Top PDF 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

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

This study answers other important questions. First, from the results it is evident that susceptibility testing can be carried out using inocula from both liquid and solid culture. Even though there was no statistically significant difference (P ⬎ 0.05) in the overall agreement (98.3% [MGIT inoculum] ver- sus 94.8% [LJ inoculum]), inoculating the MGIT PZA tube with a primary MGIT culture yielded slightly better results. This is advantageous since most of the time, PZA tubes are inoculated from liquid medium, which yields growth consider- ably earlier than does inoculation from solid medium. Second, plastic tubes were as suitable as glass tubes (agreement of 97.5% for plastic tubes versus 96.1% for glass tubes). For a clinical mycobacteriology laboratory, this finding is significant because the use of plastic tubes (currently marketed in certain countries outside the United States) increases safety for the personnel handling the tubes. Finally, compared to the radio- metric technology, the new BACTEC MGIT 960 system auto- matically interprets results as susceptible or resistant. Hence, it
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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 performance of the fully automated BACTEC MGIT 960 (M960) system for the testing of Mycobacterium tuberculosis susceptibility to streptomycin (SM), isoniazid (INH), rifampin (RMP), ethambutol (EMB), and pyrazinamide (PZA) was evaluated with 100 clinical isolates and compared to that of the radiometric BACTEC 460TB (B460) system. The agar proportion method and the B460 system were used as reference methods to resolve the discordant results for SM, INH, RMP, and EMB (a combination known as SIRE) and PZA, respectively. The overall agreements were 96.3% for SIRE and 92% for PZA. For SIRE, a total of 26 discrep- ancies were found and were resolved in favor of the M960 system in 8 cases and in favor of the B460 system in 18 cases. The M960 system produced 8 very major errors (VME) and 10 major errors (ME), while the B460 system showed 4 VME and 4 ME. No statistically significant differences were found. Both systems exhibited excellent performance, but a higher number of VME was observed with the M960 system at the critical concentrations of EMB and SM. For PZA, a total of eight discrepancies were observed and were resolved in favor of the M960 system in one case and in favor of the B460 system in seven cases; no statistically significant differences were found. The M960 system showed four VME and three ME. The mean times to report overall PZA results and resistant results were 8.2 and 9.8 days, respectively, for the M960 system and 7.4 and 8.1 days, respectively, for the B460 system. Statistically significant differences were found. The mean times to report SIRE results were 8.3 days for the M960 system and 8.2 days for the B460 system. No statistically significant differences were found. Twelve strains tested for SIRE susceptibility and seven strains tested for PZA suscep- tibility had been reprocessed because of contamination. In conclusion, the M960 system can represent a valid alternative to the B460 for M. tuberculosis susceptibility testing; however, the frequent contamination of the tests needs to be improved.
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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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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

3,200 mg/liter. One hundred microliters of each standardized bacterial inoculum, with a turbidity equivalent to that of a 1 McFarland standard, was diluted 1:20 in Middlebrook 7H9 medium, the pH was adjusted to 5.9, and then the mixture was inoculated. A PZA-free 100% growth con- trol, containing the same number of M. tuberculosis cells as the testing well with PZA, was prepared. A PZA-free 10% growth control, containing 10% of the M. tuberculosis cells of the testing well with PZA, was also prepared. The third sterile control, containing only liquid culture medium, was prepared as well (22). The plates were covered with lids, placed in a plastic bag, and incubated at 37°C. When white bacterial sediments were ob- served with the naked eye in the 10% growth control, 30 ␮ l of freshly prepared 0.01% resazurin (Sigma, St. Louis, MO) solution was added to each well, and the plates were reincubated at 37°C for 24 h. The MIC was defined as the lowest PZA concentration capable of preventing the color change from blue to pink due to resazurin reduction. M. tuberculosis strains in which the MIC was ⱕ 100 mg/liter were considered susceptible to PZA by the REMA; otherwise, the strains were considered resistant to PZA.
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Sequencing of the pncA Gene in Members of the Mycobacterium tuberculosis Complex Has Important Diagnostic Applications: Identification of a Species Specific pncA Mutation in “Mycobacterium canettii” and the Reliable and Rapid Predictor of Pyrazinamide Res

Sequencing of the pncA Gene in Members of the Mycobacterium tuberculosis Complex Has Important Diagnostic Applications: Identification of a Species Specific pncA Mutation in “Mycobacterium canettii” and the Reliable and Rapid Predictor of Pyrazinamide Resistance

ceptibilities to PZA has been complicated by the fact that this drug is active only at acidic pH; a significant number of M. tuberculosis isolates would not grow at pH 5.5 in conventional solid media (4, 20). Subsequently, a more accurate, broth- based radiometric test system (the BACTEC 460TB system) with pH 6.0 medium was described, and this has become the current recommended assay (21). However, in a study that examined 428 MTB complex strains, at least 4 (0.8%) grew in the presence of 100 ␮ g/ml PZA in the BACTEC 460TB system and yet were PZase positive, suggesting either that for some strains the radiometric assay is inaccurate or that resistance is mediated by another mechanism (15). Our results indicate that sequence analysis of pncA is also an accurate predictor of PZA resistance (99.9%). This capability, combined with the rapid turnaround time of the sequence analysis, makes it an attrac- tive new reference assay for PZA susceptibility testing.
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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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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

