摘要
目的探讨直接法固体药物敏感性试验（简称“药敏试验”）检测涂阳肺结核患者痰标本的应用效果。方法收集于2010年2月至2011年7月的抗酸染色阳性的肺结核患者的痰标本，共389例，先对痰标本进行分枝杆菌固体培养和直接法药敏试验，再用固体培养阳性的菌株进行传统比例法药敏试验和对硝基苯甲酸（PNB）培养基菌种鉴别。直接法药敏的试验方法为：视痰标本性状，将1～2倍的4％NaOH加入有痰标本的前处理管内，旋紧处理管螺旋盖，计时15min，涡旋震荡30S直至痰标本液化，室温静置至15min计时结束，用无菌吸管吸取前处理后的痰标本，分别均匀接种于酸性罗氏培养基（直接法药敏试验的对照培养基）、含利福平（RFP）的酸性罗氏培养基（RFP40.0μg／ml）、含异烟肼（INH）的酸性罗氏培养基（INH0．2μg／m1）上，每支管接种0．1～0．15ml。36℃条件下进行孵育。每周记录细菌生长情况至第8周。在对照培养基上分枝杆菌培养阳性的前提下，分别以〉1％的耐药百分比即判断为耐药和含药培养基上菌落数多于20个菌落即判定为耐药的两种耐药判定方法，计算直接法固体药敏试验的检测结果。以比例法药敏试验结果为金标准，比较直接法药敏试验的检测效果。结果剔除因污染、涂阳培阴等原因无法获得药敏试验结果的标本，最终直接法和比例法均获得RFP药敏试验结果的标本346例，均获得INH药敏试验结果的标本338例。当直接法固体药敏试验以〉1％的耐药百分比为耐药检测界限时，其检测RFP耐药率为10．4％（36／346），低于比例法检测的RFP耐药率[13．9％（48／346）]，差异有统计学意义（χ2=24．14，P〈0．01）；以比例法为标准，直接法固体药敏试验对RFP耐药检测的敏感度、特异度、一致率分别为75．0％（36／48）、82．9％（247／298）、81．8％（283／346），两种方法一致性一般（Kappa=0．43）；直接法固体药敏试验检测INH的耐药率为7．1％（24／338），低于比例法检测的INH耐药率[10．9％（37／338）]，差异有统计学意义（χ2=15．29，P〈0．01）；以比例法为标准，直接法固体药敏试验对RFP耐药检测的敏感度、特异度、一致率分别为64．9％（24／37）、86．0％（259／301）、83．7％（283／338），两种方法的一致性较差（Kappa=0．39）。当直接法固体药敏试验结果以含药培养基上的菌落数多于20个为耐药检测界限时，其检测的RFP耐药率为10．4％（36／346），低于比例法，差异有统计学意义（χ2=15．87，P〈0．01）；以比例法为标准，直接法固体药敏试验对RFP耐药检测的敏感度、特异度、一致率分别为75．oH（36／48）、86．2％（257／298）、84．7％（293／346），两种方法一致性一般（Kappa=0．49）；直接法固体药敏试验检测INH的耐药率为7．1％（24／338），低于比例法，差异有统计学意义（χ2=9．38，P〈0．01）；以比例法为标准，直接法固体药敏试验对INH耐药检测的敏感度、特异度、一致率分别为64．9％（24／37）、88．7％（267／301）、86．1％（291／338），两种方法一致性一般（Kappa=0．43）。结论直接法固体药敏试验与传统比例法药敏试验检测的耐药性结果差异较大，对耐药结核病的临床诊断效果不理想。
Objective To investigate the application value of direct solid drug sensitivity test （DST） using sputum specimens from smear-positive pulmonary tuberculosis patients. Methods A total of 389 （from Feb 2010to Jul 2011） sputum specimens isolated from patients with positive acid-fast tuberculosis were collected. The sputum samples were first used for mycobacterial solid culture and direct DST, and then the solid-culture-positive strains were subjected to conventional proportional susceptibility testing and sodium nitrobenzoate （PNB） medium growth test to differentiate strains. The direct DST was conducted as follows. Based on the specimen traits, 1 to 2 times of 4% NaOH was added to the pre-treatment tube containing sputum specimen, and the screw cap was tightened. The tube was vortexed for 30 seconds until the sputum specimen was liquefied, and then incubated for 15 minutes at room temperature. The pretreated sputum specimens were aspirated with a sterile pipette and evenly inoculated into common acidic Roche medium （control medium for DST）, rifampicin （RFP, 40.0 μg/ml）-containing acidic Roche medium and isoniazid （INH, 0.2μg/ml）-containing acidic Roche medium （0.1 to 0.15 ml per tube）. The tubes were incubated at 36 ℃. The bacterial growth on the culture tube was recorded weekly until the 8th week. On the premise of positive cultures of mycobacteria on the control medium, drug resistance was defined as a percentage of drug resistance of 〉1% or number of colonies on drug-containing medium 〉20. The results of direct DST were judged based on the above two drug resistance determination methods. The detection value of DST was analyzed using the proportional method