寡核苷酸探针膜杂交法检测耐药结核菌

目的应用寡核苷酸探针膜杂交法快速检测临床分离株中结核分枝杆菌对异烟肼(INH)、链霉素(SM)、乙胺丁醇(EMB)的耐药性。方法设计与合成用于检测结核分枝杆菌3种药物常见耐药基因的寡核苷酸探针,点于硝酸纤维素膜上,与结核分枝杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交。结果26株耐INH菌株中,16株Klb杂交阳性;7株inhla杂交阳性;23株耐SM菌株中,17株rpsl基因突变型探针Slb杂交阳性,2株突变型探针rrslb杂交阳性;31株耐EMB菌株中,13株Elb杂交阳性,1株Elc杂交阳性,5株Eld杂交阳性,1株Ele杂交阳性,11株与El探针杂交。kagG、inhA、rp-sL、rrs和embB基因膜杂交突变检出率分别为61.5%,26.9%,74%,8.7%和64.5%,与PCR-DS分析结果一致。结论寡核苷酸探针膜杂交技术可能成为检测部分结核分枝杆菌耐药基因型简便、快速的方法。     

难治性肺结核分支杆菌耐药性和药物依赖性的实验观察

目的探讨难治性肺结核分支杆菌耐药性和药物依赖性。方法对难冶性肺结核病人痰标本培养分离的分枝杆菌65株（例），进行低浓度、高浓度抗结核药物的耐药性和依赖性实验观察。结果65株分枝杆菌对SM、INH和RFP三种药物均耐药，EMB耐药率也较高（75．4％和69．2％）。分枝杆菌对sM依赖率分别为38．5％，INH为26．2％，RFP为18．5％，EMB为10．8％；同时依赖两种药物者为15．4％，三种药物为12．3％，四种药物为4．6％。结论难治性肺结核具有很高的耐药性和较高的依赖性。     

应用基因芯片分析结核分枝杆菌常见耐药基因型的研究

目的应用基因芯片快速检测结核分枝杆菌耐药基因型，建立一种新的分子药敏试验方法。方法以传统药敏试验和PCR－直接测序方法为对照，应用基因芯片快速检测157株结核分枝杆菌临床分离株异烟肼（INH）、利福平（RFP）、链霉素（SM）和乙胺丁醇（EMB）耐药基因（katG、rpoB、rpaL、rrs和embB）的带荧光素标记的PCR产物。结果PCR-直接测序和基因芯片检测36株结核分枝杆菌药物敏感株的5种耐药基因均为野生型。121株结核分枝杆菌耐药分离株中，56株耐INH分离株，katG基因突变率为62．5％，其中katG缺失率为7．5％，315位密码子突变率为55．4％，279位密码子突变率为1．8％；104株耐RFP株，rpoB基因突变率为94．2％，最常见的突变位点为531位和526位密码子（突变率分别为60．6％、15．4％），双位点突变率为5．8％，还发现511、513、515、516、517、518和533位密码子突变；62株耐SM分离株，rpsL和ITS总突变率为88．7％（两者分别为82．3％、6．5％），rpsL突变位于43位和88位密码子（突变率分别为77．4％、4．8％），rrs突变位于513位和516位碱基（突变分别为4．8％、1．6％）；57株为耐EMB株，embB基因突变率为61．4％，均为306位密码子突变，最常见的突变为ATG→GTG或剐rA（突变率分别为35．1％、15．8％），还发现306位密码子ATG→ATC、ATT和CTG突变。通过基因芯片检出的突变与基因测序结果一致。结论应用基因芯片可分析大多数结核分枝杆菌耐药基因型，弥补传统药敏试验方法的不足，指导临床治疗。     

结核分枝杆菌异烟肼耐药基因katG的检测

目的探讨结核分枝杆菌异烟肼(INH)耐药的分子机制,建立快速检测结核分枝杆菌INH耐药性的方法。方法应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测105株结核分枝杆菌临床分离株的katG基因,运用DNA测序和生物信息分析比对进行验证。结果以H37Rv标准株为对照,34株敏感株中5株(14.7%)存在KatG基因异常,105株耐药株中55株(52.4%)存在KatG基因异常。结论结核分枝杆菌对INH耐药与KatG基因突变有关,PCR-RFLP技术可快速、准确地检测结核分枝杆菌对INH的耐药性。     

