自动BACTEC960系统检测结核分枝杆菌药物敏感性的多中心评估分析

地点：巴西东南部3个分枝杆菌参比实验室。目的：评估自动化分枝杆菌生长指示管（MGIT）检测结核分枝杆菌药物敏感性的能力。设计：用自动BECTECMGIT960（M960）对95份结核分枝杆菌临床分离株进行链霉素（SM），异烟肼（INH）、利福平（RMP）、乙胺丁醇（EMB）的敏感性检测，并与BACTEC460（B460）、比例法（PM）、抗性比率法（RRM）的检测结果进行比较，其中88株以BACTEC460、比例法、抗性比率法中的至少两种方法获得一致结果作为最终结果，并与M960的结果相比较。结果：M960与B460的符合率为95．2％，与PM为96．6%,与RRM为93．4％；其中与RRM在SM上符合率最低，与B460在EMB上符合率最低。M960结果与最终结果的符合率为97．9％，INH与RMP的符合率为99．2%,SM与EMB的符合率分别为96．2%和96．9％，M960报告结果的平均时间为6．9d。结论：与比例法和抗性比率法相比，M960显示出了极大的优势，并且有助于患者的治疗。     

BACTEC MIGT960在结核分枝杆菌药物敏感性试验中的临床研究

目的 评价BACTEC MIGT960快速检测8种抗结核药物敏感性效果。方法 收集住院结核病人痰标本分离的结核分枝杆菌菌株,分别采用L-J比例法和MGIT960法进行药物敏感性检测,在完成一线4种药物的药敏检测后,选取对任何一种一线药物(异烟肼、利福平、链霉素和乙胺丁醇)耐药的菌株进行二线药物(卷曲霉素、卡那霉素、氧氟沙星和乙硫异烟胺)敏感性检测。从结果报告时间和符合率等方面进行评价。统计学分析采用kappa检验和t检验,P值为0.05。结果 对212株结核分枝杆菌临床分离株进行了4种一线药物的药物敏感性检测,随后,对耐任何一种一线药物的90株菌进行了4种二线药物的药物敏感性检测,以L-J比例法为金标准,MGIT 960法的准确度和Kappa值分别为:异烟肼96.0%和0.92,利福平99.0%和0.98,链霉素99.0%和0.97,乙胺丁醇96.0%和0.81,卷曲霉素100.0%和1.00,卡那霉素99.0%和0.85,氧氟沙星96.0%和0.88,乙硫异烟胺94.0%和0.73;完成一线/二线药物药敏试验的平均时间,MGIT 960为8.10和10d,L-J法则均为30d,两种方法培养时间的差异有统计学意义(P < 0.05)。结论 MGIT 960法与传统的L-J比例法药敏试验比较,具有符合率较好、检测时间明显缩短的特点,有利于临床及时有效合理化用药。     

BACTEC MGIT 960 system for drug susceptibility testing of Mycobacterium tuberculosis: a study using external quality assessment strains

OBJECTIVE: To evaluate the performance of the BACTEC MGIT 960 system for drug susceptibility testing (MGIT AST) of Mycobacterium tuberculosis to isoniazid, rifampin, streptomycin and ethambutol. DESIGN: Fifty external quality assessment strains of M. tuberculosis provided by the Coordinating Centers of WHO/ IUATLD were tested by BACTEC MGIT 960 system, and the results were compared with the referee results of the WHO/IUATLD Supranational Reference Laboratory Network (SRLN). RESULTS AND CONCLUSION: Overall concordance rates of the results obtained by MGIT AST and the referee results of the SRLN were 97.3% for four first-line drugs. Agreement rates were particularly high for isoniazid, rifampin, and streptomycin (agreement rate of over 97%), but somewhat lower for ethambutol, which relates to a lower sensitivity of MGIT AST. Turnaround times from inoculation to drug susceptibility results ranged from 6 to 13 days for the MGIT AST system with a median time of 7 days; this contrasted with three weeks for the proportion method using Middlebrook 7H10 agar, indicating that MGIT AST system has the potential to consistently meet with the turnaround time guidelines suggested by the Centers for Disease Control and Prevention of the United States. These results demonstrate that the fully automated BACTEC MGIT 960 AST system is useful for the rapid diagnosis of drug resistant tuberculosis.     

