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。     

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.     

超声分散比浊法处理BACTEC MGIT 960培养阳性的菌液标本用于药物敏感性试验的价值

目的分析应用超声分散比浊法处理的BACTEC MGIT 960(简称"MGIT 960")培养阳性的液体培养物菌液标本用于分枝杆菌药物敏感性试验(简称"药敏试验")的价值。方法搜集2018年1—12月西安市胸科医院经MGIT 960液体分枝杆菌培养阳性的标本,共计1086份。其中,818份菌液标本进行MGIT 960液体药敏试验,包括MGIT 960推荐方法处理的菌液标本100份、超声分散比浊法处理的菌液标本718份;268份菌液标本进行比例法药敏试验,包括采用传统磨菌法处理的菌液标本30份、超声分散比浊法处理的菌液标本238份。与传统菌液标本处理方法比较,分析超声分散比浊法处理后的菌液标本用于MGIT 960液体药敏试验和比例法药敏试验的效果。结果超声分散比浊法处理后的0.6～1.0麦氏单位菌液标本进行MGIT 960液体药敏试验报告结果的时间主要集中在8～10 d,而MGIT 960法处理后的菌液标本报告结果时间则较均匀分布在5～14 d。浊度为0.6、0.7、0.8、0.9、1.0麦氏单位菌液标本系统检测X200(标本活菌量少)报告率[分别为0.00%、0.00%、0.00%、0.72%(1/138)、1.31%(2/153)]均低于MGIT 960菌液处理法[11.00%(11/100)],差异均有统计学意义(χ~2值分别为11.41、9.94、12.43、12.79、11.28,P值均<0.01)。进行二线抗结核药物比例法药敏试验时,增菌6～7 d后,应用超声分散比浊法处理的菌液标本基本可以达到目标浊度,而传统磨菌法处理的标本在增菌第7天时仅有80%达到目标浊度。应用超声分散比浊法处理后的浊度为0.7、0.8、0.9、1.0麦氏单位的菌液标本,药敏试验成功率分别为82.61%(19/23)、82.69%(43/52)、94.00%(47/50)、96.00%(48/50),与传统磨菌法处理的菌液标本药敏试验成功率[83.33%(25/30)]相近,差异均无统计学意义(χ~2值分别为0.01、0.01、2.37、3.77,P值均>0.05)。结论应用超声分散比浊法处理MGIT 960液体培养阳性的菌液标本,可提高MGIT 960液体药敏试验成功率,缩短比例法药敏试验时间。     

应用BACTEC MGIT 960分析临床结核菌株耐药性分析

目的研究我院结核病患者的抗结核药物耐药特点。方法我院就医的结核病患者抗结核药物的敏感性试验结果,分析临床抗结核药物的耐药现状。结果 2008～2010年,耐药菌株为4825株(耐药率为66.68%,4825/7236,MDR为1142株,XDR为105株)。XDR-TB比例2008、2009、2010年分别为1.73%(30/1732)、1.33%(34/2548)、1.39%(41/2956)。结论结核病患者的抗结核药物的耐药形势依然严峻,加强抗结核药物的耐药性监测,合理使用药物非常必要。     

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。     

Differentiation of Mycobacterium tuberculosis from other mycobacteria with rho-nitrobenzoic acid using MGIT960.

SETTING: Mycobacteria growth in media with the addition of inhibitory substances has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. OBJECTIVE: To develop a rapid PNB test using the automated BACTEC MGIT960 system and to evaluate its usefulness in the screening of mycobacterial isolates. DESIGN: PNB tests were performed in 93 MTC strains and 61 NTM strains from the Instituto Adolfo Lutz Culture Collection. PNB was added to L?wenstein-Jensen (LJ) medium and to BACTEC MGIT960 medium. RESULTS: The MTC strains were all PNB-susceptible, confirming the original identification. Among 10 NTM species, all were found to be resistant to PNB, except for one strain of M. kansasii and another of M. marinum. The median time to obtain presumptive identification of MTC by inhibition test in the BACTEC MGIT960 system was 6.3 days and for NTM it was 2.5 days. The presumptive identification of MTC in LJ was mostly obtained after day 20. CONCLUSION: The key finding of this analysis was the possibility of combining the traditionally accepted method proposed by Tsukamura and Tsukamura in 1964 with the modern, safe and rapid BACTEC MGIT960 methodology.     

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。     

Multicentre evaluation of an automated BACTEC 960 system for susceptibility testing of Mycobacterium tuberculosis.

SETTING: Three mycobacteria reference laboratories in the south-eastern part of Brazil. OBJECTIVE: To evaluate the automated Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis. DESIGN: Performance of the automated BACTEC MGIT 960 (M960) system for testing M. tuberculosis susceptibility to streptomycin (SM), isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) was evaluated with 95 clinical isolates and compared to the results of the radiometric BACTEC 460TB (B460) system, the proportion method (PM), and the resistance ratio method (RRM). Judicial susceptibility profiles of 88 isolates were defined based on two or more concordant results among B460, PM and RRM, and used as a reference for comparison with M960 results. RESULTS: Agreement rates between M960 and conventional methods were 95.2% with B460, 96.6% with the PM and 93.4% with the RRM. The lowest agreement rates were obtained for SM with the RRM and for EMB with B460. When comparing M960 with judicial susceptibility profiles, the agreement rate was 97.9%. The agreement rates obtained for INH and RMP were 99.2% and for SM and EMB they were 96.2% and 96.9%, respectively. The mean time to reporting the M960 results was 6.9 days. CONCLUSION: M960 offers great improvements when compared to the proportion and resistance ratio methods and would benefit patient treatment.     

