微量液体培养基最低抑菌浓度法与罗氏比例法在结核分枝杆菌药敏试验中的价值比较

目的比较微量液体培养基最低抑菌浓度(MIC)法与罗氏比例法在结核分枝杆菌药敏试验中的临床价值。方法以100株结核分枝杆菌菌株(由辽宁省结核实验室提供)为研究样本,分别采用罗氏比例法与微量液体培养基MIC法检测结核分枝杆菌的药物敏感性。结果微量液体培养基MIC法与罗氏比例法检测结核分枝杆菌对乙胺丁醇、二卡那霉素、利福平、异烟肼、氧氟沙星、卷曲霉素药物的敏感性比较,差异无统计学意义(P 0.05)。结论微量液体培养基MIC法应用于结核分枝杆菌药敏试验中与罗氏比例法有着良好的一致性,且检测速度快,经济适用性好,能够为临床指导结核病用药提供帮助。     

结核分枝杆菌临床分离株药敏结果与耐药程度的关联分析

目的 探讨结核分枝杆菌临床分离株对12种抗结核药物的MIC值及其药敏检测结果 与耐药程度的关联规律,从而为临床制定治疗方案提供借鉴依据.方法 采用液体培养基联合MTT技术进行抗结核药物的MIC检测.对上海市肺科医院2009年1-6月间的163株结核分枝杆菌临床分离株进行RFP、INH、SM、EMB、OFLX、LVFX、MOX、AMK、CPM、PTA、CLA和PAIN的MIC检测和Bactec MGIT快速培养仪药敏检测,并进行MIC与耐药程度的关联性分析.结果 67%(42/62)的SM耐药株MIC≥16 μg/ml,63%(51/81)的INH耐药株MIC≥8 μg/ml,77%(50/65)的RFP耐药株MIC≥8 μg/ml,20%(12/60)的EMB耐药株MIC≥4 μg/ml;43%(25/58)的OFLX耐药株MIC≥8 μg/ml;41%(15/37)的AMK耐药菌株MIC≥16 μg/ml,41%(12/29)的CPM耐药菌株MIC≥4 μg/ml.OFLX耐药株的3个氟喹诺酮类药物的MIC(OFLX、LVFX和MOX的MIC分别为2～128、1～32和0.0625～1 μg/ml)差异有统计学意义(F=16.874,P<0.01);SM、INH、RFP、EMB、OFLX、AMK和CPM在任6种及7种药物同时耐药株中的MIC值(分别为0.5～128、2～64、0.25～128、1～32、1～64、0.5～128和1～128μg/ml)明显高于任1种及2种药物耐药菌株的MIC值(MIC值分别为0.25～128、0.0625～64、0.25～32、0.25～2、0.125～2、0.5～4和1～4 μg/ml,F值分别为20.066、40.499、47.197、70.373、91.432、41.840和21.547,P均<0.05);SM、INH、RFP、EMB在4种药物同时耐药菌株中MIC值(分别为1～128、2～64、0.25～128和1～32μg/ml)显著高于任1种及2种药物耐药株(MIC值分别为0.25～128、0.0625～64、0.25～64和0.25～2 μg/ml,F值分别为26.242、23.563、31.541和64.469,P均<0.05);RFP在MDR酶株的MIC值(2～64 μg/ml)显著高于非MDR的耐药菌株的MIC值(0.25 μg/ml,F=5.613,P<0.05).结论 本研究揭示了常用的12种抗结核药物的耐药程度与常规药敏检测结果 之间的关联规律,为临床医生根据常规药敏结果 制定更加有效的抗结核治疗方案提供重要的借鉴.     

变色液体培养基系统快速鉴定结核分枝杆菌和耐药性测定

由于固体培养基鉴定结核分枝杆菌(结核菌)及药敏试验均需28 d,其制作过程需加热,且培养基中蛋白质对药物有吸附作用,故仅用于常规抗结核药敏试验,不适合用于分枝杆菌最低抑菌浓度(MIC)测定.多耐药结核菌和非结核菌对常用抗结核药物耐药,因此急需一种快速测定分枝杆菌药敏的方法,筛选新的药物.尽管Bactec系统可用于分枝杆菌药敏试验和MIC测定[1],但试剂和仪器昂贵.因此,我们采用快速变色液体培养基系统鉴定结核与非结核分枝杆菌,并测定了结核菌常规药物的敏感性和非结核分枝杆菌对9种药物的MIC,结果如下. 一、材料和方法 1.标本来源:128株结核菌,34株非结核分枝杆菌(其中15株龟分枝杆菌脓肿亚种、4株龟分枝杆菌龟亚种、14株偶发分枝杆菌和1株胞内分枝杆菌).上述菌株为深圳地区和广州地区分离株. 2.结核菌快速鉴定及药敏试剂、分枝杆菌MIC测定试剂和菌株鉴定试剂:对硝基苯甲酸(PNB)培养基、5%氯化钠耐受试验、芳香硫酸酯酶(3、14     