Evaluation of the BACTEC MGIT 960 system for drug susceptibility testing of Mycobacterium tuberculosis isolates compared with the proportion method on solid media]

The methods most widely used for susceptibility testing against anti-tuberculosis drug (AST) are the proportion method on L?wenstein-Jensen egg (L-J), Ogawa egg or Middle-brook agar media, and BACTEC TB 460 system. Recently, drug concentrations have been established for AST using the automated BACTEC MGIT 960 system (aMGIT). We have evaluated the BACTEC MGIT 960 SIRE kit for AST of Mycobacterium tuberculosis to isoniazid, rifampin, streptomycin and ethambutol. Also we compared the results with the proportion methods on Middlebrook 7H10 agar (7H10), L-J and Ogawa egg, and the manual MGIT system (mMGIT). Overall concordance rates among aMGIT and the proportion method on 7H10 or Ogawa media were 98.3% and 96.9% for 4 first-line drugs, respectively. Rates were particularly high for isoniazid and rifampin between aMGIT and 7H10 (efficiency of 100%). On the other hand, overall concordance rates among two egg media, L-J and Ogawa were 99.9%. Agreement between aMGIT and mMGIT was high for the AST to isoniazid and rifampin, but lower for the AST to ethambutol (90.9%), which relates to a lower specificity of mMGIT. The mean times to aMGIT and mMGIT results of susceptibility were 7 and 6 days, respectively, contrasted with 3 weeks in 7H10 and 4 weeks in L-J and Ogawa, indicating that both MGIT systems have the potential to consistently meet the turnaround time suggested by Centers for Disease Control and Prevention (CDC) of the United States. These results demonstrate that the fully automated BACTEC MGIT 960 SIRE system for AST is useful for rapid diagnosis of drug resistant tuberculosis.     

利用960系统快速鉴定结核分枝杆菌和非结核分枝杆菌的可行性研究

目的　探讨应用BACTEC MGIT960系统的分枝杆菌生长指示管(Mycobacter Growth Indicator Tube,MGIT)加入对硝基苯甲酸(ρ-Nitro benzoic Acid,PNB)区分结核分枝杆菌复合群(MTBC)与非结核分枝杆菌(NTM)的可行性。方法 用含PNB MGIT检测5株标准分枝杆菌菌株的体外抑菌浓度,并对111株临床分离菌株与传统改良罗氏(Lwenstein-Jensen,L-J)PNB鉴定进行比较。结果 以PNB300μg/ml为界,111株临床分离菌株PNB MGIT法与L-J PNB的菌群鉴定相比较符合率为90.09%;鉴定结果报告平均7.72 d,与L-J PNB法比较有显著性差异,检测时间明显缩短(P<0.001)。结论 应用960系统PNB MGIT法鉴定分枝杆菌菌群的方法快速、准确、实用。     

Rapid radiometric susceptibility testing of Mycobacterium tuberculosis.

A 48-hour radiometric test for determining the drug susceptibility of Mycobacterium tuberculosis has been developed. The test is based on the measurement of 14CO2 produced by the oxidation of formate labeled with carbon-14. The test system uses 5 X 10(7) organisms in 1 ml of Middlebrook 7H9 medium plus albumin-dextrose-catalase enrichment and 1 muCi of [14C]formate. The 14CO2 produced is measured in an ionization chamber at 24-, 48-, and 72-hour intervals, with and without the addition of antituberculous drugs. Isoniazid, streptomycin, rifampin, and ethambutol were each tested at 3 concentrations by the radiometric method and the reference (agar dilution) method. Six standard strains and 21 patient isolates were compared by both methods. Production of 14CO2 was quantitatively decreased in the presence of drugs that inhibit the organism. The radiometric method requires 2 days; the agar dilution, 14 to 21 days.     

218例涂阳肺结核吡嗪酰胺耐药情况分析

目的 了解肺结核患者对吡嗪酰胺的耐药情况,为临床结核病防治提供参考。 方法 采用WHO推荐的BACTEC MGIT-960系统法对218例涂阳肺结核进行吡嗪酰胺耐药性检测分析。 结果 218例涂阳肺结核中耐吡嗪酰胺39例,占17.9%。其中163例初治患者耐吡嗪酰胺23例,占14.1%,55例复治患者耐吡嗪酰胺16例,占29.1%; 91例耐多药(MDR)患者耐吡嗪酰胺36例,占39.6%,127例非MDR患者耐吡嗪酰胺3例,占2.4%。复治患者吡嗪酰胺耐药率明显高于初治患者(P＜0.05),MDR患者吡嗪酰胺耐药率明显高于非MDR患者(P＜0.01)。 结论 MDR肺结核耐吡嗪酰胺率较高,应重视对复治涂阳肺结核,尤其是MDR肺结核的吡嗪酰胺耐药性检测,以便采用更合理有效的方案治疗MDR肺结核。     

