MGIT 960

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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

Test methods and media. The Mycobacteria Growth Indicator Tube (MGIT) with BACTEC MGIT 960 growth supplement for DST was used in the MGIT 960 instrument (Becton Dickinson Diagnostic Systems, Sparks, MD). The stan- dard protocol for DST in MGIT 960 was strictly followed as recommended for primary drugs. Culture suspensions for inoculation had to be well dispersed with no large clumps to avoid false-resistant results. After thorough mixing and homogenization of the culture suspensions, the tubes were allowed to rest for at least 15 min, and the supernatant was used to inoculate the drug-containing media and the control by following the manufacturer’s instructions for DST to first-line drugs (3). All inoculated drug-containing MGIT 960 tubes were placed * Corresponding author. Mailing address: Forschungszentrum
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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

new nonradiometric BACTEC MGIT (mycobacterial growth indicator tube) 960 system (9, 15, 20), designed for the detec- tion of mycobacterial species commonly found in human clin- ical samples (5, 15, 17). The system uses an oxygen-quenching fluorescent sensor in conjunction with software algorithms to determine when tubes are “positive,” i.e., when significant bac- terial growth has occurred. This system has been adapted for the detection of M. paratuberculosis in veterinary clinical sam- ples by using a new culture medium specific for M. paratuber- culosis, called MGIT ParaTB medium (Becton Dickinson, Sparks, MD), and a modified algorithm built into the MGIT 960 instrument for the interpretation of fluorescence measure- ments made hourly for each culture tube (4).
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Testing of Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Nonradiometric BACTEC MGIT 960 System

Testing of Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Nonradiometric BACTEC MGIT 960 System

The reliability of the novel BACTEC MGIT 960 pyrazinamide (PZA) kit (Becton Dickinson Microbiology Systems, Sparks, Md.) was assessed for testing of susceptibility of Mycobacterium tuberculosis to PZA. Results generated by the BACTEC MGIT 960 system (Becton Dickinson) were compared with those obtained with the BACTEC 460TB system. Extensive proficiency testing (phase I) and reproducibility testing (phase II) as well as susceptibility testing of blinded strains of M. tuberculosis from the Centers for Disease Control and Prevention (phase III) were performed prior to testing 58 strains isolated from clinical specimens (phase IV). After resolution of discrepant results obtained by the two BACTEC methods by two other laboratories which acted as independent arbiters (phase V), overall agreement of the BACTEC MGIT 960 system with the BACTEC 460TB system for PZA testing of phase IV strains was 96.6%. Between the two systems there was no statistically significant difference in time until results were obtained, i.e., 6.8 days (BACTEC MGIT 960) versus 5.4 days (BACTEC 460TB), the latter not counting the time required for a subculture with a growth index of 200, however. The new BACTEC MGIT PZA susceptibility testing procedure works equally well for inocula prepared from liquid (MGIT) and solid (Löwenstein-Jensen) cultures. PZA MGIT medium in plastic tubes yielded results equivalent to medium dispensed in glass tubes.
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Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures

Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures

Capilia TB assay is an immunochromatographic method that detects MPB64 protein secreted from MTC bacilli into the culture medium [12]. Originally found in M. bovis, similar proteins (i.e. orthologous to MPB64) have been detected in all MTC species and are report- edly rare in NTM. Capilia TB assay is rapid, simple and does not require special equipment [19,20]; it has been found efficient for identification of MTC in South Africa, Thailand and Zambia [20,21]. In this study, the performance of Capilia TB assay was evaluated for rapid detection of MTC from BACTEC MGIT 960 and BAC- TEC 9120 systems in Kampala, Uganda. Additionally, the performance of Capilia TB on blood cultures was evaluated since previous studies mainly dealt with respiratory samples. The overall Sensitivity, Specificity, PPV and NPV of Capilia TB assay were high and in agreement with values obtained elsewhere.
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Use of BACTEC MGIT 960 for Recovery of Mycobacteria from Clinical Specimens: Multicenter Study

Use of BACTEC MGIT 960 for Recovery of Mycobacteria from Clinical Specimens: Multicenter Study

The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by Mycobacterium tuberculosis complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the M. tuberculosis complex, Mycobacterium xenopi, and the Mycobacterium avium complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonra- diometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the “gold standard,” the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.
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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