结核分枝杆菌链霉素耐药性的噬菌体检测技术研究

目的 建立结核分枝杆菌（结核菌）链霉素耐药性的噬菌体快速检测技术，并探讨其临床应用价值。方法 应用分枝杆菌噬菌体感染结核菌建立链霉素耐药性的噬菌体检测技术，并对最佳测定条件进行探讨；将所建立的方法用于38株世界卫生组织药敏质控株和372株临床分离株链霉素耐药性测定，并与绝对浓度法和Bactec 960药敏结果比较，对不符合菌株进行最低抑菌浓度（MIC）和rpsL耐药基因检测。结果 链霉素终浓度5μg／ml作用24h、噬菌体10^8噬菌体形成单位／ml感染60min、杀毒剂5％室温作用5min为选择的检测条件。噬菌体法检测38株药敏质控株结果完全符合。372株临床分离株链霉素耐药性检测结果表明，如以绝对浓度法药敏结果为判断标准，则本法敏感性为97.4％、特异性为91．0％、阳性预测值为81.7％、阴性预测值为98.9％、准确性为92.9％；如以Bactec 960药敏结果为判断标准，则本法敏感性为97.2％、特异性为97.1％、阳性预测值为94.6％、阴性预测值为98.5％、准确性为97．1％。有22株噬菌体法药敏结果与常规药敏试验结果不符，其中的11株噬菌体法检测结果与MIC和rpsL耐药基因检测结果相同。结论 噬菌体法检测链霉素耐药性快速、简便，具有很高的敏感性和特异性，可作为结核菌链霉素耐药性的快速检测方法。     

黄芩苷对结核分枝杆菌抑菌作用的研究

目的探索黄芩苷对结核分枝杆菌的体外抑制作用。方法采用改良罗氏药敏绝对浓度法对107株结核分枝杆菌进行黄芩苷的体外抑菌试验。结果 107株结核分枝杆菌的体外抑菌试验结果表明,黄芩苷对结核分枝杆菌有明确的抑制作用,最低抑菌浓度(MIC)值为0.75~12 mg/mL之间,MIC为12 mg/mL的菌株占8.41%,6 mg/mL的菌株占46.73%,3 mg/mL的菌株占37.38%,1.5 mg/mL的菌株占6.54%,0.75 mg/mL的菌株占0.93%,MIC_(50)和MIC_(90)均为6 mg/mL。结论 6 mg/mL黄芩苷具有较强的体外抗结核分枝杆菌作用。     

结核分枝杆菌对氟喹诺酮类药物敏感性的实验研究

目的 获得四种氟喹诺酮（FQ）对结核分枝杆菌的最小抑菌浓度（MIC）,评价PCR探针熔解曲线法（PMAA）检测结核分枝杆菌对氟喹诺酮药物耐药性的临床价值.方法 124例对氧氟沙星敏感的菌株和78例对氧氟沙星耐药的菌株,在96孔细菌培养板中,用Middlebrook 7H9液体培养基将药物进行连续倍比稀释后,加入5×10^-2 mg/ml菌液100 μl,用刃天青显色法测定MIC;并用PCR探针熔解曲线法（PMAA）检测结核分枝杆菌对氟喹诺酮（FQ）药物的耐药突变.结果 氧氟沙星折点浓度为2 μg/ml,加替沙星和莫西沙星对结核分枝杆菌的MIC90比氧氟沙星和左氧氟沙星要低4-8倍,具有比氧氟沙星和左氧氟沙星更好的抗结核分枝杆菌的活性.以罗氏比例法为金标准,PCR-PMAA检测敏感度、特异度、阳性预测值、阴性预测值和符合率分别为96.2％,97.6％,96.2％,97.6％和97.0％.结论 利用微量MIC方法较常规药敏方法可以获得更多的耐药信息.用PCR-PMAA法能高效快速的检出耐药突变菌株.两种方法结合应用,对临床肺结核疾病早期用药具有更好的指导价值.     