The reliability of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system for testing of Mycobacterium tuberculosis susceptibility to the three front-line drugs (isoniazid [INH], rifampin [RIF], and ethambutol [EMB]) plus streptomycin (STR) was compared to that of the BACTEC 460 TB system. The proportion method was used to resolve discrepant results by an independent arbiter. One hundred and ten strains were tested with an overall agreement of 93.5%. Discrepant results were obtained for seven strains (6.4%) with INH (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for one strain (0.9%) with RIF (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for seven strains (6.4%) with EMB (six resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB; one susceptible by BACTEC MGIT 960 and resistant by BACTEC 460 TB), and for 19 strains (17.3%) with STR (resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB). After resolution of discrepant results, the sensitivity of the BACTEC MGIT 960 system was 100% for all four drugs and specificity ranged from 89.8% for STR to 100% for RIF. Turnaround times were 4.6 to 11.7 days (median, 6.5 days) for BACTEC MGIT 960 and 4.0 to 10.0 days (median, 7.0 days) for BACTEC 460 TB. These data demonstrate that the fully automated and nonradiometric BACTEC MGIT 960 system is an accurate method for rapid susceptibility testing of M. tuberculosis.
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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

Conventional indirect drug susceptibility testing of Mycobacterium tuberculosis with liquid medium is well established and of- fers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substan- tial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed speci- mens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA] pro- tocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time sav- ings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three differ- ent countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% con- cordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.
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Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

Strain selection. Strains were made available by six TB National/Supra- national Reference and partner laboratories within the TB-PANNET Consortium to provide wide coverage for most of the lineages observed for the M. tuberculosis complex. Strains were tested for PZA susceptibility and included in the study regardless of testing for other antitubercular drugs. PZA drug susceptibility testing (DST) was performed by using a Bactec MGIT 960 mycobacterial detection system and MGIT 960 PZA kits (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instruc- tions. A total of 1,950 clinical isolates were incorporated in the database. Whenever available, genotyping information (spoligotyping and/or my- cobacterial interspersed repetitive-unit ⫺ variable-number tandem-repeat [MIRU-VNTR] typing [25]) were collected. The MIRU-VNTRplus web tool (26, 27) was used to define lineage information (similarity search settings for identification: 0.17; distance measure for MIRU-VNTR: cate- gorical, weighting 1; distance measure for spoligotyping: categorical, weighting 1).
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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

Only a few publications reported in the literature deal with MICs of second-line drugs in the MGIT medium. These, how- ever, are not multicenter studies, and in addition, they focus on one or two drugs only (2, 6). Our multicenter study was de- signed to establish the critical test concentrations of second- line and newer antituberculosis drugs. Previously, a similar kind of study was carried out for the radiometric BACTEC 460 TB system (9). Since the MGIT 960 uses a richer medium, it was anticipated that different critical concentrations might be required for the new system, as it is well known that the MIC of a drug may vary due to many factors such as medium components, pH, and inoculum size. Several concentrations for each drug were tested to establish MICs and breakpoints for the critical concentrations. The critical concentration was considered the concentration that results in the least number of discrepant results upon testing of a large number of suscep- tible and resistant cultures.
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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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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 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

Delayed diagnosis, inadequate treatment regimens, and mortality characterize drug-resistant and multidrug-resistant TB. The importance of rapidly available results of M. tubercu- losis susceptibility testing is universally acknowledged. Tradi- tional drug susceptibility testing procedures, such as the pro- portions methods on Lo ¨wenstein-Jensen or agar medium, are time-consuming. Liquid systems have been introduced as an alternative method for mycobacterium susceptibility determi- nations. The major disadvantages of the BACTEC 460TB sys- tem are that it is semiautomated and that it uses radioactive material, with the need for the disposal of the radioactive waste. A number of automated, nonradiometric detection sys- tems able to perform susceptibility testing are presently com- TABLE 2. Resolution of discrepant results from the MB/BacT and BACTEC MGIT 960 systems in comparison with results from
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Use of Cost Effective Semi Automated  (Mannual/Micro) MGIT System over BACTEC 960 to Perform First Line Anti Tuberculosis Drugs Sensitivity Testing

Use of Cost Effective Semi Automated (Mannual/Micro) MGIT System over BACTEC 960 to Perform First Line Anti Tuberculosis Drugs Sensitivity Testing

