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 susceptibility 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 heteroresistance 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.     

Antimicrobial susceptibility testing of Mycobacterium tuberculosis to first-line drugs: comparisons of the MGIT 960 and BACTEC 460 systems

The reliability of the Mycobacteria Growth Indicator Tube (MGIT) 960 system for rapid antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis was evaluated. Forty-seven isolates, including 10 fully susceptible and 37 resistant strains, were tested for susceptibility to the critical concentrations of streptomycin (STR), isoniazid (INH), rifampin (RMP), and ethambutol (EMB), as recommended by the manufacturer. Strains resistant to the critical concentrations were tested with higher concentrations. The results were compared to those obtained by a radiometric method (BACTEC 460TB) and by a conventional agar dilution method, which served as the reference method. Based on these data, we suggest that the following antibiotic concentrations give satisfactory results with the MGIT 960 system: STR, 4.0 microg/ml; INH, 0.1 microg/ml; RMP, 1.0 microg/ml; and EMB, 5.0 microg/ml. The time required to obtain susceptibility results averaged 6.9 days by the MGIT 960 system and 5.4 days by the BACTEC 460TB system; these intervals were not significantly different. This study shows that the MGIT 960 system is a reliable, rapid, automated method for testing the susceptibility of M. tuberculosis isolates to first-line drugs.     

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

The reliability of the BACTEC MGIT 960 system, an automated version of the Mycobacteria Growth Indicator Tube (MGIT), for antimicrobial susceptibility testing of Mycobacterium tuberculosis was evaluated on 78 clinical isolates. Rifampin (RMP), isoniazid (INH), streptomycin (SM), and ethambutol (EMB) were tested at the following concentrations: 1.0 microg/ml for RMP, 0.1 and 0.4 microg/ml for INH, 1.0 and 4.0 microg/ml for SM, and 5.0 and 7.5 microg/ml for EMB. Results were compared with those obtained by the BACTEC 460 TB radiometric system. Initially the reproducibility study showed 99.5% agreement on repeat testing with all the four drugs. With susceptibility testing of clinical isolates, excellent agreement between the two systems was found for all the drugs. A total of nine major errors were observed for only three isolates, resistant according to BACTEC MGIT 960 and susceptible according to BACTEC 460 TB, to SM (4.0 microg/ml), INH (0.1 microg/ml), and EMB (5.0 microg/ml) (one isolate) and to SM (1.0 microg/ml), INH (0.4 microg/ml), and EMB (5.0 microg/ml) (two isolates). When these isolates were tested by using the conventional proportion method on L?wenstein-Jensen medium, agreement with BACTEC MGIT 960 was found for five results and with BACTEC 460 TB for the remainder. The time to report results was 7.9 days by MGIT 960 and 7.3 days by BACTEC 460 TB, which was not found statistically significant (P > 0.05). In conclusion, the performance of BACTEC MGIT 960 was found similar to that of BACTEC 460 TB and this new system can be considered a good alternative to the radiometric method for routine susceptibility testing of M. tuberculosis.     

Multicenter Evaluation of the Mycobacteria Growth Indicator Tube for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs

In a multicenter study involving three reference centers for mycobacteria, the reliability of the Mycobacteria Growth Indicator Tube (MGIT) for rapid antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis was evaluated and compared to the radiometric method (BACTEC 460TB). Test cultures for which the results of the MGIT and BACTEC 460TB tests were discordant were checked by the conventional proportion method on solid medium. Four hundred forty-one isolates have been tested for susceptibility to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM). Discrepant results were obtained for three isolates (0.7%) with INH (susceptible by MGIT, resistant by BACTEC 460TB), for four isolates (0.9%) with RMP (susceptible by MGIT, resistant by BACTEC 460TB), for six isolates (1.9%) with EMB (four susceptible by MGIT, resistant by BACTEC 460TB; two resistant by MGIT, susceptible by BACTEC 460TB), and for four isolates (0.9%) with SM (two susceptible by MGIT, resistant by BACTEC 460TB; two resistant by MGIT, susceptible by BACTEC 460TB). When cultures with discordant results were tested by the conventional proportion method, about half of the cultures yielded results similar to the BACTEC 460TB results, while the other half yielded results similar to the MGIT results. Turnaround times were 3 to 14 days (median, 8.8 days) for MGIT and 3 to 15 days (median, 7.8 days) for BACTEC 460TB. There was no statistically significant difference between the susceptibility testing results of the two methods (P > 0.05). These data demonstrate that the MGIT system is an accurate, nonradiometric alternative to the BACTEC 460TB method for rapid susceptibility testing of M. tuberculosis.     