BACTEC MGIT960在二线抗结核药物药敏试验应用中的效果评价

目的 评价BACTEC MGIT 960进行4种二线抗结核药物药敏试验的效果.方法 用MGIT 960对结核分枝杆菌临床分离菌株进行二线抗结核药物卷曲霉素、卡那霉素、氧氟沙星和乙硫异烟胺的药敏检测,并将结果与L-J比例法结果进行比较分析.结果 111 株结核分枝杆菌临床分离株用MGIT960法与L-J比例法卷曲霉索、卡...     

焦磷酸测序技术检测耐多药结核分枝杆菌

目的利用焦磷酸测序技术,建立结核分枝杆菌菌株耐多药检测方法,并与Bactec MGIT 960药敏法进行比较。方法设计rpoB、katG基因PCR扩增引物和焦磷酸测序引物,建立结核分枝杆菌菌株焦磷酸测序检测方法。利用新确立的焦磷酸测序法检测202株临床分离的耐多药结核分枝杆菌的利福平、异烟肼耐药性。结果与BACTEC MGIT 960法检测药物耐药性结果比较,焦磷酸测序检测多耐药结核分枝杆菌临床分离株的敏感性与特异性为72.8%[95%可信区间(confidence in-terval,CI):66.3%~78.4%]及90.0%(95%CI:80.1%~95.1%)。结论新确立的焦磷酸测序技术可快速检测结核分枝杆菌对利福平、异烟肼的耐药性,其可作为耐多药结核分枝杆菌药敏有效的检测工具。     

显微镜观察药物敏感度检测技术在二线抗结核药耐药性检测中应用

目的 探讨MODS同时快速检测4种二线抗结核药耐药性的效果.方法 用24孔细胞培养板建立显微镜观察药物敏感性技术,进行二线抗结核药物耐药性检测,并对最佳检测条件进行探讨.将该技术用于上海市肺科医院2007-2008年住院肺结核患者痰标本分离60株结核分枝杆菌对丙硫异烟胺、阿米卡星、卷曲霉素、左氧氟沙星耐药性检测,并与传统罗氏药敏结果进行比较,对2种方法检测结果不符的菌株进行MIC测定.结果 MODS检测丙硫异烟胺、阿米卡星、卷曲霉素、左氧氟沙星药敏结果与罗氏药敏结果符合率分别为96.7%(58/60)、98.3%(59/60)、91.7%(55/60)、96.7%(58/60).以传统罗氏药敏结果为判断金标准,MODS检测敏感度、特异度、阳性预测值和阴性预测值及准确性丙硫异烟胺分别为100%、87.5%、87.5%、100%、96.7%,阿米卡星分别为100%、90.0%、90.0%、100%、98.3%,卷曲霉素分别为76.9%、95.7%、83.3%、93.8%、91.7%,左氧氟沙星分别为100%、96.0%、83.3%、100%、96.7%.结论 MODS检测丙硫异烟胺、阿米卡星、卷曲霉素、左氧氟沙星耐药性具有快速、操作简便、价廉等优点,与传统罗氏药敏结果有较高的符合率,可作为MTB耐药性的快速检测方法之一.     

中药结核丸对结核分枝杆菌体内外抗菌效果的观察

目的检测中药结核丸的体内外抗结核分枝杆菌效果.方法建立结核分枝杆菌H37Rv株小鼠感染模型.以乙胺丁醇为对照药,采用二倍试管稀释法测定中药结核丸对结核分枝杆菌H37Rv株、牛分枝杆菌bovis株、草分枝杆菌phlie株的体外最低抑菌浓度(MIC);以半数存活时间(ST50)为指标,检测各药物对结核分枝杆菌H37Rv株感染小鼠的体内抗菌效果.结果中药结核丸对结核分枝杆菌H37Rv株、牛分枝杆菌bovis株、草分枝杆菌phlie株的MIC分别为1024、1024和2048μg/ml,对照药乙胺丁醇MIC则均分别为1μg/ml.小鼠感染对照组ST50为13d.中药结核丸对结核分枝杆菌感染小鼠的ST50分别为16d(32mg/d/mouse)、15d(16或8mg/d/mouse)、14d(4mg/d/mouse)和13d(2或1mg/d/mouse),对照药乙胺丁醇对结核分枝杆菌感染小鼠的ST50分别为大于28d(1～4mg/d/mouse)、24d(0.5mg/d/mouse)和19d(0.25mg/d/mouse).结论中药结核丸体外抑制结核分枝杆菌的作用较弱,但剂量≥4mg/只小鼠时有明显的体内抗结核分枝杆菌活性.     