as gold standard. Results Specimens that could not obtain susceptibility test results due to pollution or smear-positive but culture-negative were excluded. As a result, 346 specimens were available for RFP susceptibility test by both the direct method and the proportional method, and 338 specimens were available for INH susceptibility test. When using a resistance percentage of 〉1%as the definition for drug resistance, the detec tion rate of RFP resistance by the direct method was 10.4% （36/346）, which was lower than that detected by the proportional method （13.9 %, 48/346）, and the difference was statistically significant （χ2 = 24. 14, P〈 0.01）. Using the proportional method as the standard, the sensitivity, specificity, and accuracy of the direct solid DST for RFP resistance testing were 75.0% （36/48）, 82.9% （247/298）, and 81.8% （283/346）. No strong consistency between the two methods was shown （Kappa=0. 43）. For INH resistance, the detection rate by the direct method was 7.1% （24/338）, lower than that detected by the proportion method （10. 9%, 37/338）, and the difference was statistically significant （χ2= 15.29, P〈0.01）. Taking the proportion method as the standard, the sensitivity, specificity, and accuracy of the direct solid method for INH resistance testing were 64.9% （24/37）, 86.0% （259/301）, and 83.7% （283/338）, respectively. The consistency between the two methods was poor （Kappa- 0.39）. When using number of colonies on the drug-containing medium more than 20 as the definition for drug resis- tance, the RFP resistance rate tested by the direct method was 10.4% （36/346）, which was lower than that of the proportional method. The deference was statistical significance （χ2 = 15.87, P〈0. 01）. Using the proportional method as the standard, the sensitivity, specificity, and accuracy of the direct solid DST for RFP resistance testing were 75.0% （36/48）, 86.2% （257/298）, and 84.7%（293/346）, respectively. No strong consistency between the two methods was shown （Kappa=0. 49）. The INH resistance rate detected by the direct method was 7.1% （24/338）, which was lower compared with the proportion method, and the difference was statistically significant （χ2 =9.38, P〈0.01）. Using the proportional method as the standard, the sensitivity, specificity, and accuracy of the direct solid DST for INH resistance testing were 64.9%（24/37）, 88. 7% （267/301）, and 86.1% （291/338）. No strong consistency between the two methods was shown （Kappa = 0.43）. Conclusion The direct solid DST shows quite different drug resistance results compared with the traditional proportional DST. The clinical diagnosis of drug resistant tuberculosis by direct solid method is not ideal.
作者
陈丽
邬剑
秦玉宝
唐鹭
苏欣
张艳
CHEN Li, WUJian, QIN Yu-bao, TANG Lu, SU Xin, ZHANGYan.(Department of Provincial Reference Laboratory, Center for Tuberculosis Control of Heilongjiang Province, Harbin 150030, Chin)
出处
《结核病与肺部健康杂志》
2018年第1期33-36,共4页
Journal of Tuberculosis and Lung Health
关键词
分枝杆菌
结核
抗药性
细菌
微生物敏感性试验
对比研究
Mycobacterium tuberculosis
Drug resistance, bacterial
Microbial sensitivity tests
Compara- tive study