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 (L?wenstein-Jensen) cultures. PZA MGIT medium in plastic tubes yielded results equivalent to medium dispensed in glass tubes.     

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 discrepancies 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 susceptibility 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.     

Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan

We reevaluated the BACTEC MGIT 960 antimicrobial susceptibility testing system (MGIT 960 AST) by using 1,112 isolates of Mycobacterium tuberculosis. When the results of MGIT 960 AST were compared with that of the proportion method using Ogawa medium (Ogawa PM), discrepant results were obtained for 30 strains with isoniazid, all resistant by MGIT 960 AST but susceptible by Ogawa PM. For 93% of the strains that produced discrepant results, the MIC was 0.4 or 0.8 microg/ml, showing resistance by the proportion method using Middlebrook agar plates. Furthermore, it was also established by analyses of the katG and inhA genes that strains resistant only by MGIT 960 AST have a low level of isoniazid (INH) resistance, indicating that MGIT 960 AST is a reliable method. Ninety-six strains were resistant to 0.1 microg/ml INH by MGIT 960 AST. When they were divided into three groups, Low-S (susceptible at 0.2 microg/ml), Low-R (resistant at 0.2 microg/ml), and High-R (resistant at 1.0 microg/ml), by Ogawa PM, 43.3% of the Low-S strains had mutations in the promoter region of inhA and no mutations were detected in katG codon 315, while 61.7% of the High-R strains had katG codon 315 mutations or a gross deletion of katG. These results suggest that mutations in inhA are associated with low-level resistance to INH and katG codon 315 mutations are associated with high-level resistance to INH. In addition, the analyses demonstrated some relationship of mutations in the inhA gene with ethionamide resistance for the Low-S strains, but not for the High-R strains.     

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

In this multicenter study, the reliability of two nonradiometric, fully automated systems, the MB/BacT and BACTEC MGIT 960 systems, for testing the susceptibilities of 82 Mycobacterium tuberculosis strains to isoniazid, rifampin, ethambutol, and streptomycin was evaluated in comparison with the radiometric BACTEC 460TB system. The arbitration of discrepant results was done by the reanalysis of the strain, the determination of the MIC, and the molecular characterization of some resistance determinants. The overall level of agreement with BACTEC 460TB results was 96% with the MB/BacT test and 97.2% with the BACTEC MGIT 960 system. With both methods, the level of agreement with BACTEC 460TB results was 96.3% for isoniazid, 98.8% for rifampin, and 98.8% for ethambutol. The level of agreement for streptomycin was 90.2% with MB/BacT and 97.5% with BACTEC MGIT 960. Overall, there were 11 very major errors and 2 major errors with the MB/BacT method and 5 very major errors and 2 major errors with the BACTEC MGIT 960 system. In general, the MB/BacT and BACTEC MGIT 960 systems showed good performance for susceptibility testing with first-line antituberculosis drugs.     

Evaluation of automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to four major antituberculous drugs: comparison with the radiometric BACTEC 460TB method and the agar plate method of proportion

We evaluated the performance of BACTEC MGIT 960 for automated testing of the susceptibility of 133 strains of Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, and ethambutol. The BACTEC MGIT 960 results were compared with those obtained with the radiometric BACTEC 460TB system, and when there was disagreement, the method of proportion on agar plates was used as a reference method. Strains resistant to the critical concentration of streptomycin, isoniazid, or ethambutol were also tested with a second, higher concentration. The overall agreement between the two systems was 96.7%, and the 18 discrepancies were resolved in favor of BACTEC 460TB in 11 cases and in favor of BACTEC MGIT 960 in 7, a difference which was not statistically significant. Apart from the assay's low specificity for ethambutol, which was low for the radiometric assay as well, good sensitivity and specificity values characterized BACTEC MGIT 960. The average time required for completion of the test was 2.5 days shorter with BACTEC 460TB. In conclusion, BACTEC MGIT 960 appears to be a suitable replacement for the radiometric method of antimicrobial susceptibility testing of M. tuberculosis. The problem of frequent contamination of BACTEC MGIT 960 tests needs to be quickly resolved; in fact, 14 strains had to be reprocessed because of contamination.     

