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.     

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.     

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

The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 μg/ml, that established for capreomycin was 3.0 μg/ml, and that established for ethionamide was 5.0 μg/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.The increase in the incidence of multidrug-resistant tuberculosis (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 methods 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 initiation of effective regimens. Various drug susceptibility testing (DST) methods that use solid media, including the agar proportion method (AP) and other methods, have the drawback of prolonged turnaround times (TATs). The World Health Organization and the U.S. Centers for Disease Control and Prevention 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 Systems, Sparks, MD), a radiometric liquid system, provided an excellent alternative for testing of the susceptibilities of Mycobacterium tuberculosis complex (MTBC) isolates to streptomycin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE) and to pyrazinamide (PZA) with improved TATs. The MGIT 960 liquid, nonradiometric SIRE DST (Becton Dickinson Diagnostic 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 implemented 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, levofloxacin (LVX), amikacin (AMK), capreomycin (CAP), and ethionamide (ETH), was initiated in November 2004. The study was conducted at two laboratories: MDL and the TB Reference Laboratory of the Veteran Affairs Medical Center (VA) in West Haven, CT. Here we report the results of the multicenter study, in which the critical concentrations of the test drugs were established, the performance of the MGIT 960 system was compared to that of AP, and the interlaboratory reproducibility of the method was evaluated.(Part of this work was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2006, San Francisco, CA.)     

Performance of BACTEC 960 Mycobacteria Growth Indicator Tube in the Susceptibility Testing of Genetically Characterized<Emphasis Type="Italic"> Mycobacterium tuberculosis</Emphasis> Isolates

In this study, the 7H10 agar proportion method was compared with the BACTEC TB-460 and BACTEC MGIT 960 systems (BD Biosciences, USA) for the susceptibility testing of 22 genetically characterized Mycobacterium tuberculosis isolates for isoniazid, rifampin, streptomycin, and ethambutol. The 7H10 agar proportion method agreed with the resistant genotype in 87.3%, BACTEC TB-460 in 92.7%, and the MGIT in 96.4% of the cases, showing the high sensitivity of MGIT in the detection of resistant isolates.     

Use of the BACTEC Mycobacteria Growth Indicator Tube 960 automated system for recovery of Mycobacteria from 9,558 extrapulmonary specimens, including urine samples

The BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT 960) system was applied for recovery of mycobacteria from extrapulmonary specimens and compared with solid media (L?wenstein-Jensen and Stonebrink). A total of 9,558 specimens were investigated, comprising 3,074 body fluids, 1,878 tissues, and 2,069 urine samples, from which the recovery of mycobacteria was not yet established for MGIT 960. In total, the MGIT 960 was able to detect 446 (90.3%) of the 494 isolates of Mycobacterium tuberculosis complex (MTBC) and 223 (86.0%) out of the 259 isolates of nontuberculous mycobacteria (NTM). In comparison to this, culture on solid medium revealed 358 (72.6%) MTBC isolates and 164 (66.8%) NTM isolates. While 136 (27.6%) of the MTBC isolates and 95 (19.2%) of the NTM isolates were recovered from the MGIT 960 only, 48 (9.7%) of the MTBC isolates and 36 (13.9%) NTM isolates grew only on solid media. Thus, the overall sensitivities for the recovery of mycobacteria from extrapulmonary specimens with MGIT 960 and solid media were 88.8% and 69.3%, respectively. However, the efficiency of the MGIT 960 system can be maximized with additional culture on solid media.     

Evaluation of the BD Bactec MGIT 320 System for Detection of Mycobacteria and Drug Susceptibility Testing of Mycobacterium tuberculosis

Two different laboratories evaluated growth and detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis by the BD Bactec MGIT 320 using the BD Bactec MGIT 960 (BD Diagnostics, Sparks, MD) as a reference method. Out of 359 processed sputum specimens for detection of mycobacteria, 99.7% were in agreement between the MGIT 320 and MGIT 960. Streptomycin (STR), isoniazid (INH), rifampin (RIF), ethambutol (EMB) (collectively known as SIRE), and pyrazinamide (PZA) drug susceptibility testing was performed on 89 clinical strains, prepared from both liquid and solid inocula. The results of SIRE and PZA were 100% reproducible between the two instruments tested at both laboratories.     

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, nonradiometric 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.     

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

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.     