结核分枝杆菌耐乙胺丁醇分离株emb B基因突变研究

目的探讨结核分枝杆菌对乙胺丁醇（EMB）产生耐药的分子机制,建立直接快速检测结核分枝杆菌EMB药物敏感性的实验方法。方法采用聚合酶链反应（PCR）扩增技术对临床耐乙胺丁醇结核分枝杆菌进行PCR扩增,扩增产物经纯化后,直接进行DNA测序分析。结果 54例临床分离株中,19例为EMB敏感株,35例为EMB耐药株。35例耐药株中13例（37.1%）发生基因突变。13例基因突变皆为错义突变,且有1例同时发生310位和313位突变。结论 embB306位氨基酸Met被置换是结核分枝杆菌对乙胺丁醇产生耐药性的重要机制,测序分析可以确认耐药基因的突变位点,是快速检测耐乙胺丁醇结核分枝杆菌的快速、有效的方法。     

基因芯片快速检测结核分枝杆菌katG基因突变及其与异烟肼耐药相关性

目的了解结核分枝杆菌katG基因S315突变与异烟肼（isoniazid,INH）耐药相关性,建立快速简便的katG基因S315突变基因芯片检测方法。方法采用二倍稀释法检测123株结核分枝杆菌临床分离菌株对INH的耐药性。PCR及测序确定上述结核分枝杆菌katG基因S315位点突变率及突变类型。根据PCR及测序结果,设计Cy3荧光标记探针并制备katG基因S315位点突变检测基因芯片。基因芯片检测结果与PCR及测序结果进行对比分析。结果 123株结核分枝杆菌临床菌株中,39.0%（48/123）菌株对INH敏感,61.0%（75/123）菌株对INH耐药。结核分枝杆菌H37Rv株及所有临床菌株均能扩增出katG基因片段。75株INH耐药菌株中,69.3%（52/75）菌株katG基因出现S315位突变,其中25株突变类型为S315N（AGC→AAC）、12株为S315T（AGC→ACC）、7株为S315I（AGC→ATC）、各有3株分别为S315T（AGC→CGC）和S315R（AGC→AGA）、2株为S315G（AGC→GGC）。所制备的基因芯片对123株结核分枝杆菌katG基因S315野生型或突变型检测结果与PCR及测序结果完全一致。结论结核分枝杆菌katG基因S315突变与INH耐药密切相关。本研究中制备的基因芯片可快速、简便、准确、敏感和特异地检测结核分枝杆菌katG基因S315突变及其类型。     

耐多药结核分枝杆菌基因突变分析

目的对临床分离的耐多药结核分枝杆菌株进行基因突变分析。方法对耐多药结核分枝杆菌临床分离菌株进行耐药基因的PCR检测,利用基因序列测定方法分析耐药基因突变情况。结果从耐多药结核病（MDR-TB）患者临床分离菌株中快速检测到rpoB、katG、rpsL、embB基因及其突变。耐多药菌株、全耐及单耐利福平分离菌株均检测rpoB基因点突变,突变位点主要为第516、526和531常见密码子,1例MDR-TB出现第479位和第531位密码子同时突变。耐多药菌、全耐菌及单耐异烟肼的菌株均检测有katG基因点突变,突变位点均为第2066位碱基C突变为Go全耐菌和对乙胺丁醇（EMB）耐药的耐多药菌株均检测有embB基因点突变,突变位点均为第306位密码子ATG突变为ACG。全耐菌和对链霉素（SM）耐药的MDR-TB菌株均检测有rpsL基因点突变,突变密码子为CCT突变为CTT,其中从1株对SM敏感的MDR-TB中也检测到突变。全敏感株、标准株及利福平（RIF）、异烟肼（INH）或EMB敏感的耐药株均无rpoB、katG或embB基因突变。结论PCR及基因序列测定可快速检测耐多药结核基因,结核分枝杆菌耐多药性与多个基因突变相关。     

耐药基因芯片对初治菌阳肺结核治疗指导价值研究

目的研究结核分枝杆菌(Mtb)耐药基因芯片法检测对初治菌阳肺结核治疗指导价值。方法应用Mtb耐药基因芯片检测100例初治肺结核感染菌株对利福平(RFP)、异烟肼(INH)、链霉素(SM)、乙胺丁醇(EMB)的耐药性,并与BACTEC MGIT960系统检测结果比对,经鉴别罗氏培养基(PNB)除外非结核分枝杆菌(NTM)结果 Mtb耐药基因芯片法检测耐RFP、INH、SM、EMB的敏感性和特异性分别为64.29%、75.00%、52.94%、62.5.%;93.02%、82.5%、93.98%、97.83%。结论结核分枝杆菌耐药基因芯片对于5种基因相关突变所致耐药性的敏感性略低,特异性较高,对初治菌阳肺结核临床治疗有一定参考价值。     