Reevaluation of the critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide using wild-type MIC distributions and pncA gene sequencing

Pyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosis strains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosis isolates and compared the results to pncA sequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n = 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤ 8 to 64 mg/liter), and no pncA gene mutations were detected. In strains resistant in both Bactec systems (n = 18), the PZA MICs ranged from 256 to ≥ 1,024 mg/liter. The discordances between pncA sequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncA mutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤ 64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.     

Sensititre MycoTB Plate Compared to Bactec MGIT 960 for First- and Second-Line Antituberculosis Drug Susceptibility Testing in Tanzania: a Call To Operationalize MICs

MIC testing for Mycobacterium tuberculosis is now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first- and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.     

Evaluation of mycobacteria growth indicator tubes for susceptibility testing of Mycobacterium tuberculosis to isoniazid and rifampin

The reliability of mycobacteria growth indicator tubes (Mgit) for determining the susceptibility of Mycobacterium tuberculosis to isoniazid (0.1 μg/ml) and rifampin (2.0 μg/ml) was evaluated by comparing Mgit results to those obtained by the radiometric Bactec TB method. We tested 29 isolates, many selected for resistance. The turnaround times were 3–8 days (median 6) for Mgit and 4–10 days (median 6) for Bactec. Isoniazid results by both methods agreed for all isolates tested: 20 resistant and nine susceptible. Rifampin results agreed for 28 isolates: 10 resistant and 18 susceptible. One isolate yielded discrepant results: resistant to rifampin by Bactec, but susceptible by Mgit. This isolate was also rifampin-resistant by the traditional Method of Proportion and when tested by the Mgit method using 1.0 μg/ml of rifampin. The Mgit system is a nonradiometric alternative to the Bactec for rapid susceptibility testing of M. tuberculosis to isoniazid and rifampin; however, additional testing is needed to determine the optimal concentration of rifampin.     

Rapid, automated, nonradiometric susceptibility testing of Mycobacterium tuberculosis complex to four first-line antituberculous drugs used in standard short-course chemotherapy

The increasing prevalence of drug-resistant tuberculosis necessitates rapid and accurate susceptibility testing. The nonradiometric BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) system for susceptibility testing was evaluated on 222 clinical Mycobacterium tuberculosis complex isolates for isoniazid, rifampin, and ethambutol. Fifty-seven of the isolates were tested for pyrazinamide. Results were compared to those of radiometric BACTEC 460 system and discrepancies were resolved by the agar proportion method. We found an overall agreement of 99.0% for isoniazid, 99.5% for rifampin, 98.2% for ethambutol, and 100% for pyrazinamide. After resolution of discrepancies, MGIT yielded no false susceptibility for rifampin and isoniazid. Although turnaround times were comparable, MGIT provides an advantage as inoculation can be done on any weekday as the growth is monitored automatically. The automated MGIT system is a rapid and reliable alternative for susceptibility testing of M. tuberculosis complex to first-line drugs.     

焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性,并对其临床应用进行评价.方法 收集上海市肺科医院2008-2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株.所有菌株按<结核病诊断实验室检验规程>进行分枝杆菌培养、菌种鉴定.另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库.利用焦磷酸测序技术,以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株,探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、rrs耐药基因的最佳模式,并用该条件检测89株结核分枝杆菌临床分离株,检测结果与Bactec 960药敏法进行比较.结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式,katG、rrs基因检测宜采用焦磷酸测序单核苷酸多态性模式.以Bactec 960药敏法测定结果为判断标准,则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98.0%、64.1%、79.5%、78.3%,特异度分别为97.5%、100.0%、90.0%、100.0%,准确性分别为97.8%、77.5%、85.4%、94.4%,该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactec 960药敏检测结果一致性较好,Kappa值均≥0.7.结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点,是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法.

Abstract：

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.     

Comparison of the BBL-mycobacteria growth indicator tube method with culture in the diagnosis of tuberculosis and evaluation of the resistance patterns of isolated strains to four major drugs