Rapid susceptibility testing of Mycobacterium tuberculosis by the Mycobacteria Growth Indicator Tube (MGIT AST SIRE)

Objective   To evaluate the reliability of the Mycobacteria Growth Indicator Tube (MGIT AST) for susceptibility testing of Mycobacterium tuberculosis.
Methods   Seventy strains of M. tuberculosis were tested for susceptibility to streptomycin, isoniazid, rifampicin and ethambutol by comparing MGIT AST results to those obtained by the method of proportion (MOP) on Lowenstein–Jensen (LJ) and Middlebrook 7H10 media. The 7H10 MOP was considered the method of reference.
Results   The turnaround time for MGIT AST was 6.2 days (5–10 days) and for MOP it was 18–21 days. With rifampicin, MGIT AST agreed for all isolates with both MOP. For streptomycin, MGIT AST and 7H10 MOP agreed for 64 isolates (91.4%); 61 were susceptible and three resistant. LJ MOP and 7H10 MOP agreed for 64 isolates (92.2%); 62 were susceptible and three resistant. With isoniazid, both MOP agreed for all isolates, while MGIT AST and 7H10 MOP had two discrepancies. For ethambutol, MGIT AST and 7H10 MOP were concordant for 66 isolates; 65 were susceptible and one resistant. Both MOP were concordant for 67 isolates; 66 were susceptible and one resistant.
Conclusions   Based on these results, MGIT AST is a time-saving method and can be used as an alternative to the BACTEC System. MGIT AST is reliable as far as rifampicin and isoniazid are concerned; however, additional studies are needed for streptomycin and ethambutol.     

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 Bactec 960 Automated Nonradiometric System for Isolation of Mycobacteria from Clinical Specimens

 The Bactec MGIT 960 system (Becton Dickinson, USA), designed for the culture of mycobacteria, was compared with the Bactec 460 instrument and culture on two solid egg-based media using a total of 1024 clinical specimens. Mycobacteria could be identified from 99 (9.7%) specimens, 89 (90%) of which were identified by the Bactec 960 system, 90 (91%) by the Bactec 460 system, and 82 (83%) by culture on the two egg-based media. The Bactec 960 cultures became positive an average of 16.7 days after specimen collection, the Bactec 460 cultures 14.9 days after collection, and the cultures on egg-based media 26.2 days after collection. The Bactec 960 is a compact and highly automated nonradiometric system that may replace the Bactec 460 system.     

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

Since numbers of drug-resistant Mycobacterium tuberculosis strains are on the rise, the simple classification into “susceptible” and “resistant” strains based on susceptibility testing at “critical concentrations” has to be reconsidered. While future studies have to address the correlation of phenotypic resistance levels and treatment outcomes, a prerequisite for corresponding investigations is the ability to exactly determine levels of quantitative drug resistance in clinical M. tuberculosis isolates. Here we have established the conditions for quantitative drug susceptibility testing for first- and second-line agents using MGIT 960 instrumentation and EpiCenter software equipped with the TB eXiST module. In-depth comparative analysis of a range of well-characterized susceptible and resistant clinical isolates has allowed us to propose conditions for testing and to develop criteria for interpretation.Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Developing countries are the most vulnerable, with more than 95% of the cases (32, 33). The present trend is characterized by an alarming emergence of drug resistance (7, 8, 30). Much attention has focused on the burden of multidrug-resistant (MDR) TB, i.e., resistance to the first-line drugs isoniazid and rifampin (rifampicin) (32, 33), and the emergence of extensively drug resistant TB (6, 26). The rise of drug-resistant TB and the increased susceptibility of the human population to TB due to coinfection with human immunodeficiency virus are driving the worldwide TB pandemic and will worsen the situation in the years ahead, with devastating effects in poor countries, whose economies suffer most from this development (19, 20).In the diagnostic laboratory, testing of mycobacteria for drug susceptibility is substantially different from the general testing procedures used in bacteriology. Rather than determining MICs, a single drug concentration, termed the critical concentration, is usually used to categorize a clinical isolate as susceptible or resistant. This “critical concentration” is more an epidemiological parameter (to distinguish “wild-type” strains from “non-wild-type” strains that are able to grow in the presence of higher drug concentrations [5]) than a clinical cutoff value established to guide treatment decisions (14). With growing knowledge about the mechanisms that underlie drug resistance, it has become evident that drug resistance is multifaceted and that different mutations may lead to different levels of resistance. The acquisition of a resistance mutation leading to a decrease in drug susceptibility should not inevitably exclude an anti-TB drug from a treatment regimen, since low-level resistance does not necessarily imply clinical resistance (3). However, until now, different levels of phenotypic resistance have only rarely been taken into account in the procedures used for in vitro drug susceptibility testing (DST) of mycobacteria (4).“Critical concentration”-based DST of primary and secondary drugs has been established for the radiometric Bactec 460 instrumentation (Becton Dickinson Microbiology Systems, Sparks, MD) and is considered the “gold standard” in the testing of second-line drugs (22, 25). However, the Bactec 460 system has several drawbacks: (i) it involves the use of sharps and radioisotopes with the need for disposal; (ii) it is only semiautomated; and (iii) it needs considerable hands-on time. The nonradiometric MGIT (mycobacterial growth indicator tube) 960 platform (Becton Dickinsion) has been evaluated extensively for DST of first-line drugs (1, 2, 15, 29) and has recently also been evaluated for second-line DST (16, 25). In contrast to the Bactec 460 instrumentation, the MGIT 960 platform is a fully automated system that uses a fluorescence-quenching-based oxygen sensor for growth detection.We have previously characterized quantitative drug resistance levels in clinical strains of drug-susceptible and drug-resistant Mycobacterium tuberculosis using radiometric Bactec 460 measurements (28). However, for widespread implementation of quantitative DST in diagnostic mycobacteriology, the technique chosen should be fully automated, compatible with a computerized expert system for interpretation (so as to avoid individual errors and subjectivity), and safe and reliable (e.g., if possible, the system should not use radioactive material nor needles, such as syringes, for inoculation nor needles inside the instrument). To this end, we have subjected a carefully chosen subset of a previously described and well-characterized collection of clinical M. tuberculosis isolates (28) to quantitative measurements of drug susceptibility using the MGIT 960 platform in conjunction with EpiCenter software equipped with the TB eXiST module.     