Rapid identification of Mycobacterium tuberculosis in BACTEC MGIT960 cultures by in-house loop-medicated isothermal amplification

Definitive diagnosis of tuberculosis (TB) by conventional culture, followed by bacterial identification based on biochemical tests is time-consuming and tedious. Simple loop-mediated isothermal amplification (LAMP) specific for Mycobacterium tuberculosis complex, targeting the M. tuberculosis 16S ribosomal RNA gene, termed TB-LAMP, was evaluated as an alternative for rapid culture confirmation. TB-LAMP was assessed for its ability to detect M. tuberculosis complex in BACTEC MGIT 960-positive cultures. Of the 103 cultures evaluated, 100 were identified to contain M. tuberculosis complex by TB-LAMP and had concordant results with standard biochemical tests of niacin accumulation, nitrate reductase, lack of heat-stable catalase, and susceptibility to para-nitrobenzoic acid. These results indicate that TB-LAMP in combination with BACTEC MGIT 960 is a specific, reliable, and technically feasible method for rapid and accurate identification of M. tuberculosis complex.     

A study on inoculum density and reproducibility of drug susceptibility testing by BACTEC MGIT 960

OBJECTIVE: The BACTEC MGIT 960 drug susceptibility system (MGIT AST) has been recently introduced in Japan. The issue of discordant MGIT results compared with the conventionally used Ogawa method has been raised. It has been speculated that discordant results might be due to MGIT inoculum density since there is no standardization step other than dilution of growth for tubes beyond 2 days after MGIT turns out to be positive. In this study, we examined the reproducibility of the MGIT AST system. MATERIALS AND METHODS: Nineteen sputum specimens from drug-resistant and susceptible pulmonary tuberculosis patients were processed with CCE pretreatment reagent (Japan BCG), inoculated into 3 MGIT tubes, and loaded into the MGIT 960. Inocula for MGIT AST were prepared 1, 3, and 5 days after MGIT tubes became positive. Cultures on day 3 and 5 were diluted 1: 5 with saline. Ten-fold dilutions from each positive culture were plated on Middlebrook 7H11 agar plates for CFU determination. MGIT AST results were compared with those of the conventional proportion method on Ogawa egg and Vite-spectrum (Kyokuto), or Pyrazinamidase (Pzase) assay and Kyokuto PZA test. RESULTS AND CONCLUSION: A total of 15 specimens were culture positive in all 3 tubes. Four of 19 cases were removed from the analysis because of negative cultures in one or more tubes. Three of 4 culture negative cases were MDR-TB. Colony counting showed the mean CFU/ml of inocula prepared from tubes 1, 3, and 5 days after MGIT tube became positive were 3.6 x 10(6), 1.6 x 10(6), 3.1 x 10(6), respectively. There was no significant difference although the CFU range was wide (8 x 10(4)-2 x 10(7)). MGIT AST results were consistent among 3 inocula. Moreover, overall concordance rates between MGIT AST and the conventional methods were over 90% for 5 first-line antituberculosis drugs. These results indicate that the BACTEC MGIT 960 system is very useful for rapid diagnosis of drug resistant tuberculosis.     

Comparative evaluation of the BACTEC MGIT 960 system with solid medium for isolation of mycobacteria.

SETTING: The utilisation of new, rapid methods of diagnosis of tuberculosis is currently of great interest for tuberculosis control. This study was carried out in a teaching hospital in the eastern region of Taiwan. OBJECTIVE: The BACTEC MGIT 960 system was evaluated and compared with Lowenstein-Jensen (LJ) medium and Middlebrook 7H11 plate for recovery rate and time to detection of mycobacteria. DESIGN: A total of 1396 sputum samples were tested for the presence of mycobacteria. Specimens were processed and inoculated separately in the BACTEC MGIT 960 system, on LJ medium and 7H11 for comparative study. RESULTS: The BACTEC MGIT 960 detected 235 isolates (100%), followed by LJ with 205 isolates (87.2%) and 7H11 with 178 isolates (75.7%). The mean time to detection of Mycobacterium tuberculosis complex was 11.6 days with MGIT 960, 20.1 days with LJ, and 18.7 days with 7H11. The contamination rates were 15.1% with MGIT 960, 10.1% with LJ and 9.7% with 7H11. CONCLUSION: The BACTEC MGIT 960 system is a sensitive, rapid mycobacterial culturing system. However, the high contamination rate is a concern that should be carefully evaluated in the clinical setting.     