应用硝酸盐还原试验快速检测结核分枝杆菌药物敏感性

目的评价硝酸盐还原试验(Nitrate Reducrase Assay,NRA)方法检测结核分枝杆菌分离株的药物敏感性的应用价值。方法通过应用NRA方法和绝对浓度法同时检测了492株结核分枝杆菌临床分离株和30株质控菌株对INH、RFP、SM、EMB的药物敏感性,以牛结核分枝杆菌标准株、结核分枝杆菌标准菌株H37Rv做阴阳性对照。并随机选择62株菌株进行重复试验。结果与绝对浓度法比较,NRA检测对INH、RFP、SM、EMB的耐药性的敏感性分别为91.6%、96.5%、92.2%、90.7%;特异性分别为96.0%、98.2%、97.3%、97.3%;一致率分别为95.0%、97.9%、96.2%、96.7%。重复试验符合率为100%。结论NRA是一种成本低、操作简单、快速、与绝对浓度法有较高一致率的药敏试验方法,具有良好的临床应用前景。     

应用硝酸盐还原试验快速检测结核分枝杆菌药物敏感性

目的评价硝酸盐还原试验(Nitrate Reducrase Assay,NRA)方法检测结核分枝杆菌分离株的药物敏感性的应用价值。方法通过应用NRA方法和绝对浓度法同时检测了492株结核分枝杆菌临床分离株和30株质控菌株对INH、RFP、SM、EMB的药物敏感性,以牛结核分枝杆菌标准株、结核分枝杆菌标准菌株H37Rv做阴阳性对照。并随机选择62株菌株进行重复试验。结果与绝对浓度法比较,NRA检测对INH、RFP、SM、EMB的耐药性的敏感性分别为91.6%、96.5%、92.2%、90.7%;特异性分别为96.0%、98.2%、97.3%、97.3%;一致率分别为95.0%、97.9%、96.2%、96.7%。重复试验符合率为100%。结论NRA是一种成本低、操作简单、快速、与绝对浓度法有较高一致率的药敏试验方法,具有良好的临床应用前景。     

Evaluation of Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis.

SETTING: Mycobacteria Supranational Reference Laboratory. OBJECTIVE: To evaluate the Mycobacteria Growth Indicator Tube (MGIT) method for drug susceptibility testing of Mycobacterium tuberculosis. DESIGN: One hundred and one clinical strains of M. tuberculosis were evaluated for their susceptibilities to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (SM) by MGIT and by the proportion method on L?wenstein-Jensen (L-J) medium. The concentrations of drugs in MGIT were: 0.1, 1.0, 3.5 and 0.8 microg/ml for INH, RIF, EMB and SM, respectively. RESULTS: The results for individual drugs showed a good correlation: the specificity was 100% for INH, RIF and EMB and 92% for SM; the sensitivity was 100%, 94.6%, 96.1% and 89.7% for INH, RIF, EMB and SM, respectively, and the accuracy values 1.0, 0.98, 0.99 and 0.91. CONCLUSION: The MGIT system appears to be a reliable and simple non-radiometric method for the drug susceptibility testing of M. tuberculosis.     

显微镜观察药物敏感性检测在分枝杆菌分离培养和药物敏感性测定中的应用研究

目的评价显微镜观察药物敏感性检测(MODS,microscopic observation drug suscepti-bility assay)方法用于分枝杆菌分离培养和直接药物敏感性测定的应用前景。方法应用MODS方法对113例涂片镜检阳性患者痰标本进行分离培养,同时进行异烟肼、利福平、链霉素、乙胺丁醇4个药物的直接药物敏感性测定,以罗氏法分离培养和间接绝对浓度法作为对照,对分离培养和药敏结果进行对比分析,并通过DNA序列分析对不一致药敏结果进一步验证。结果 MODS与罗氏法分离培养的阳性率均为83.2%,污染率分别为0%和2.7%,两方面差异无统计学意义,两者的符合率为93.6%(kappa值:0.75),培养的敏感性、特异性分别为95.7%、81.3%。MODS的分离培养中位数时间为10 d(第95百分位数为21.5 d),罗氏分离培养的中位数时间为25 d(第95百分位数为39.3d)。MODS与间接绝对浓度法的耐药株检出率分别为49.5%、47.8%,符合率为95.5%(kappa值:0.91)差异无统计学意义,MODS检测的敏感性、特异性分别为97.7%、93.3%。MODS对结核分枝杆菌检测的敏感性、特异性、符合率分别为异烟肼(100%、84.5%、89.8%);利福平(100%、100%、100%);链霉素(94.6%、98.0%、96.6%);乙胺丁醇(100%、73.8%、75.0%)。对于利福平、链霉素的药物敏感性检测两种方法有相当高的一致性,而对异烟肼、乙胺丁醇的检测一致性较低。结论 MODS方法是一种操作简单、成本低廉、快速、敏感的诊断和药物敏感性检测方法。适合发展中国家和经济欠发达地区,在我国结核病防控工作中,具有广泛的应用前景。     

Treatment of tuberculosis in HIV infection.

