Top PDF Multicenter Evaluation of Bactec MGIT 960 System for Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Complex

Multicenter Evaluation of Bactec MGIT 960 System for Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Complex

Multicenter Evaluation of Bactec MGIT 960 System for Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Complex

As a routine practice, only primary drugs are tested initially. When strains test resistant to rifampin or any two of the pri- mary drugs, second-line drugs are then tested. When resistance to primary drugs is detected, the growth on LJ slants is usually adequate for the testing of second-line drugs. In this study, we used the growth from LJ slants as the main source for the inoculum preparation. With advances in molecular technolo- gies, isoniazid and/or rifampin resistance can be detected by rapid methods, such as those that use molecular beacons, line probes, or microchips (2, 3, 6, 7, 11, 13). Once rifampin resis- tance or MDR TB is detected, testing for susceptibility to second-line drugs should be performed along with testing for susceptibility to first-line drugs as early as possible, and testing should not wait for growth from LJ slants. Therefore, we in- cluded a subset of 23 specimens for validation of this assay using inocula prepared from positive MGIT tubes. Our proto- col prepares inocula from MGIT tubes by standardization of the growth to a 0.5 McFarland standard, as is done for LJ slants. DST can be performed from a primary MGIT tube and not subject to the manufacturer’s time schedule, as stated in the package insert for SIRE DST. This has two advantages: the inoculum does not contain coarse clumps, which helps to pro- duce more consistent results, and it does not require seeding of a MGIT tube and waiting for its growth for DST, thus improv- ing the TAT and reducing the cost for medium.
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Multicenter Evaluation of the BACTEC MGIT 960 System for Recovery of Mycobacteria

Multicenter Evaluation of the BACTEC MGIT 960 System for Recovery of Mycobacteria

The increasing incidence of tuberculosis and other mycobac- terial diseases has made it essential for laboratories to quickly detect and identify mycobacteria from human clinical material. When conventional culture media are used, as many as several weeks of incubation and substantial technical labor may be necessary for the recovery of organisms. Since it was first in- troduced, the BACTEC 460 TB system (Becton Dickinson Microbiology Systems, Sparks, Md.) has been the benchmark for rapid detection of Mycobacterium tuberculosis complex (8, 9). In recent years, however, a number of new systems which provide similar times to detection, with fully automated instru- ments or without the need for any instrumentation, have been developed. The BACTEC MGIT 960 system is a fully auto- mated, high capacity, nonradiometric, noninvasive instrument which requires neither needles nor other sharp implements to simultaneously incubate and monitor 960 7-ml culture tubes. To monitor microbial growth, the BACTEC MGIT 960 uses the same oxygen-quenching fluorescent sensor technology as both the manual Mycobacteria Growth Indicator Tube (BBL MGIT) and the BACTEC 9000MB system, in conjunction with unique on-board algorithms to determine the positivity of the culture tubes. This multicenter study evaluated the perfor-
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Comparison of line probe assay to BACTEC MGIT 960 system for susceptibility testing of first and second line anti tuberculosis drugs in a referral laboratory in South Africa

Comparison of line probe assay to BACTEC MGIT 960 system for susceptibility testing of first and second line anti tuberculosis drugs in a referral laboratory in South Africa

This was a prospective descriptive study comparing the performance of the LPA (Hain Lifescience, Nehren, Germany) to BACTEC MGIT 960 system (Becton Dickin- son Microbiology System, Sparks, MD, USA) for suscepti- bility testing of first and second-line anti-TB drugs. All testing was done in a routine diagnostic referral labora- tory. We collected 100 consecutive non-repetitive M. tu- berculosis cultures that were resistant to either isoniazid or rifampicin or both. Resistance was confirmed using the MGIT 960 system (Fig. 1). All samples were supplied by the National Health Laboratory Services, Tshwane Aca- demic Division TB laboratory (NHLS/TAD) in the De- partment of Medical Microbiology University of Pretoria, South Africa. The NHLS/TAD is a high throughput diag- nostic laboratory that receives specimens for microscopy, culture and drug susceptibility testing from surrounding Gauteng Province clinics and hospitals and other referring provinces such as Limpopo and Mpumalanga. The cul- tures were drawn from specimens of patients who were seeking care at primary facilities. The cultures used in the study were collected from January to June 2014. Agar pro- portion method was conducted at the South African Med- ical Research Council (SAMRC), Pretoria, TB Platform Unit, which is a Bio-safety level 2 laboratory. The HIV sta- tus of patients was not collected.
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Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation

Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation

Existing procedures for DST of mycobacteria are adequate for screening but require complementation with quantitative DST measures, in particular for those drugs where heteroge- neity in phenotypic resistance is present. We have established the conditions for quantitative DST using the MGIT 960 sys- tem in combination with EpiCenter software equipped with the TBeXiST module, thus providing a fully automated walk-away system for quantitative DST of M. tuberculosis. This platform allows electronic data management and is compatible with expert systems for interpretation. The MGIT 960 platform in conjunction with the EpiCenter software shows high consis- tency with Bactec 460 test results over a wide range of con- centrations tested for first- and second-line anti-TB drugs. While we note that further studies are needed to address the correlation of phenotypic resistance levels and treatment out- come, we have summarized our recommendations for quanti- tative DST of M. tuberculosis in Table 5. Widespread imple- mentation of MGIT 960 protocols for quantitative DST should provide standardized data to enable the correlation of results from quantitative DST with clinical outcomes by high-through- put statistical analysis in order to address the issue of pheno- typic drug resistance levels and treatment failure. In addition, data sets obtained by an automated standardized procedure based on agreed guidelines provide optimal input for monitor- ing the epidemiology of resistance at a supranational level.
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Multicenter Laboratory Validation of the BACTEC MGIT 960 Technique for Testing Susceptibilities of Mycobacterium tuberculosis to Classical Second Line Drugs and Newer Antimicrobials

Multicenter Laboratory Validation of the BACTEC MGIT 960 Technique for Testing Susceptibilities of Mycobacterium tuberculosis to Classical Second Line Drugs and Newer Antimicrobials

Phase II data clearly indicated the working critical concen- trations of the test drugs in MGIT 960, which would yield results equivalent to those in BACTEC 460 (Table 6). AK at 1.0 ␮ g/ml yielded satisfactory results for both methods. CM at 1.25 ␮ g/ml in BACTEC 460 compared best with 2.5 ␮ g/ml in MGIT 960, and ETH at 2.5 ␮ g/ml in BACTEC 460 compared best with 5.0 ␮ g/ml in MGIT 960. With PTH, the inhibitory concentration was lower than that of ETH, but MGIT 960 required a higher concentration (2.5 ␮ g/ml) than BACTEC 460 (1.25 ␮ g/ml). Critical concentrations were found to be the same for OFX (2.0 ␮ g/ml), RIF (0.5 ␮ g/ml), and LIN (1.0 ␮ g/ml). It was found that the majority of the differences in the DST results among the test sites were related to the presence of a low level of resistance for a particular drug, as the results indicated when the drug was tested at a level lower than the critical concentrations. Applying the tentative critical concen- trations, among 441 tests and three sites in phase II, there were only 3 (0.7%) false-susceptible and 15 (3.4%) false-resistant results with MGIT 960. The highest discrepancies were seen with ETH and PTH. The data also indicate high interlabora- tory reproducibility of MGIT 960 DST for these drugs.
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Evaluation of the Fully Automated BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide, Streptomycin, Isoniazid, Rifampin, and Ethambutol and Comparison with the Radiometric BACTEC 460TB Method

Evaluation of the Fully Automated BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide, Streptomycin, Isoniazid, Rifampin, and Ethambutol and Comparison with the Radiometric BACTEC 460TB Method

The objective of a laboratory’s QC program is to evaluate the precision and accuracy of test procedures, monitor reagent performance, and evaluate the proficiency of personnel per- forming tests. This is particularly important because the clini- cal mycobacteriology laboratory is responsible for providing accurate and reliable information that is necessary for man- agement of the patient’s therapy. A critical element of QC is the selection and use of reference strains that are genetically stable and for which susceptible or resistant results are well documented. We have used the fully susceptible M. tuberculosis H37Rv (ATCC 27294) strain for quality control of susceptibil- ity testing, as recommended by NCCLS. Furthermore, when testing both the critical concentration and a higher concentra- tion of a drug, a strain of M. tuberculosis that consistently demonstrates resistance to the low concentration but is sus- ceptible to the higher concentration should be an ideal refer- ence strain for quality control. However, at present, reference strains that perform optimally for this purpose are not avail- able. Alternatively, in-house isolates with the same charac- teristics may be used for QC programs, but for safety con- siderations, the use of multiple-drug-resistant strains is not recommended (6). In the absence of these possibilities, for QC testing we have used strains of M. tuberculosis (American Type Culture Collection strains) that are resistant to INH, RMP, ETB, and PZA; however, these strains are resistant to high concentrations of the respective drugs and are not ideal for QC
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Rapid Detection of Mycobacterium tuberculosis Resistance to Second Line Drugs by Use of the Manual Mycobacterium Growth Indicator Tube System