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.     

External quality assessment of drug susceptibility testing for Mycobacterium tuberculosis

OBJECTIVE: The Committee for Mycobacterial Examinations has planned and implemented the third external quality assessment of drug susceptibility testing for Mycobacterium tuberculosis to the hospital and private laboratories. METHOD: The Committee delivered 20 M. tuberculosis strains, exactly pairs of 10 strains, which were evaluated and standardized for the drug resistance pattern in the WHO/ IUATLD supra-national laboratory network (SRLN). The agreement of the majority of SRLN laboratories was considered as the gold standard of susceptibility result for each strain tested. Each laboratory performed the drug susceptibility testing (DST) with its own routine method. The sensitivity to detect drug resistance, the specificity for susceptible strain, the efficiency of overall agreement, the reproducibility for each pair, and kappa coefficient were calculated to evaluate their performance. DST was performed for isoniazid (INH), rifampicin (RFP), streptomycin (SM) and ethambutol (EB). RESULTS: A total of 48 results has been collected. The overall sensitivity, specificity, efficiency, reproducibility and kappa coefficient for each anti-tuberculosis drug tested were as follows respectively; 100%, 99.0% (62.5-100), 99.6% (85.0-100), 99.6% (90-100) and 0.991 for INH; 97.7% (83.3-100), 100%, 98.6% (90-100), 99.0% (90-100) and 0.972 for RFP; 87.5% (66.7-100), 99.0% (87.5-100), 92.1% (80-100), 97.5% (70-100) and 0.84 for SM; 99.5% (75.0-100), 97.9% (75.0-100), 98.5% (85.0-100), 97.9% (70-100) and 0.97 for EB. Regarding private laboratories, all indicators showed 100% for INH but 2 hospital laboratories showed less than 90% in sensitivity. As for RFP, one private laboratory (4.3%) and 3 hospital laboratories showed less than 90% in sensitivity. The major difference between private and hospital laboratories was seen in EB. One hospital laboratory (4.0%) showed less than 90% in sensitivity and three (12.0%) showed less than 90% in specificity, compared to none in private laboratories. Additionally, six hospital laboratories (24.0%) showed less than 90% in reproducibility. As for quality improvement, two private laboratories that showed poor performance in 2003 have improved their quality up to 100% in 2004. DISCUSSION: The overall efficiency by private and hospital laboratories satisfied WHO criteria. However, it diverged in each category of laboratories and hospital laboratories tended to show poor performance compared to the private ones. The reason for the difference was not clear, but the routine workload, allocated time and cost for the panel testing might contribute to it. The sensitivity of SM was relatively low compared to the other drugs as it was observed in 2003. It was mainly due to two strains to which the participating laboratories showed poor agreement to the gold standard. The difference of critical drug concentration for SM in L?wenstein-Jensen and in 1% Ogawa medium might contribute to the discrepancies. As for quality improvement, two private laboratories with poor performance in 2003 have shown marked improvement after on-site evaluation. The results indicated the usefulness of external quality assessment for the maintenance and improvement of the quality of the test.     

110株结核分支杆菌对喹喏酮类药物的药敏试验分析

目的　分析110株结核分支杆菌对环丙沙星(CPLX)、氧氟沙星(OFLX)、左氧氟沙星(LVFX)、司帕沙星(SPFX)的药敏试验结果。方法 采用2倍稀释法,改良罗氏培养基进行药敏试验。结果 49例初治病人耐药率:CPLX4%,OFLX8%,LVFX2%,SPFX6%;52例耐多药菌株耐药率:CPLX52%,OFLX44%,LVFX22%,SPFX10%;110株结核分支杆菌总耐药率:CPLX26.2%,OFLX24.7%,LVFX10.5%,SPFX7.2%。交叉耐药率23.6%。结论 (1)初治结核病人,抗炎治疗尽量避免使用喹喏酮类药物;(2)复治结核病人要选择合理治疗方案,避免假联合用药;(3)应用喹喏酮类药物,剂量要足,疗程要够;(4)建议将喹喏酮类药列入结核分枝杆菌常规药敏试验中。     

结核分枝杆菌对氨基甙和喹诺酮类药物耐药性调查

目的分析了解近年来结核病患者对氨基甙和喹诺酮类药物的耐药情况。方法采用绝对浓度间接法进行了三种常用抗结核药物的敏感试验。结果每种抗结核药物的年耐药发生率呈波浪式变化,但是总的发生率却是呈现逐步上升趋势。结论目前对于丁胺卡那霉素,卷曲霉素,左氧氟沙星这类药物的耐药率仍处于较低水平,我们在临床工作中必须规范用药,以避免或减少广泛耐多药的产生。     

Rapid and simple MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis.

The MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis was developed by using bacterial suspension prepared from colonies on solid media. The MTT tube assay in 1 ml Middlebrook 7H9 broth was completed within 4 days for rifampicin (RMP) and within 7 days for isoniazid (INH). When MTT assay results with 279 M. tuberculosis clinical isolates were compared with those of the conventional proportion method on L?wenstein-Jensen medium, high specificity and sensitivity values of 100% and 94.1%, respectively, for RMP susceptibility testing, and 99.5% and 89.2%, respectively, for INH susceptibility testing were obtained. The accuracy of the MTT method for RMP and INH was > 0.97 concordance with the proportion method. The MTT method is simple, inexpensive and rapid. The high level of agreement with the conventional proportion method suggests a potential to rapidly detect drug-resistant M. tuberculosis in developing countries, as only basic microbiological equipment is need.     

Comparative study of bactec MGIT 960 AST and conventional proportion method using Ogawa medium for the drug susceptibility testing of Mycobacterium tuberculosis to isoniazid

OBJECTIVE: To evaluate the accuracy of drug susceptibility testing to isoniazid with BACTEC MGIT 960 (MGIT AST) comparing with the standard proportion method using Ogawa medium. METHOD: A total of 1109 M. tuberculosis strains, which were selected from the collection of RYOKEN drug resistance survey in 2002, were selected and subjected to the susceptibility testing to isoniazid using MGIT AST and 1% Ogawa standard methods. The results from MGIT AST were compared with the judicial diagnosis by Ogawa. The sensitivity to detect drug resistance, the specificity for susceptible strain, the efficiency of overall agreement, and kappa coefficient were calculated to evaluate the performance. The treatment process, outcome and prognosis were analysed for the patients on whom the tests showed discrepant results. RESULTS: Compared with the judicial results, the sensitivity, specificity, efficiency, and kappa coefficient of MGIT AST were 100%, 97.1%, 97.3%, and 0.798, respectively. The strains, which showed discrepant results between MGIT AST and Ogawa, were all susceptible by Ogawa and resistant by MGIT AST. A total of 11 out of 30 discrepant cases were followed clinically and no relapse cases were identified, irrespective of the modification of the treatment regimen. As for the proportion of primary INH drug resistance in the present study, it was 5.3% with MGIT AST but was 2.7% with Ogawa, and the difference was statistically significant (p = 0.005). DISCUSSION: The discrepancies on the results of drug susceptibility testing of M. tuberculosis strains to isoniazid between MGIT AST and 1% Ogawa proportion method have been reported. In the present study, the sensitivity, specificity, and overall efficiency of MGIT AST on the prevalent strains in Japan were all beyond 95%, and considered sufficient as the anti-tuberculosis drug susceptibility testing (AST), though 2.7% of discrepancy was observed. Even for the discrepant cases, there was no difference in the treatment outcome and prognosis. Thus, MGIT AST was confirmed as a reliable AST method comparable to Ogawa standard. However, MGIT AST might increase the proportion of INH resistance if it was used as a major AST method, compared with Ogawa.     

BACTEC MGIT960系统快速培养分枝杆菌的效果评价

目的与传统罗氏培养法相比较,评价BACTECMGr／960快速培养系统在基层结控实验室的应用效果及前景。方法采用改良罗氏培养基、BACTECMGIT960液体培养基对521份临床痰标本进行平行接种培养,比较两者的阳性率、培阳时间、污染率。结果传统罗氏培养法的阳性率为47．1％（81／172）,BACTECMGIT960系统的阳性率为68．0％（117／172）;传统罗氏培养法最早培阳时间为12d,平均26d,BACTECMGr1960快培系统最早培阳时间为4d,平均12d;传统罗氏培养法污染率为2．5％（13／521）,BACTECMGr1960快培系统的污染率为3．5％（18／521）;结论BACTECMGIT960系统可以提高分枝杆菌的培养阳性率和明显缩短培养时间,污染率与传统罗氏培养相较无显著性差异,是一种快速,有效的检测方法,对基层结控机构早期发现肺结核病人和疗效观察将发挥重要作用。