Whether tuberculosis patients received short-course chemotherapy with treatment of isoniazid (INH) and rifampicin (RIF), combined or not with pyrazinamide (PZA) and ethambutol (EMB) or streptomycin (SM), or long term chemotherapy with INH, SM and thiacetazone (Tb1), the rate of sputum culture conversion was similar in HIV-positive and HIV-negative patients. To prevent relapses it was recommended to treat patients for a minimum of 9 months and for at least 6 months after culture conversion, or even to administer INH for life after the end of treatment. However, no difference was observed in the percentage of relapses between HIV-positive and HIV-negative patients. Side-effects were observed in approximately 20% of HIV-positive patients treated with INH + RIF + PZA + EMB (or SM) or with INH + SM + Tb1, Tb1 being responsible for epidermal necrolysis, in some cases fatal. The mean survival of HIV-patients with tuberculosis was from 10 to 18 months after the diagnosis of tuberculosis. Other opportunistic infections could have been the main cause of death. Acquired drug resistance is not a common complication of tuberculosis treatment in HIV-positive patients, but several epidemics of nosocomial transmission of multiple drug-resistant tuberculosis have recently been observed in the USA. Sparfloxacin, a new fluoroquinolone with a long half-life and low MIC (0.25-0.50 mg/l) against Mycobacterium tuberculosis, is a promising drug against tuberculosis.     

中国河南WHO结核病耐药监测报告(续)

结果与分析一、耐药监测一般资料统计监测点覆盖面：地区覆盖面８８－２％（１５／１７）,县区数覆盖面为１９－１％（３０／１５７）,人口为２７－２％（２４７４－８万／９１００万）。１４县为卫生部结控项目县,另１６县为非项目县。收集痰标本例数为２３１８例。其中有耐药监测结果的为１４２２例,非结核分枝杆菌５０例外,入选作耐药监测的结核分枝杆菌为１３７２例。后者占１９９６年各监测县同期涂阳病例的４９－８％和全省１９９６年同期涂阳病例的１５－６％。无抗结核用药史及用药史＜１月者９１６例,占６６－８％;有抗结核…     

Blood agar for susceptibility testing of Mycobacterium tuberculosis against first-line drugs.

OBJECTIVE: To evaluate the performance of blood agar for the susceptibility testing of 50 Mycobacterium tuberculosis clinical isolates against isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB). DESIGN: The activity of the drugs was determined by the proportion method on blood agar instead of Middlebrook 7H10 agar according to Clinical Laboratory Standard Institute recommendations. The final concentrations of INH, RMP, SM and EMB were 0.2 microg/ml, 1 microg/ ml, 2 microg/ml and 5 microg/ml, respectively. RESULTS: The results were compared with the radiometric proportion method as the reference, and the agreements were determined as 100% for INH and RMP, 92% for SM and 96% for EMB. The specificity, sensitivity, positive predictive value and negative predictive value were 90.4% and 97.5%, 100% and 90%, 66.6% and 90% and 100% and 97.5% for SM and EMB, respectively, while these values were 100% for INH and RMP. The results of susceptibility testing were obtained on the 14th day of incubation. CONCLUSION: According to this preliminary study, our results suggest that blood agar can be used as an alternative medium for the susceptibility testing of M. tuberculosis strains against INH, RMP, SM and EMB in resource-limited countries. However, further studies are needed before implementating the method in diagnostic laboratories.     