According to our results, encompassing more than 4,400 individual susceptibility test results generated by testing 247 M. tuberculosis clinical isolates, MGIT 960 is appropriate for drug susceptibility testing of most of the drugs examined, including five first-line and five second-line drugs. In particular, the com- parative results for MGIT 960 and the RRM for rifampin and isoniazid were in excellent agreement for drug-susceptible and MDR strains. The pyrazinamide results were also highly con- cordant. The PZA susceptibility results generated by MGIT 960 disagreed in 8% of the cases with the semisolid method. Very similar results were reported by Scarparo et al. for first- line drugs (31). The concordance of INH and RIF susceptibil- ity results obtained in the studies that compared the MGIT 960 and BACTEC 460 methods were also similar to our findings (1, 4, 32). However, in the case of EMB, fewer than 85% (65% for the critical concentration and 84% for the high concentration) of the results obtained by the MGIT method were in agree- ment with the RRM.
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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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Use of Molecular Methods To Identify the Mycobacterium tuberculosis Complex (MTBC) and Other Mycobacterial Species and To Detect Rifampin Resistance in MTBC Isolates following Growth Detection with the BACTEC MGIT 960 System

Use of Molecular Methods To Identify the Mycobacterium tuberculosis Complex (MTBC) and Other Mycobacterial Species and To Detect Rifampin Resistance in MTBC Isolates following Growth Detection with the BACTEC MGIT 960 System

The guidelines of the Centers for Disease Control and Pre- vention (CDC) recommends that the turnaround times for the growth detection, identification, and susceptibility testing of the MTBC should be 2 to 3 weeks and 2 to 4 weeks, respec- tively, after receipt of the specimen (6, 31, 32). Previous studies have shown that the fully automated MGIT 960 is comparable in sensitivity and rapidity to the BACTEC 460 TB system (10, 34). Therefore, it is a viable alternative to replace the semiau- tomated radiometric system. However, the rapidity of the MGIT or any other new broth-based system is of limited ben- efit if it cannot be correlated with accurate and rapid species identification. Rapid identification is best supported by the application of appropriate molecular biologic methods, but only a limited number of evaluations have been reported on the identification of the MTBC from MGIT broth by molecu- lar or other rapid methods (1–3, 12, 14, 17, 27; S. Rusch- TABLE 1. Mean times in days (range) from inoculation to detection of mycobacteria in the MGIT and Bactec 460 TB systems and to detect
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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

There were only 3 strains, out of a total 78 tested, with nine discordant results: one isolate for SM (4.0 ␮g/ml), INH (0.1 ␮g/ml), and EMB (5.0 ␮g/ml) and two isolates for SM (1.0 ␮g/ml), INH (0.4 ␮g/ml), and EMB (5.0 ␮g/ml). All the dis- cordant results showed resistance with BACTEC MGIT 960 and were found susceptible by the radiometric method. The arbiter results by MOP confirmed resistance and resolved the discrepancies in five cases, to high-concentration SM for the first strain and to low-concentration SM and high-concentra- tion INH for the other two. Interestingly, the genetic analysis performed on these strains in order to detect mutations in the genes involved in the resistance against SM and INH rein- forced the finding of resistant strains obtained by BACTEC MGIT 960 and MOP (in LJ medium). In fact, we found an S315T mutation in the katG gene of the R077 and R078 strains and a K43R mutation in the rpsL gene of the R076 strain. These mutations are known to be associated with resistance to INH and SM, respectively (10, 16, 17, 30). On the other hand, no mutations were observed in the embB gene of the three strains, in the rrs and rpsL genes of R077 and R078 strains, and in the inhA, oxyR-ahpC, and katG genes of strain R076; these findings were in agreement with the MOP results. These data indicate that it is possible to define the R077 and R078 strains as truly resistant to INH and the R076 strain as truly resistant to SM, while the three strains can be considered truly suscep- tible to low-concentration EMB and the R076 strain can be considered truly susceptible to INH. After resolution by the arbiter results, the specificity of BACTEC MGIT 960 in- creased. The turnaround times for reporting results for the two systems were also found very close, with no significant differ- ence.
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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

There were no changes in the initial isoniazid sensitivity categorization by the BACTEC 460TB method after discrep- ancy resolution. Therefore, there were three false susceptibility results, or VME, by the BACTEC MGIT 960 system, whereas the MB/BacT test had one VME and two false resistance results, or ME. However, the MIC of isoniazid for the strain corresponding to one of the three BACTEC MGIT 960 VME was 0.125 ␮ g/ml, and the strain had no mutations at the studied targets. The MIC for another BACTEC MGIT 960 VME strain was 0.25 ␮ g/ml, and this strain had a Trp728Tyr muta- tion. Both MICs were very close to the critical concentration used. This was not the case for the MIC for the remaining BACTEC MGIT 960 VME strain (059R), also associated with the MB/BacT VME, which was 1 ␮ g/ml. MICs for all the ME strains were ⬍ 0.05 ␮ g/ml.
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Evaluation of the Invader Assay with the BACTEC MGIT 960 System for Prompt Isolation and Identification of Mycobacterial Species from Clinical Specimens