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

Given the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosis susceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.     

Comparison of BACTEC MGIT 960 system and BACTEC 460 TB system for growth and detection of Mycobacteria from clinical specimens

This study was carried out to compare the performance of BACTEC MGIT 960 with the BACTEC 460 TB for growth and detection of Mycobacteria from human clinical specimens. The BACTEC MGIT 960 instrument is a fully automated system that utilizes MGIT tubes containing an oxygen sensor embedded in silicon at the bottom and filled with 7 mL of modified Middlebrook 7H9 broth. Identical samples were inoculated into the two automated systems and incubated for six weeks. Over a period of three months, 279 specimens were decontaminated and processed according to the standard CDC NALC/NaOH method, using the commercial MycoPrep kit. Forty-two specimens (15%) yielded Mycobacterium tuberculosis; 37 (88%) were detected by the fluorescent BACTEC MGIT 960 and 35 (83%) detected by the radiometric BACTEC 460 TB. Fifteen specimens (5%) yielded Mycobacterium Other Than Tuberculosis (MOTT); 10 (66%) were detected by BACTEC MGIT 960 and 11 (73%) detected by BACTEC 460 TB. The average time to detection and contamination rates and the average time to obtain results of antimicrobial susceptibility tests between the two systems were compared. The performance of the BACTEC MGIT 960 was comparable to the BACTEC 460 TB system which has been the "Gold Standard" for automated detection of TB. The former was more rapid, as sensitive and less labour intensive than the BACTEC 460. Our data demonstrates that the BACTEC MGIT 960 system is an accurate, automated and a non-radioactive alternative to the BACTEC 460 TB for the culture and susceptibility testing of M. tuberculosis.     

Rapid colorimetric method for testing susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin in liquid cultures

We have developed a rapid colorimetric method for testing the susceptibility of M. tuberculosis to isoniazid (INH) and rifampin (RIF) based on incorporation of nitrate in broth cultures containing growth supplements. The performance of this colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) test was compared with that of the radiometric BACTEC 460TB system in determining the susceptibilities of 74 M. tuberculosis strains to INH and RIF. By using the BACTEC 460TB system as the "gold standard," the sensitivity (i.e., the ability to detect true drug resistance) and specificity (i.e., the ability to detect true drug susceptibility) of the CONRAS test were 100 and 95% for INH and 94 and 100% for RIF, respectively. The repeatability of the CONRAS test was excellent (for INH, kappa = 1 and P < 0.001; for RIF, kappa = 0.88 and P < 0.001). For the majority of strains, results were obtained within 5 days. The CONRAS test is rapid, accurate, and inexpensive and is an adequate alternative, particularly for resource-poor countries.     

Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials

The BACTEC MGIT 960 system, a fully automated, nonradiometric, noninvasive system for detection and drug susceptibility testing of mycobacteria, was evaluated for the ability to test susceptibilities to second-line drugs. In this study, which was carried out in three phases (phase I, mostly susceptible strains; phase II, mostly resistant strains; phase III, final testing of the optimal drug concentrations found in phases I and II), we established the critical concentrations for seven drugs to be tested in the BACTEC MGIT 960 system compared to the BACTEC 460TB system. The critical concentrations for the seven drugs used in the MGIT 960 system are as follows: amikacin, 1.0 microg/ml; capreomycin, 2.5 microg/ml; ethionamide, 5.0 microg/ml; protionamide, 2.5 microg/ml; ofloxacin, 2.0 microg/ml; rifabutin, 0.5 microg/ml; linezolid, 1.0 microg/ml. Our results demonstrate that the BACTEC MGIT 960 system is an accurate method for rapid testing of the susceptibilities of Mycobacterium tuberculosis to second-line drugs.     