Rapid Detection of Mycobacterium tuberculosis Resistance to Second Line Drugs by Use of the Manual Mycobacterium Growth Indicator Tube System

The emergence of multidrug-resistant tuberculosis (MDR- TB) caused by Mycobacterium tuberculosis and, recently, exten- sively drug-resistant tuberculosis caused by an MDR strain that is also resistant to any fluoroquinolone and at least one of the three injectable second-line drugs (kanamycin [KAN], amika- cin [AK], and/or capreomycin [CM]) is a real threat for TB control programs (28). It is obvious that there is a great ne- cessity for rapid, reliable, and economical methods for testing the susceptibility of M. tuberculosis not only to first-line drugs but also to second-line drugs. Access to drug susceptibility testing (DST) is a priority, and TB culture is an essential component of TB management. Using the standardized con- ventional DST methods, it takes a minimum of 3 to 8 weeks to identify resistant or susceptible strains on solid media (6, 7). The introduction of liquid culture media such as the manual mycobacterium growth indicator tube (MGIT) reduces the turnaround time compared to that of solid media, taking an average of 15 days to get results (1, 5, 19, 23, 25). In June 2007, the World Health Organization issued a recommendation for the use of liquid media for culture and DST in middle- and low-income countries to address challenges due to the epi- demic of human immunodeficiency virus-associated TB and drug-resistant TB, especially in resource-limited settings (29). Fully automated commercial systems such as the BACTEC MGIT 960 (Becton Dickinson) have shown their usefulness for the rapid detection of resistance to second-line drugs (12, 24);
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Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

imens are needed. Some of the noncommercially available direct tests include the nitrate reductase assay (NRA) and microscopic observation drug susceptibility (MODS) assay. These tests have been developed as “in house” assays with the aim to overcome the high costs of the commercially available techniques. In NRA, the addition of the NRA reagent requires tubes to be regularly opened, which poses significant risk of aerosol generation. Reading of the MODS plates has to be performed on a daily basis and is laborious and time-consuming (34). Commercially available molecular as- says, such as Genotype MTBDR Plus (Hain Lifescience, Nehren, Germany), can be applied directly to smear-positive specimens and have less turnaround time, thus saving several weeks. How- ever, none of the established molecular tests target all possible genes involved in resistance, and thus a variable proportion of resistant strains may not be detected (17, 33). Liquid culture has been established as a gold standard and is most rapid for pheno- typic DST. This was a research study designed to establish time savings with the direct DST approach compared to the routine indirect approach in liquid medium, and thus molecular testing was not included in the study.
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Evaluation of Methods for Testing the Susceptibility of Clinical Mycobacterium tuberculosis Isolates to Pyrazinamide

Evaluation of Methods for Testing the Susceptibility of Clinical Mycobacterium tuberculosis Isolates to Pyrazinamide