中国疾病控制体系实验室第1轮结核杆菌药物敏感试验室间质量评估分析

目的评估中国疾病控制体系结核病实验室药物敏感性测试能力。方法国家结核病参比实验室下发30株结核杆菌至受评估的实验室,受评估实验室按照WHO推荐的罗氏培养基药物敏感性试验方法(比例法),进行药物敏感性试验,检测药物包括异烟肼(INH)、利福平(RFP)、链霉素(SM)、乙胺丁醇(EMB),并报告国家参比室,国家参比室将其结果与正确结果比对。结果 4种药物的4个评估指标分别为,符合率:INH 97%,RFP 95%,SM 89%,EMB 93%;重复性:INH 96%,RFP98%,SM 86%,EMB 94%;特异性:INH 97%,RFP 99%,SM 89%,EMB 96%;敏感性:INH 97%,RFP 65%,SM 91%,EMB 47%;各实验室4个评价指标均数比较,除敏感性差异有统计学意义外,其他4个检测值均高于85%,差异无统计学意义。结论除个别实验室在耐药检出能力上需提高外,其他参与实验室的药敏试验水平达到设定要求,INH、RFP药物检测稳定,SM和EMB需要进一步标化。     

Pattern of mycobacterial resistance to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the state of Qatar after the implementation of DOTS and a limited expatriate screening programme.

OBJECTIVE: To determine the resistance pattern of Mycobacterium tuberculosis to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the State of Qatar after the implementation of DOTS and an expatriate screening programme on arrival. METHOD: A state-wide, population-based, retrospective analysis of all cases of pulmonary tuberculosis with positive M. tuberculosis culture reported to the Division of Public Health TB Unit from January 1996 to December 1998. M. tuberculosis sensitivity testing was done by the Bactec method for isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB). The results were interpreted as a daily change of the growth index of test vials (with drug) compared with controls. RESULTS: There were 406 isolates with positive M. tuberculosis culture. Sixty-one (15%) were resistant to one or more of the four anti-tuberculosis drugs, of which 58 (95%) were from newly diagnosed cases (primary) and three (5%) were from previously treated cases (acquired). Primary resistance was as follows: any resistance 15%, INH 12.4%, RMP 2%, SM 5.2%, EMB 0.8% and multidrug resistance (MDR, resistance to INH and RMP at least) was found in 0.8%. Acquired resistance was as follows: any resistance 15%, INH 15%, RMP 5%, SM 5% and MDR 5%. CONCLUSION: The prevalence of resistance to four anti-tuberculosis drugs is strikingly low due to the limited expatriate screening programme (chest radiography) and implementation of DOTS. The four-drug regimen is recommended for the initial phase of therapy until the results of sensitivity testing are known.     

Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Reference Laboratory Network: five rounds of proficiency testing, 1994-1998.

SETTING: Quality assurance for the WHO/IUATLD Global Tuberculosis Drug Resistance Surveillance Programme. OBJECTIVE: To implement an ongoing proficiency-testing programme for drug susceptibility testing (DST) of Mycobacterium tuberculosis within the WHO/IUATLD Supranational Reference Laboratories Network (SRLN). DESIGN: Five culture panels, each consisting of 10 duplicate drug-susceptible and drug-resistant clinical isolates (100 strains) of M. tuberculosis were tested for resistance to streptomycin (SM), isoniazid (INH), rifampicin (RMP) and ethambutol (EMB). DST procedures included the proportion, absolute concentration and resistance ratio methods, as well as the radiometric BACTEC 460 method. RESULTS: The efficiency, sensitivity and specificity of M. tuberculosis DST as well as the intra-laboratory reproducibility showed that the laboratories tested susceptibility to RMP and to INH very reliably, with values ranging from 97% to 99%. The testing of SM and EMB was less dependable, with values ranging from 90% to 95%. The sensitivity of testing of EMB increased from 60% in Round 1 to 98% in Round 5, without a concomitant decrease in specificity. CONCLUSIONS: This study has shown that regular proficiency testing can significantly improve the quality of DST, even in the most sophisticated TB laboratories. Mean DST efficiency levels of 92% for both SM and EMB and 97% and 99% for INH and RMP, respectively, are proposed as reasonable performance goals for the SRL network. Efficiency, consistently lower than these values, would require remedial action. Efficiency levels lower than mean -1 standard error, i.e., 80% for SM and EMB, 89% for INH and 95% for RMP, should always be considered as sub-standard performance for DST.     

174株结核分枝杆菌耐药情况分析

目的分析我所183株痰培养阳性菌株的耐药情况。方法采用药敏比例法对183株阳性菌株进行菌种鉴定及药物敏感性试验。结果共分离出174株结核分枝杆菌,且4种一线抗结核药物异烟肼(INH)、利福平(RFP)、乙胺丁醇(EMB)、链霉素(SM)的总耐药率为25.3%,耐多药率为8.0%。4种抗结核药物的耐药率由高到低依次为SM、INH、EMB、RFP。结论陕西省结核耐药情况仍较严重。