Evaluation of the Invader Assay with the BACTEC MGIT 960 System for Prompt Isolation and Identification of Mycobacterial Species from Clinical Specimens

Rapid and accurate identification of mycobacterial species is essential for patient management. We describe the use of the Invader assay in conjunction with the BACTEC MGIT 960 system that together provide an efficient procedure for clinical use. This assay discriminates single-base differences (e.g., genotyping single- nucleotide polymorphisms) under homogeneous and isothermal conditions and can measure directly on genomic DNA without prior target DNA amplification. To identify a wide variety of mycobacterial species, 20 Invader probes were designed to target the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer 1 (ITS-1) region. To validate the Invader probes, we used 78 ATCC strains, and 607 clinical mycobac- terial strains, which were identified by DNA sequencing of the 16S rRNA gene and ITS-1. The Invader assay could accurately identify and differentiate these strains according to target sequences. Moreover, it could detect and identify 116 (95.1%) of 122 positive liquid cultures from the BACTEC MGIT 960 system and did not react to 83 contaminated MGIT cultures. Species identification takes 6.5 h by the Invader assay: 2.0 h for DNA extraction, 0.5 h for handling, and up to 4 h for the Invader reaction. The Invader assay has the speed, ease of use, and accuracy to be an effective procedure for the bacteriological diagnosis of mycobacterial infections.
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Susceptibility Testing with the Manual Mycobacteria Growth Indicator Tube (MGIT) and the MGIT 960 System Provides Rapid and Reliable Verification of Multidrug Resistant Tuberculosis

Susceptibility Testing with the Manual Mycobacteria Growth Indicator Tube (MGIT) and the MGIT 960 System Provides Rapid and Reliable Verification of Multidrug Resistant Tuberculosis

The objective of the study was to compare the manual Mycobacteria Growth Indicator Tube (MGIT) method and the BACTEC MGIT 960 system to the BACTEC 460 method for susceptibility testing of Mycobacterium tuberculosis. The evaluation was based on testing of 36 M. tuberculosis strains with various susceptibilities to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM). In addition, five of the strains generating discrepant results in testing for EMB were analyzed for heteroresistance. For INH, the suscepti- bility test results obtained by the MGIT 960 and the manual MGIT systems agreed with the BACTEC 460 results in 94 and 97% of the cases, respectively. The results of susceptibility to RMP were all in agreement. For SM, 78 and 72% of the results obtained by the MGIT 960 and the manual MGIT systems, respectively, agreed with the BACTEC 460 results. In contrast, less than 80% of the results for susceptibility to EMB obtained by the two MGIT methods agreed with the BACTEC 460 results. All five strains analyzed for EMB heteroresis- tance were found to consist of resistant and susceptible subpopulations. The average turnaround times were 6.4 days for the MGIT 960 system, 6.5 for the manual MGIT system, and 8.7 days for the BACTEC 460 method. Both MGIT methods can be regarded as accurate and rapid alternatives to the BACTEC 460 method for detection of strains resistant to INH and RMP. However, more studies are needed for solving the problems associated with susceptibility testing to EMB and SM.
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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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Accelerated Detection and Identification of Mycobacteria with MGIT 960 and COBAS AMPLICOR Systems

Accelerated Detection and Identification of Mycobacteria with MGIT 960 and COBAS AMPLICOR Systems

The current epidemiology of mycobacterial infections in high- and medium-income countries is characterized by epi- demic bursts of tuberculosis in high-risk minority groups (14). There has also been an increase in the number of infections caused by mycobacteria other than Mycobacterium tuberculosis (MOTT), which pose a particular threat to the growing popu- lations of immunocompromised patients. Consequently, the major targets for improvement of the laboratory diagnosis of mycobacterial infections center on speeding up detection and identification of mycobacteria and also increasing the sensitiv- ity of detection of MOTT. Molecular methods have not been able to replace cultivation for the detection of mycobacteria from clinical specimens (1), and attention is now being paid on ways to improve cultivation. When routine molecular methods for identification of mycobacteria are combined, one could predict accelerated laboratory diagnosis of mycobacterial in- fections. Automated blood culture systems for cultivation of mycobacteria have recently been introduced (3, 16, 17). Their major drawbacks are that they require space-demanding cab- inets and the use of a needle and syringe for inoculation and sampling. A more recent approach is a modification of a pre- vious manual technique, the mycobacteria growth indicator tube (MGIT) technique (11, 12), which has been modified to permit the use of an automated cultivation and detection sys- tem (the MGIT 960 system [MGIT system]) (2).
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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