P yrazinamide (PZA) is a first-line antituberculosis (anti-TB) drug. It is often used in combination with isoniazid, ethambutol, and rifampin for the treatment of TB (1). PZA can shorten TB treatment time from the 9 to 12 months required prior to its introduction to the current standard treatment time of 6 months. This is often referred to as short-course chemotherapy (2). PZA is more effective against non- replicating, persistent Mycobacterium tuberculosis than any other conventional anti-TB drug (3). However, reliable testing of the sus- ceptibility of M. tuberculosis to PZA in vitro is challenging since PZA has no apparent effect on actively growing TB bacilli under normal culture conditions at neutral pH. PZA is effective against M. tubercu- losis only at an acidic pH, and acidic media inhibit the growth of M. tuberculosis (4, 5). The Bactec 460 and MGIT 960 are the only two methods recommended by the World Health Organization (WHO) for susceptibility testing ofM. tuberculosis to PZA, due to the difficulty in standardizing its susceptibility to PZA on solid media (6). Drug susceptibility testing in liquid media is costly, especially in some re- gions that do not have enough economic capabilities (6). Several other drug susceptibility-testing methods have been developed, in- cluding the molecular drug susceptibility test (mDST) based on the detection of a pncA mutation, the pyrazinamidase (PZase) activity assay, and colorimetric methods based on a minimal inhibitory con- centration (MIC) or redox indicator (7, 8, 9, 10, 11, 12). In this study, we used 432 clinical M. tuberculosis isolates to compare five methods for determination of the susceptibility of M. tuberculosis to PZA: the MGIT 960 system, the mDST, the PZase activity assay, and two colorimetric methods [the resazurin microtiter assay (REMA) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test].
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Evaluation of MGIT 960 Based Antimicrobial Testing and Determination of Critical Concentrations of First  and Second Line Antimicrobial Drugs with Drug Resistant Clinical Strains of Mycobacterium tuberculosis

Evaluation of MGIT 960 Based Antimicrobial Testing and Determination of Critical Concentrations of First and Second Line Antimicrobial Drugs with Drug Resistant Clinical Strains of Mycobacterium tuberculosis

Reagents and drug concentrations. (i) MGIT 960. Lyophilized drugs (BACTEC MGIT 960 SIRE kit, MGIT 960 STR4.0 kit, MGIT 960 INH0.4 kit, MGIT 960 EMB7.5 kit, and MGIT 960 PZA kit; Becton Dickinson, Baltimore, MD) were dissolved in diluent according to the manufacturer’s instructions. From the dissolved drug solutions, 100 ␮ l was pipetted into a 7-ml MGIT 960 tube. All drugs used in phase II were obtained from the manufacturers in a chemically pure form. The drugs used were AMI from Bristol-Myers Squibb, Syracuse, N.Y., dissolved in deionized (DI) water; CAP sulfate from Sigma, St. Louis, Mo., dissolved in DI water; OFL from Ortho/R. W. Johnson Pharmaceutical, Raritan, N.J., dissolved in 1/10 N NaOH solution; CIP from Miles, West Haven, Conn., dissolved in 1/10 N NaOH solution; MOX, generously provided by Bayer Health- Care AG, Leverkusen, Germany, dissolved in 1/10 N NaOH solution; PRO, a gift from Fatol Arzneimittel GmbH, Schiffweiler, Germany, dissolved in ethylene glycol; and RIFB from Pharmacia, Spa, Italy, dissolved in methanol. The subse- quent dilutions for all drugs were made in DI water, and aliquots of the stock solution were stored at ⫺ 70°C for 6 months. The final drug concentrations used were 1.0 and 4.0 ␮g/ml for STR; 0.1 and 0.4 ␮g/ml for INH; 5.0 and 7.5 ␮g/ml for EMB; 1.0 ␮ g/ml for RIF; 100 ␮ g/ml for PZA; 1.0 ␮ g/ml for AMI; 1.25 ␮ g/ml for
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Canadian Multicenter Laboratory Study for Standardized Second Line Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis

Canadian Multicenter Laboratory Study for Standardized Second Line Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis

Currently it is recommended that after systematic testing against first-line anti-TB agents, isolates that are found to be monoresistant to rifampin or to demonstrate resistance to any two of the first-line antimicrobials be tested against a panel of second- line antimicrobials (3, 7, 21). Though the majority of resistant isolates in Canada show monoresistance to isoniazid (INH), re- cently published Clinical and Laboratory Standards Institute (CLSI) document M24-A2 recommends that second-line antimi- crobial testing also be performed on isolates that are INH monoresistant in cases where fluoroquinolones may be added to the therapy. The current standard method for first-line antimi- crobial sensitivity testing (AST) of M. tuberculosis in Canada is the Bactec MGIT 960 (M960) system (BD, Sparks, MD). Although the M960 system has been approved for first-line AST (2–4, 20), the Bactec 460 (B460; BD, Sparks, MD) and the agar proportion method are currently used for second-line AST since the M960 system had not been validated for this purpose (3, 8). Due to the
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Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis

Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis

With three million deaths and ten million people infected each year, tuberculosis is still the infectious disease with the highest morbidity and mortality. In addition, multidrug-resis- tant Mycobacterium tuberculosis strains have been emerging worldwide in both high- and low-income countries. The need for rapid methods of diagnosis and determination of drug susceptibility is particularly important. The Centers for Dis- ease Control and Prevention in Atlanta, Ga., recommend that susceptibility test results for M. tuberculosis complex isolates be available 28 to 30 days from receipt of a specimen in the laboratory (16). The most widely used methods for antimyco- bacterial susceptibility testing are the proportion method on solid medium and the radiometric procedure on liquid broth. The former procedure cannot provide results before 21 days of inoculation. The radiometric BACTEC 460TB requires less than 10 days of incubation before results are available (13). The BACTEC 460TB system, however, is semiautomated and entails disposal of a radioactive substance.
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Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa

All MDR-TB isolates were sub-cultured on Middlebrook medium prior to testing. Drug susceptibility testing of MDR-TB isolates using the agar proportion method was done for two first-line (EMB and STR) and two second- line drugs (KAN and OFX). The agar proportion method was performed on Middlebrook 7H11 medium accord- ing to the Clinical and Laboratory Standards Institute (CLSI) procedures and recommended critical concentra- tions (Table 3) [38]. Briefly, six-welled Petri plates of Middlebrook7H11 medium (TB Diagnostic Services, South Africa) was used. Two quadrants in each plate contained drug-free medium, one was used as the pro- portional control and the other was used as a quality control. Fully susceptible M. tuberculosis H37Rv refer- ence strain and a known MDR M. tuberculosis isolate were used as quality controls. The other four quadrants contained the drug concentrations (Table 3). Each quad- rant was inoculated with a standard inoculum of 0.1 ml of mycobacterial suspension and the inoculum was dis- tributed by tilting the plate. An aliquot of 0.1 ml of the 1:100 dilutions was used to inoculate the proportional control. The plates were sealed in a plastic bag and incu- bated at 37 °C. The plates were examined 7, 10, 14 and 21 days of incubation. An isolate was classified as resist- ant when the colonies on the drug-containing quadrant were more than 1% compared to the colonies present on the drug-free control quadrant. An XDR-TB was defined as MDR-TB with additional resistance to KAN and OFX.
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Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) Automated System for Drug Susceptibility Testing ofMycobacterium tuberculosis

Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) Automated System for Drug Susceptibility Testing ofMycobacterium tuberculosis

Drug-resistant Mycobacterium tuberculosis strains represent a serious public health problem. Resistance to the four primary drugs, streptomycin (SM), isoniazid (INH), rifampin (RMP), and ethambutol (EMB) (a combination known as SIRE), makes tuberculosis difficult to treat (19). Multidrug-resistant strains have emerged within the last decade, and the rapid detection of these isolates is critical for the effective treatment of patients (28). As recommended by the National MDR TB Task Force, to combat multidrug-resistant tuberculosis (7), antimicrobial susceptibility testing (AST) must be performed on all initial and follow-up M. tuberculosis isolates from each patient. Among the methods used for drug susceptibility test- ing, the agar proportion method (MOP) is universally accepted as the “gold standard” (18, 33). However, it requires a long time to report (generally 21 days after the test is set up). Since 1980 the BACTEC 460 TB radiometric system (Becton Dick- inson Diagnostic Instruments, Sparks, Md.), which is based on the modified version of the proportion method (26), has been introduced to perform AST. The BACTEC 460 TB method provides results within 5 to 6 days, with a significant time savings. Several studies (25) have demonstrated that AST re- sults obtained by BACTEC 460 TB were comparable with those of MOP, thus suggesting that the former method could be adopted for routine laboratory purposes. In 1995, the My- cobacteria Growth Indicator Tube (MGIT, 4 ml) (Becton
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Multicenter Laboratory Evaluation of the MB/BacT Mycobacterium Detection System and the BACTEC MGIT 960 System in Comparison with the BACTEC 460TB System for Susceptibility Testing of Mycobacterium tuberculosis

Multicenter Laboratory Evaluation of the MB/BacT Mycobacterium Detection System and the BACTEC MGIT 960 System in Comparison with the BACTEC 460TB System for Susceptibility Testing of Mycobacterium tuberculosis

