4周后菌落形成单位（CFU）的数量差异。结果异烟肼、利福平和乙胺丁醇经绝对浓度法测定的浓度界限分别为0．2μg／ml、40μg／ml、2μg／ml；不同接种量（10-3mg与10-4mg）接种相同含药培养基，其药敏试验结果差异无统计学意义（3c2值分别为0．57、0．00、0．00，P值分别为0．45、1．00、1．00）；使用接种环接种菌悬液的菌落形成单位数[（40．60±34．54），个，95％CI=35．08～46．12个]明显多于使用滴管的菌落形成单位数Ⅸ11．27±11．11），个，95％CI=9．50～13．05个]（t=11．58，P〈0．01）。结论异烟肼、利福平、乙胺丁醇的耐药界限采用比例法药物浓度界限更适宜；接种菌量10-3mg和10-4mg对药敏试验结果无明显影响；接种环接种效果较滴管接种效果好。

Objective To evaluate the drug concentration criteria of drug susceptibility test （DST） of Mycobacterium tuberculosis （MTB） to isoniazid, rifampicin, ehambutol by using absolute concentration method based on Lowenstein-Jensen media, and to evaluate the effects on DST results when different inoculums and inoculation methods were adopted. Methods According to the methods recommended by World Health Organization （WHO）, the MTB strains were isolated from the sputum specimens collected from the TB patient who were likely drug susceptible cases and likely drug resistant cases respectively, and then the minimal inhabitation concentration （MIC） of isoniazid, rifampicin, ethambutol to the two kinds of MTB strains were tested. The cumulative percentage of the two kinds of MTB strains were calculated and the MIC of which the greatest difference of cumulative percentage between the two kinds of MTB strains was regarded as the cut-off value of drug resistance. 10.3 mg and 10.4 mg MTB were inoculated on the drug contained media with isoniazid, rifampicin and ethambutol respectively and their drug-resistance cut-off values were the criteria of proportion method. The drug resistance results were compared after 4 weeks incubation; MTB suspicion were inoculated on Lowenstein-Jensen with loop or pipette respectively （inoculums were both 10.6 rag）, colony-forming units （CFU） were compared after four weeks incubation. Results The concentration cut- off values of isonazid, rifarnpicin and ethambutol tested by absolute concentration method were 0.2 μg/ml, 40 μg/ml and 2 Ixg/ml respectively. The DST results showed no statistically significant difference between the different inoculums （10-3 mg vs. 10-4 mg） （ 2 was 0.57, 0.00, 0.00; P value was 0.45, 1.00, 1.00 respectively）. The number of CFU inoculated with loop （40.60 ＋ 34.54, 95% C1 =35.08 - 46.12） was significantly higher than the number of CFU inoculated with pipette （11.27 ± 11.11, 95% CI= 9.50 - 13.05） （t=11.58, P 〈 0.01）. Conclusion The drug resistance cut-off values of isonazid, rifampicin and ethambutol tested by the proportion method are more reasonable. There is no obvious difference between the DST results with the inoculums of 10.3 mg and 104 mg. It is better to inoculate with loop than with pipette.