Comparison of the BACTEC MGIT 960 and BACTEC 460TB systems for detection of mycobacteria in clinical specimens

The reliability of the Mycobacteria Growth Indicator Tube (MGIT) 960 system for rapid detection of mycobacteria in clinical specimens was evaluated and compared to the radiometric method (BACTEC 460TB) and to mycobacterial culture on Lowenstein-Jensen (LJ) medium. Clinical specimens (n = 590) were tested without selection. A total of 121 (20.5%) isolates of mycobacteria were recovered; 98 (81.0%) of them were recovered with the BACTEC 460TB system, 86 (71.1%) were recovered with the BACTEC MGIT 960 system, and 55 (45.5%) were recovered with LJ medium (MGIT 960 versus BACTEC 640TB, p >0.05; MGIT 960 or BACTEC 460TB versus LJ, p <0.001). The mean time to detection (TTD) was 18 da for BACTEC 460 TB, and 13 da for BACTEC MGIT 960. The mean time to detection in each system, based upon data where both systems were culture positive, was significantly different (16.6 da for BACTEC 460TB and 13 da for BACTEC MGIT 960, p<0.001). The contamination rate of the BACTEC MGIT 960 system was 13.2%, which was intermediate between the BACTEC 460TB system (11.7%) and the LJ medium (14.7%). These data indicate that the fully automated MGIT 960 system is an accurate, non-radiometric alternative to the BACTEC 460TB method for rapid detection of mycobacteria in a clinical setting.     

Comparison of recoveries of mycobacterium tuberculosis using the automated BACTEC MGIT 960 system, the BACTEC 460 TB system, and Löwenstein-Jensen medium

Using two different liquid media and one conventional solid medium, a total of 57 mycobacterial isolates (Mycobacterium tuberculosis, n = 55; nontuberculous mycobacteria, n = 2) were recovered from 377 clinical specimens. The rates of recovery of M. tuberculosis were 96. 4% with the BACTEC MGIT 960 liquid medium, 92.7% with BACTEC 12B liquid medium, and 81.8% with the L?wenstein-Jensen (LJ) medium. The mean time to detection of M. tuberculosis in smear-positive specimens was 12.6 days for BACTEC MGIT 960 medium, 13.8 days for BACTEC 12B medium, and 20.1 days for LJ medium, and in smear-negative specimens it was 15.8 days for BACTEC MGIT 960 medium, 17.7 days for BACTEC 12B medium, and 42.2 days for LJ medium. The rates of contamination were 3.7, 2.9, and 1.2% for the BACTEC MGIT 960, BACTEC 12B, and LJ media, respectively. In conclusion, the nonradiometric, fully automated 7-ml BACTEC MGIT 960 system can be considered a viable alternative to the semiautomated, radiometric BACTEC 460 TB system.     

Performance of tuberculosis drug susceptibility testing in U.S. laboratories from 1994 to 2008

We present a statistical summary of results from the Model Performance Evaluation Program (MPEP) for Mycobacterium tuberculosis Drug Susceptibility Testing, 1994 to 2008, implemented by the U.S. Centers for Disease Control and Prevention (CDC). During that period, a total of 57,733 test results for culture isolates were reported by 216 participating laboratories for the first-line antituberculosis drugs used in the United States-isoniazid (INH), rifampin (RMP), ethambutol (EMB), and pyrazinamide (PZA). Using Clinical Laboratory and Standards Institute (CLSI)-recommended concentrations for one or more of three methods, agar proportion (AP), BACTEC460 (Bactec), and MGIT-960 (MGIT), yielded overall agreement of 97.0% for first-line drugs. For susceptible strains, agreement was 98.4%; for resistant strains, agreement was 91.0%, with significantly lower accuracy (chi-square test, P < 0.0001). For resistant strains, overall agreement by methods was 91.3% for AP, 93.0% for Bactec, and 82.6% for MGIT and by drugs was 92.2% for INH, 91.5% for RMP, 79.0% for EMB, and 97.5% for PZA. For some strains, performance by method varied significantly. Use of duplicate strains in the same shipment and repeat strains over time revealed consistent performance even for strains with higher levels of interlaboratory discordance. No overall differences in performance between laboratories were observed based on volume of testing or type of facility (e.g., health department, hospital, independent). By all methods, decreased performance was observed for strains with low-level INH resistance, RMP resistance, and EMB-resistant strains. These results demonstrate a high level of performance in detection of drug-resistant M. tuberculosis in U.S. laboratories.     

Multicenter evaluation of ethambutol susceptibility testing of mycobacterium tuberculosis by agar proportion and radiometric methods

Reproducibility of ethambutol (EMB) susceptibility test results for Mycobacterium tuberculosis has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems, M. tuberculosis drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 micro g/ml in addition to the standard 2.5 micro g/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.     

Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin

Levofloxacin, the active l-isomer of the quinolone ofloxacin, is now widely accepted for treatment of multidrug-resistant tuberculosis. Because the drug is now widely used, we sought to establish susceptibility test conditions for Mycobacterium tuberculosis against levofloxacin by the traditional reference method, agar proportion (AP), the commonly used BACTEC 460 radiometric system, and the newer BACTEC MGIT 960 method. To determine the stability of levofloxacin in the two newer test systems (BACTEC 460 and BACTEC MGIT 960), media containing subinhibitory levels of levofloxacin were prepared and stored at 4 and 37 degrees C for 14 days. The stored media were inoculated with H37Rv, and the drug activity was compared to freshly prepared media. Results show that levofloxacin is stable over the course of testing. Next, optimum levofloxacin test concentrations were determined for AP, BACTEC 460, and BACTEC MGIT 960 methods. MICs were determined for 32 pan-susceptible isolates of M. tuberculosis obtained from presumably untreated patients and 14 quinolone-resistant isolates. The levofloxacin-resistant strains either were isolated from patients who remained culture-positive despite treatment with a quinolone agent (six strains) or contained known mutations in gyrA (eight strains). Levofloxacin MICs resulted in a bimodal pattern with values for resistant strains consistently higher than those for pan-susceptible strains. Results show that levofloxacin concentrations of 2 microg/ml (BACTEC 460 and BACTEC MGIT 960) and 1 microg/ml (AP) inhibited the growth of all pan-susceptible strains while permitting the growth of all levofloxacin-resistant strains. Confirmatory tests with a subset of pan-susceptible and levofloxacin-resistant isolates validated the selected test concentrations.     

Evaluation of BACTEC MGIT 960 and BACTEC 460TB systems for recovery of mycobacteria from clinical specimens of a university hospital with low incidence of tuberculosis

Clinical samples obtained over a period of 8 months (n = 2,624) were processed in parallel with the BACTEC 460TB system, with the MGIT 960 system, and in L?wenstein-Jensen (LJ) medium, resulting in the recovery of 127 mycobacteria. Recovery rates in combinations of the BACTEC 460TB or MGIT 960 system with LJ were, respectively, 94.7 and 94.7% for Mycobacterium tuberculosis complex (n = 57) and 91.4 and 70.0% for nontuberculous mycobacteria (n = 70). Contamination rates, elevated in the MGIT 960 system, were associated with patients (cystic fibrosis) and type of material but not with transport time. Detection time was reduced in the MGIT 960 system.     

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

We evaluated the BACTEC MGIT 960 system, which is a fully automated, noninvasive system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 7-ml culture tubes. We studied 3,330 specimens, 2,210 respiratory and 1,120 nonrespiratory specimens, collected from 2,346 patients treated at six sites. Processed specimens were inoculated into the BACTEC MGIT 960 and BACTEC 460 TB systems, as well as onto Lowenstein-Jensen slants and Middlebrook 7H11/7H11 selective plates. From all culture systems, a total of 362 isolates of mycobacteria were recovered; these were recovered from 353 specimens collected from 247 patients. The greatest number of isolates of mycobacteria (289, or 80% of the 362 isolates) was recovered with the BACTEC MGIT 960, followed by the BACTEC 460 TB (271, or 75%) and solid media (250, or 69%). From all culture systems a total of 132 isolates of Mycobacterium tuberculosis complex were recovered. The greatest number of isolates of M. tuberculosis complex was recovered when liquid medium was combined with conventional solid media; the number recovered with BACTEC 460 TB plus solid media was 128 (97%), that recovered with BACTEC MGIT 960 plus solid media was 121 (92%), that recovered with BACTEC 460 TB was 119 (90%) and that recovered with all solid media combined was 105 (79%). The recovery with BACTEC MGIT 960 alone was 102 (77%). The mean times to detection (TTD) for M. tuberculosis complex were 14.4 days for BACTEC MGIT, 15.2 days for BACTEC 460 TB, and 24.1 days for solid media. The numbers of isolates of Mycobacterium avium complex (MAC) recovered were 172 (100%) for all systems, 147 (85%) for BACTEC MGIT 960, 123 (72%) for BACTEC 460 TB, and 106 (62%) for all solid media combined. The TTD for MAC in each system were 10.0 days for BACTEC MGIT 960, 10.4 days for BACTEC 460 TB, and 25.9 days for solid media. Breakthrough contamination rates (percentages of isolates) for each of the systems were 8.1% for BACTEC MGIT 960, 4.9% for BACTEC 460 TB, and 21.1% for all solid media combined.