The MB/BacT system presented one VME (false suscepti- bility) for isoniazid. The MIC for this strain was 1 ␮ g/ml, and the molecular characterization of codon 315 in the katG gene and the mabA-inhA regulatory region showed wild-type se- quences. False susceptibility results represent a serious draw- back, as they can lead to the failure of anti-TB chemotherapy (14). False susceptibility to isoniazid as determined using the MB/BacT system in comparison with the BACTEC 460TB method has been reported previously (6, 23). However, this finding is infrequent in the literature. The two ME (false re- sistance) observed label as ineffective a very important drug that could be successfully used. This fact, although considered a less serious problem than false susceptibility results (14), considerably complicates the tuberculosis treatment.
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Multicenter Evaluation of Fully Automated BACTEC Mycobacteria Growth Indicator Tube 960 System for Susceptibility Testing of Mycobacterium tuberculosis

Multicenter Evaluation of Fully Automated BACTEC Mycobacteria Growth Indicator Tube 960 System for Susceptibility Testing of Mycobacterium tuberculosis

Among the primary drugs, EMB is considered a difficult drug to be tested that often yields less reproducible results. For the BACTEC 460 TB, Roberts et al. (14) observed a sensitivity value that did not exceed 66%, when it was compared with the proportion method. In 1994, a quality assurance program for drug susceptibility testing of M. tuberculosis was initiated by the World Health Organization in 16 laboratories across the world. The specificity values of EMB (mean, 98%) were significantly higher than its sensitivity values (mean, 66% [9]). As a conse- quence, the sensitivity of EMB leads to underreporting of drug resistance. With the MB/BacT System, Brunello and Fontana (4) found five ME of 120 strains and Diaz-Infantes et al. (5) found five ME of 83 strains in EMB testing. Ru¨sch-Gerdes et al. (15) found four ME with the manual MGIT. In our study, resolved results showed only three ME with the critical con- centration and two ME at the high concentration of EMB in the BACTEC MGIT 960 system. A specificity of almost 97% at the critical concentration with 100% sensitivity indicates that EMB testing in the BACTEC MGIT 960 system is very reli- able.
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Current Perspectives on Drug Susceptibility Testing of Mycobacterium tuberculosis Complex: the Automated Nonradiometric Systems

Current Perspectives on Drug Susceptibility Testing of Mycobacterium tuberculosis Complex: the Automated Nonradiometric Systems

method required longer times to report, new broth-based methods with a shortened incubation time were introduced into clinical laboratories to fulfill CDC recommendations. The B460 system is currently regarded as an excellent method able to provide rapid and reliable results of susceptibility to first- line drugs. This half-automated system, however, lacks com- puterized data management and presents some well-recog- nized limitations, among which the growing expense related to the radioactive waste to be disposed of and a potential risk of cross-contamination due to the invasive reading represent the most relevant. Recently, some new liquid medium-based sys- tems have been commercially introduced as alternatives to the radiometric system and are being evaluated for MTC suscep- tibility testing. Primary antituberculous drugs currently recom- mended by the Clinical and Laboratory Standards Institute (CLSI) (formerly NCCLS) for liquid systems include INH (two concentrations) and single concentrations of RMP, EMB, and PZA (27). Today, despite a pressing demand for susceptibility testing in order to limit the expansion of drug-resistant TB, a full correlation between results from the newly introduced NRM methods and patients’ outcome still remains to be fully demonstrated (17). In fact, studies reported herewith clearly showed that the discriminatory power between resistant and susceptible strains was more reliable for INH and RMP than for other drugs. For this reason, it is recommended to deter- mine in vitro criteria, which could be used to predict clinical resistance and susceptibility with acceptable accuracy, by test- ing a well-defined and soundly representative number of clin- ical isolates. In this context, it must be pointed out that most of the discrepant results observed with MGIT 960 and MB/BacT methods are related to strains with a low level of resistance that are difficult to classify as being composed of different mycobacterial subpopulations. A better concordance of results could be achieved by adjusting the drug concentrations accord- ing to those used in the conventional AP method, provided that this method can still represent the “gold standard” in the presence of MTC strains showing borderline susceptibility re- sults. In addition, systematic use of double concentrations (low and high) for SM, INH, and EMB seems to be a useful skill to drastically reduce ME. Similarly, procedural test complexity (especially for the MB/BacT system) and poor standardization TABLE 8. Summary of reviewed studies dealing with Versa TREK system a
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Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System

Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System

Phenotypic methods remain the gold standard for DST in clin- ical trials, and past and current trials depend on phenotypic test- ing of anti-TB drugs to ensure that study participants are suscep- tible to the drugs that they are receiving. Having reliable susceptibility results for the study drugs within the screening pe- riod, e.g., 2 to 3 days, would be a significant advancement for clinical trials. Currently, the mechanism or molecular basis of drug resistance is not known for some of the second-line drugs and new TB drugs like bedaquiline, sutezolid, pretomanid (PA- 824), and delamanid. Furthermore, not all gene targets associated with resistance are known (e.g., INH, fluoroquinolones, and in- jectables). Therefore, until current molecular tests are improved or new ones are developed, a rapid phenotypic method like the direct MGIT system would be preferable to indirect MGIT testing. Phenotypic methods may be replaced in the future with molecular tests; however, until we know the relationship between resistance mutations, MICs, and clinical outcomes, there will be a need for phenotypic testing to determine MICs. Rapid MIC determina- tions are possible with the direct MGIT method (K. Eisenach, unpublished data).
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Discordance across Several Methods for Drug Susceptibility Testing of Drug Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

Discordance across Several Methods for Drug Susceptibility Testing of Drug Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

We think it is both intuitive and logical to use a quantitative susceptibility method for MDR-TB detection, such as the Trek MycoTB plate or the phage qPCR method. The clinician may seek to maximize dosing for an isolate with a MIC or quantitative result indicating a borderline susceptible range, as discussed for the fluo- roquinolone, or even continue a medication if the MIC or quan- titative result is at the lower end of the resistance range in the setting of extensively drug-resistant TB or within a limited for- mulary. Both of these methods were readily established in the laboratory, provided information for a broad range of drugs, were relatively rapid, and could be useful for DST surveillance or individualization of multidrug regimens. Of course, interpre- tation of a quantitative range is new territory for the TB field, but so are the issues of complex drug resistance that we are now facing. Some operational aspects of the methods should be men- tioned. MTBDRplus and the Xpert MTB/RIF can of course be performed on sputum samples (preferably smear positive) as a direct DST, which saves time over methods requiring culture. The Xpert MTB/RIF method was the fastest (2 h 40 min) and required the fewest repeats, but it is limited to RIF susceptibility testing. The MTBDRplus line probe assay additionally yields INH suscep- tibility information, but it required more repeats due to missing control bands. All other methods (L-J proportion, MGIT 960, MycoTB, and phage qPCR), being culture based, required time to obtain an adequate isolate. With the MGIT 960 SIRE AST method, when a valid result could be obtained using a seed tube from the primary MGIT culture, the turnaround time was good; however this method required the most repeat testing due to contamina- tion. The MycoTB MIC plate method required the least special- ized equipment of all the methods: an incubator and a multichan- nel pipette. When this test was valid on the first try, the turnaround time was 21 days when we used growth from solid medium. However, contamination or no growth may not be ap- parent for 21 days with this method; thus, when repeat testing is required, the turnaround time doubles to 42 days. For the D29 phage method, contamination was less problematic, due to the short incubation times and specificity of the D29 phage, and this is the easiest method to customize for laboratory-specific drug pan- els. We found that this and other culture-based methods per- formed best when our slow-growing MDR and extremely drug- resistant isolates were subcultured on 7H11 agar.
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Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System

Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System

K-10 are type strains; those with the JTC prefix are clinical isolates recovered from bovine fecal or tissue samples by the Johne’s Testing Center (School of Veterinary Medicine, University of Wisconsin—Madison). Strains UCF-5 and UCF-7 are M. paratuberculosis strains of human origin that were recently isolated from Crohn’s disease patients. These strains and four bovine strains, B213, B236, B238, and B244, were kindly provided by Saleh A. Naser (University of Central Florida, Orlando, FL). All strains were cultivated in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-catalase (Difco Laboratories, MD) and 2 ␮g/ml of mycobactin J (Allied Monitor, Fayette, MO) for 1 month at 37°C. The identity of the organisms was verified by multiplex PCR (2, 3) for the insertion elements IS900, IS901, IS1311, and IS1245 as well as by high-performance liquid chromatography of cell wall mycolic acids by a reference laboratory (State Lab- oratory of Hygiene, Madison, WI).
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