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.     

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。     

Detection of mycobacteria in patients with pulmonary tuberculosis undergoing chemotherapy using MGIT and egg-based solid medium culture systems.

SETTING: National Minami-Yokohama Hospital, Kanagawa, Japan. OBJECTIVE: To compare the performance of a liquid medium system using the Mycobacteria Growth Indicator Tube (MGIT) with that of the conventional Japanese culture system using egg-based Ogawa medium, equivalent to Lowenstein-Jensen medium, in cases with pulmonary tuberculosis on chemotherapy. DESIGN: A single-centre prospective case study of 61 hospitalised patients from 1 May to 31 July 1998 on a standard 6-month regimen of anti-tuberculosis chemotherapy including isoniazid, rifampin, streptomycin or ethambutol, and pyrazinamide. Sputum cultures using both culture systems were performed bi-weekly up to week 16 of treatment, and were further monitored by MGIT alone at the end of chemotherapy and every 6 months after the end of chemotherapy up to 2 years. RESULTS: The detection time by MGIT gradually became longer with the progression of chemotherapy. The recovery rate at weeks 2, 4, 6, 8, and 10 by MGIT were significantly higher (P < 0.05) than on the Ogawa slants. Although one case was microbiologically diagnosed as a relapse, using the more sensitive MGIT system did not increase the relapse rate. CONCLUSION: The gradual prolongation of detection time with the progression of treatment and an attainment of negativity of sputum culture at 4 months after chemotherapy could be a useful intermediate marker to monitor the efficacy of treatment for patients with pulmonary tuberculosis by the MGIT system. Further evaluation is necessary to establish the utility of MGIT in monitoring the treatment process.     

BACTEC960培养阳性530株分枝杆菌的形态学特征探讨

目的探讨分枝杆菌在BACTEC960中的生长特点和涂片抗酸染色形态特征,为菌群鉴定提供初步意见。方法收集福州肺科医院临床标本经BACTEC960培养阳性530例,进行直接涂片抗酸染色、菌群鉴定和药敏试验。结果结核分枝杆菌在MGIT培养管中呈絮状沉淀生长,镜检呈绳索状排列。非结核分枝杆菌（NTM）呈混浊生长,镜检呈散在、团粒状、块状、碎片状和松疏束状等不规则排列。结论结核分枝杆菌在BACTEC960中培养形成有特点的缠结,在显微镜下很容易识别,对鉴定分枝杆菌有初筛作用。     

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。     

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.     

MGIT 960系统行二线抗结核药物敏感性试验的实用性评价

目的 评价全自动BACTEC MGIT 960系统在结核分枝杆菌二线抗结核药物（second-line anti-TB drugs,SLD）药敏试验中的实用性。 方法 本实验纳入患者435例。该纳入患者均通过痰涂片抗酸染色（萋-尼法）镜检,L-J固体和MGIT 960系统液体培养及鉴定,共筛查出阳性菌株212例。212例阳性菌株分别进行一线抗结核药物异烟肼（INH）、利福平（RFP）、链霉素（S）和乙胺丁醇（EMB）的耐药性试验,其中耐药菌株（所有耐药株）118例。以118例耐药株作为SLD药敏试验实验株,应用L-J和MGIT 960两种方法分别进行卷曲霉素（Cm）、卡那霉素（Km）、氧氟沙星（Ofx）和乙硫异烟胺（Eto）的药物敏感性试验,并计算出两方法药敏试验所需的时间和符合率;L-J和MGIT960两种培养方法的比较采用配对设计的卡方检验;两种方法检测结果的一致性用Kappa值检验判别。 结果 MGIT 960系统药敏试验所需时间平均10.0 d（1183/118）,L-J固体培养法所需时间平均28.5 d（3360/118）,两者相差18.5 d。MGIT 960与L-J培养法的SLD Cm、Km、Ofx和Eto敏感及耐药符合率和一致性检验Kappa值分别为88.1%（104/118）和0.520;95.8%（113/118）和0.815;91.5%（108/118）和0.824;71.2%（84/118）和0.375。 结论 MGIT 960系统液体培养法常用SLD药敏试验与金标准（L-J培养）比较除Eto外符合率高、一致性好;该方法检测快速、准确,具有很强的实用性,可作为结核分枝杆菌快速培养和药敏试验的新方法。     

Validity of measuring time to detect growth of M. tbc by BACTEC MGIT960 system for quantitation and prediction of mycobacterial growth

STUDY DESIGN: Time to detect growth of M. tbc by BACTEC MGIT960 system was examined in sputum specimens collected from 114 patients with active pulmonary tuberculosis before and during antituberculosis therapy. By measuring TTD under chemotherapy, we tried to quantify mycobacterial growth and determine the sensitivity of MGIT system. RESULTS: The mean TTD significantly decreased in response to an increment in the range of the quantitation scale for solid media. Moreover, the TTD negatively correlated with colony counts (rho = - 0.636, P < 0.01). When automated monitoring continued until Day 28 after incubation, MGIT system had been capable of detecting 98% of Ogawa-positive specimens. The receiver operating characteristic (ROC) curve was plotted to determine the sensitivity and specificity in MGIT system, indicating the sensitivity of 98.3% corresponding cutoff level for TTD of Day 28. CONCLUSION: Measuring TTD in MGIT system could allow estimating the mycobacterial growth in similarly quantitative manner. The appropriate endpoint of monitoring could be decided as 4 weeks, accurately reflecting an outcome of cultivation with solid media.