Introduction: Multi-drug resistant tuberculosis (MDR-TB) that is the tuberculosis that is resistant to at least 2 of the first line anti-tuberculosis drugs is fatal infectious disease. Cases of MDR-TB are now increasing with 30,000 cases of MDR-TB re- ported in 2013 by national TB programme. Rapid diagnosis of MDR-TB is extremely important for rapid treatment of patient and to prevent spread of MDR-TB to other. BACTEC 960 system helps in rapid diagnosis but purchase of expensive instrument for the same is the limitation. However, the same purpose can be solved by use of semi-automated MGIT system. Aims and Objectives: Aim of this study is to do drug sensitivity testing of the first line anti-tuberculosis drugs with the use of semi- automated MGIT systems. 350 newly registered and suspected cases of tuberculosis in tertiary care hospital were included. Samples were processed for digestion and decontamination and inoculated in MGIT tubes and also on LJ medium. Reading was taken using semi-automated MGIT system. Positive tubes were confirmed by rapid test for M. tuberculosis and then drug sensitivity was performed. Result: Out of 350 samples, 62% were sputum; 33% were pleural fluid and rest 5% were lymph node, Ascetic fluid, CSF, pus. Average day of positivity by MGIT was 13 - 20 days as compared to 25 - 37 days by solid medium, which was statistically significant with p value < 0.01. MDR cases were 2% out of 350 samples. Conclusion: Manual MGIT System is a simple, efficient, safe to use diagnostic system. It does not require any expensive/special instrumentation other than the UV lamp for detection of fluores- cence. The rapidity by which mycobacteria are detected is the most important ad- vantage of the Manual MGIT. In areas with limited resources where purchase of ex- pensive instruments such as the MGIT960 is out of scope, the use of manual MGIT for rapid susceptibility testing for MDR-TB could be a possibility.
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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

Although a variety of molecular biological methods have been shown to have the potential to provide direct detection of Mycobacterium tuberculosis complex from clinical specimens within a few hours (3, 5), culture still represents the corner- stone on which a definitive diagnosis of tuberculosis and other mycobacterioses relies. In recent years, the development of rapid, reliable methods for culture detection of acid-fast bacilli has been regarded as worthy of absolute priority (12, 13). Reasons for this renewed concern include the serious public health risk due to the reemergence of tuberculosis, the appear- ance of multidrug-resistant strains of M. tuberculosis, and the high incidence of Mycobacterium avium complex disease in patients with AIDS. Currently, mycobacterial culture can be performed with conventional solid media and by one of the available broth-based methods. Of these, the radiometric semi- automated BACTEC 460TB system (Becton Dickinson, Sparks, Md.), which was the first system to permit the signifi- cantly earlier detection of mycobacteria, is now widely ac- cepted as the “gold standard” (4). It has several drawbacks, however: it involves the use of radioactive material, and read- ing of cultures is labor-intensive and is associated with a po- tential risk of cross-contamination. Furthermore the use of needles for inoculation of the vial involves the risk of stick injury. In recent years, several new nonradiometric technolo-
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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

timely matter. In resource poor settings that have many specimens for DST testing, confirmation of discordant results may be a challenge since new molecular rapid as- says are not easily accessible and costly. In RIF resistant isolates, we observed missense mutations at codons 531, 516 and 526 of the rpoB gene. The S531 L missense mu- tation was most common in the rpoB gene accounting for 55.4% (46/83) of all RIF resistant isolates. This muta- tion pattern is similar to other studies [11–15]. High levels of RIF mono-resistance was confirmed by both MGIT 960 (33/97, 34%) and LPA (43/97, 44%) in this study. Increasing levels of RIF mono-resistant has been reported in South Africa at rates ranging from 8 to 13% [16–19]. In South Africa, RIF mono-resistance is in- creasingly observed in HIV-infected people [17]. Unfor- tunately, no data was collected on HIV status, limiting our interpretation of results. Due to the continuous in- crease in RIF mono-resistance in South Africa, RIF re- sistance is no longer a reliable marker for MDR-TB, making DST for the identification of MDR-TB even more important. In countries with high RIF mono- resistance, the use of Xpert MTB/RIF as a base-line rapid screening method for MDR-TB should be revised. This may avoid unnecessary exposure to other toxic TB drugs. We suggest DST using LPA from direct positive smear specimens in high burden countries. The disad- vantage of LPA are longer turnaround times when high volumes of specimens need to be processed since the LPA requires separate DNA extraction before PCR and amplicon detection unlike the Xpert MTB/RIF assay that extracts, amplifies and detects resistance in real time.
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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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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

In contrast, both EMB and SM testing pointed out some problems in use of the two MGIT methods as well as the BACTEC 460 system. In case of EMB, less than 80% of the results obtained by the MGIT methods agreed with those ob- tained by the BACTEC 460 system. The MGIT results agreed best with the BACTEC 460 results when an EMB concentra- tion of 7.5 ␮g/ml was used in all methods. However, the MGIT results agreed better with the expected results given by the WHO when an EMB concentration of 3.5 ␮g/ml was used. Our results indicated that the EMB concentrations commonly ap- plied are suboptimal for both the MGIT methods and the BACTEC 460 method. In addition, our results indicated that the five isolates tested for heteroresistance to EMB truly con- sisted of resistant and susceptible subpopulations. This prob- ably explains at least partly the discordant results obtained. The heteroresistance also indicates that these isolates are not optimal for use as quality control strains.
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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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