The BBL-mycobacteria growth indicator tube system (MGIT) is used for a rapid detection of the presence of mycobacteria. Our study aimed to compare MGIT with the L?wenstein-Jensen (LJ) reference method in clinical samples with suspected pulmonary and extrapulmonary tuberculosis, and to evaluate the primary and secondary resistance patterns by determining the resistances of the isolated strains to four major antimycobacterial drugs. 648 clinical samples from different clinics, with suspected pulmonary or extrapulmonary tuberculosis based on clinical, radiological, histopathological and immunological findings, were included in the investigation. The samples were first stained with Ziehl-Neelsen (ZN) and then cultured in LJ medium according to the standard bacteriological procedure and in the MGIT as recommended by the manufacturer. Conventional biochemical tests and p-nitro-alpha-acethylamino-beta-hydroxypropiophene of the Bactec system were used to identify the isolated mycobacterial strains. The susceptibilities to streptomycin, isoniazid, rifampicin, ethambutol were tested by the BBL-MGIT antibiotic susceptibility test and the resistances of the strains found to be resistant to any of the drugs were confirmed by the agar proportion method. Mycobacterium spp. were isolated in 61 (9.4%) out of 648 samples. Eventually, 58 out of 61 strains were classified as Mycobacterium tuberculosis and the other 3 as Mycobacterium tuberculosis complex. 32 of these were ZN positive. The growth time was determined as 12.2 days by the MGIT method and 24.1 days by the LJ method (p < 0.001). 29 strains were ZN negative. Their growth time was 23 days by the MGIT method and 37 days by the LJ method (p < 0.001). Drug resistance was detected in 23 (37.7%) of 61 cases (of whom 39 were new and 22 were former patients); of these resistances, 8 (20.51%) were primary and 15 (68.18%) were secondary. In double drug resistance, secondary resistance was found only to isoniazid + rifampin (4 cases) whereas both primary and secondary resistances were found to one drug. The highest cumulative drug resistance - both primary and secondary - was found to isoniazid. In conclusion, the MGIT was found to be advantageous because it enables rapid bacterial identification of tuberculosis and detection of antimicrobial resistance due to its high sensitivity and specificity. It is quicker than the LJ method. Its antibiotic susceptibility can be tested and it is easy to perform. We recommend to include it in routine laboratory work. In addition, our study suggests that the high ratio of secondary resistance in the public might be related to inappropriate and insufficient treatment of tuberculosis, and noncompliance, which appear to cause an important increase in primary tuberculosis as a result of new contaminations.     

微量液体培养硅胶显色法快速检测结核分枝杆菌吡嗪酰胺耐药性

目的建立用24孔微量液体培养硅胶显色板对结核分枝杆菌(MTB)吡嗪酰胺(PZA)药物敏感性判断的方法,并评价该方法临床应用价值。方法以MGIT960药敏结果作为标准对照,应用硅胶显色板对30株已知PZA药敏结果 MTB临床分离株进行PZA药敏检测,观察不同pH值,不同接种浓度对其结果的影响,并对最佳检测条件进行探讨。最后应用硅胶显色板和MGIT960同时对98株未知PZA药敏结果的MTB临床分离株进行检测,判断灵敏度、特异度、准确度。结果 24孔微量液体培养显色板,液体培养基最佳pH值为5.8~5.9,最佳接种菌量为2.5×10-1 mg/mL,7~14d即可报告结果。PZA临界浓度100μg/mg时灵敏度达95.50%,特异度为96.30%,准确度为98.21%;PZA临界浓度200μg/mg时灵敏度均可达90.90%,特异度为92.59%,准确度为91.84%。结论使用24孔微量液体培养硅胶显色板能对MTB的PZA药物敏感性进行快速鉴定,结果准确度高,操作简便,成本低廉。     

Evaluation of BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA): compared with the results of pyrazinamidase assay and Kyokuto PZA test

The fully automated BACTEC MGIT 960 PZA medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide (PZA) was evaluated using 101 Mycobacterium tuberculosis clinical isolates. The results obtained with the system were compared with those of the pyrazinamidase (PZase) assay and the Kyokuto PZA test based on a broth culture, which is commercially available in Japan. The overall concordance rate was 90.1% (91/101) among the three methods in the initial test. The concordance rates between the BACTEC MGIT 960 PZA medium vs the PZase assay, the BACTEC MGIT 960 PZA medium vs the Kyokuto PZA test, and the PZase assay vs the Kyokuto PZA test were 93.1, 91.1, and 96.0%, respectively. On the repeat test of the 10 strains with discrepant results among the three methods, the concordance rates reached over 97% between each of the two systems. The results of the repeat test were confirmed by MIC testing and sequencing analysis of the pncA gene encoding PZase of M. tuberculosis. The mean turnaround times from incubation for PZA susceptibility testing were almost similar for the two methods based on liquid media, the BACTEC MGIT 960 PZA medium and the Kyokuto PZA test (7.7 and 7.4 days, respectively). These results indicate that both methods based on liquid media, the fully automated BACTEC MGIT 960 PZA medium and the Kyokuto PZA test for susceptibility testing to PZA, are useful for rapid diagnosis of PZA resistant tuberculosis.