The increase in the incidence of multidrug-resistant tuber- culosis (MDR TB) and the emergence of extensively drug- resistant tuberculosis present tremendous challenges to the global efforts to combat tuberculosis (1, 5, 16, 21). Rapid meth- ods enabling accurate susceptibility testing of first-line and second-line drugs are critical for the early diagnosis of MDR TB and extensively drug-resistant tuberculosis and the initia- tion of effective regimens. Various drug susceptibility testing (DST) methods that use solid media, including the agar pro- portion method (AP) and other methods, have the drawback of prolonged turnaround times (TATs). The World Health Or- ganization and the U.S. Centers for Disease Control and Pre- vention have recommended the use of liquid culture systems for the diagnosis of tuberculosis and DST to improve TATs (22, 25). The Bactec 460 (Becton Dickinson Diagnostic Sys- tems, Sparks, MD), a radiometric liquid system, provided an excellent alternative for testing of the susceptibilities of Myco- bacterium tuberculosis complex (MTBC) isolates to streptomy- cin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE) and to pyrazinamide (PZA) with improved TATs. The MGIT 960 liquid, nonradiometric SIRE DST (Becton Dickinson Di- agnostic Systems), whose performance is comparable to that of the Bactec 460 system, has been commercially available since April 2002 (4, 20, 23). The Microbial Diseases Laboratory (MDL) of the California Department of Public Health imple- mented SIRE DST with the MGIT 960 system in 2004. With confidence in the SIRE DST with the MGIT 960 system, a study that used the same platform to test the susceptibilities of MTBC isolates to four classes of second-line drugs, levofloxa- cin (LVX), amikacin (AMK), capreomycin (CAP), and ethio- namide (ETH), was initiated in November 2004. The study was conducted at two laboratories: MDL and the TB Reference
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Meta Analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without Solid Media, for Detection of Mycobacteria

Meta Analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without Solid Media, for Detection of Mycobacteria

The present analysis was aimed to provide an overall sum- mary of diagnostic accuracy from the multitude of sometimes conflicting results from reports on the comparative diagnostic yield of the BACTEC 960/MGIT and BACTEC 460 systems. Using meta-analysis, we provide data from the available liter- ature, which include a larger number of specimens than can be encompassed by individual studies. However, as the individual TABLE 2. Sensitivity of culture systems according to mycobacterial species 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

tocol was used instead of the standard 4- to 13-day protocol for the INH and RIF indirect tests, to allow more time for the growth control tube to reach the required 400 growth units for a valid test. The indirect PZA test protocol is 4 to 21 days long; the extended incubation time allows more time for the M. tuberculosis bacteria to grow if the growth rate in the slightly acidified MGIT PZA medium is lower. The same protocol was used for the direct PZA test since it was not possible to adjust the instrument protocol, i.e., extend it beyond 21 days, using the Bactec MGIT Epicenter sys- tem, which was not available in these laboratories. Slow growth of some M. tuberculosis strains in PZA medium may have been a cause for growth failures. It is more likely that the reason for in- sufficient growth in the control was that the inoculum density was too low. Although the inoculum for the control tube is a 1/10 dilution of the sputum pellet, instead of the 1/100 dilution used in the indirect test, the concentration of viable M. tuberculosis bacte- ria may have been very low in some sputum specimens despite these specimens being smear positive. Furthermore, it is possible that some strains had a delayed lag time before the beginning of replication and did not reach the threshold of detection before the end of the protocol.
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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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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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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

Our findings carry obvious implications for the laboratory, clinician, and patient. First, DST remains expensive and techni- cally challenging, and so a laboratory usually will only have one validated method available for use. Thus, a high discrepancy rate of one’s chosen method will have direct clinical consequences. If our Bangladesh laboratory in this high MDR-TB setting were to use only the MGIT 960 SIRE method, up to 49% of all EMB- susceptible findings might be false. The possibility of false suscep- tibility to EMB with the MGIT 960 method has been reported since the scale-up of the platform (9) and has been postulated to be a function of isolates with MICs at or just above the critical concentration, or to be due to heteroresistance, where growth rates of resistant mutants are slowed in liquid medium but can be improved by late growth on solid agar. The latter explanation is possible, but the former seems insufficient, as we did not note any statistical difference in the MICs (among the MGIT “false” sus- FIG 3 Drug susceptibility testing discrepancies by drug and methodology. Discrepancies versus the consensus gold standard were enumerated for INH, RIF, EMB, and STR by methodology. Discrepancy rates for EMB were higher for MGIT 960 than with other methods (29%, versus 4 to 5% for other methods; P ⬍ 0.05). Discrepancies were further delineated as susceptible (boxed outline indicating the majority were susceptible) or resistant (dashed outline indicating the majority were resistant). There were 0 discrepancies for INH by L-J. nd, not done (GeneXpert only evaluates RIF and the Hain MTBDRplus method only evaluates INH and RIF).
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