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.     

Potential for Erroneous Results Indicating Resistance When Using the Bactec MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

During susceptibility testing of 743 isolates of Mycobacterium tuberculosis to pyrazinamide (PZA) using the Bactec 960 system, 57 (7.7%) isolates showed PZA resistance. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (4.4%) of these isolates as resistant, and 24 (3.2%) were susceptible. Erroneous results for resistance with the Bactec 960 were confirmed by testing the 24 discordant isolates for pyrazinamidase and mutations in the pncA gene.Pyrazinamide (PZA) is an important component of the multidrug regimen used to treat tuberculosis (TB). With increasing worldwide prevalence of drug-resistant TB, it is vital for laboratories to accurately detect resistance to first-line antimicrobials.The CLSI-recommended method for PZA testing (4) is the Bactec 460TB radiometric system (Becton Dickinson, Sparks, MD). Most laboratories have now replaced the 460TB system with the nonradiometric Bactec MGIT 960 (BT960) system (Becton Dickinson, Sparks, MD). Both methods utilize an acidified Middlebrook broth and a critical concentration of 100 μg/ml.PZA is a prodrug which in Mycobacterium tuberculosis is converted to its active form, pyrazinoic acid (POA), by the enzyme pyrazinamidase (PZase) (5, 7). The absence of a functional PZase enzyme in an M. tuberculosis strain therefore indicates resistance to PZA. The pncA gene coding for PZase in M. tuberculosis has been sequenced, and mutations in this gene have been shown to be responsible for resistance to PZA (5, 8, 12). Tests both for PZase activity and for the detection of mutations in pncA may be utilized as alternative methods for the detection of PZA resistance in M. tuberculosis.All new M. tuberculosis isolates are tested for susceptibility to first-line drugs, including PZA, at the Public Health Laboratory, Toronto. During the report period, any isolate demonstrating PZA resistance by the BT960 was retested using the 460TB. If the 460TB PZA result was discordant, these isolates were further tested for the presence of PZase activity and mutations in the pncA gene, and testing in the BT960 was repeated.PZA susceptibility testing in the BT960 system was performed according to the manufacturer''s instructions (2). Briefly, isolates of M. tuberculosis in Mycobacteria Growth Indicator Tubes (MGITs) were used as the test inocula. A drug-free control tube was inoculated with a 1:10 dilution of the inoculum, and the PZA test tube was inoculated with 0.5 ml of the inoculum and 0.1 ml of PZA. The tubes were monitored with the BT960 instrument until the growth control tube flagged positive. At that time, the instrument read the PZA test tube as either resistant (growth unit [GU] ≥ 100) or susceptible (GU < 100). A blood agar purity plate from the inoculum was incubated for 3 days.PZA testing in the BT 460TB system was performed according to the manufacturer''s instructions (13). Briefly, a drug-free control vial and the PZA test vial were each inoculated with 0.1 ml of inoculum from an actively growing culture. The vials were incubated and were read on the BT460 instrument daily until the growth index (GI) in the control vial was ≥200. At that time, the GI in the test vial was calculated as a percentage of the GI in the control. A result of ≥11% indicated resistance, <9% indicated susceptibility, and from 9 to 11% was borderline. A blood agar purity plate from the inoculum was incubated for 3 days.Isolates with results that were discordant between the two systems were tested for the presence of PZase activity and for mutations in the pncA gene at the National Reference Centre for Mycobacteriology, Winnipeg, Manitoba, Canada, by standard methodologies (12, 14) and with repeat testing in the BT960.During the report period, 743 PZA susceptibility tests were performed using the BT960. Of these, 57 (7.7%) showed PZA resistance. Thirty-three of these isolates were confirmed to be PZA resistant using the Bactec 460 system and were eliminated from the discordant-data analysis. The remaining 24 PZA-resistant isolates (3.2% of the total) were susceptible to PZA with the 460TB and were considered discordant. Follow-up testing of these isolates showed the presence of PZase in all isolates, and all were negative for mutations in the pncA gene. Repeat PZA testing of the discordant isolates with the BT960 gave a second resistant result for 10 isolates and a susceptible result for 14 isolates, indicating lack of reproducibility. The 460TB result was considered the gold standard for reporting results.There are reports citing technical problems with in vitro testing of M. tuberculosis with PZA. None of the methods described give 100% agreement when compared with the 460TB reference method, and most cite problems with false resistance (1, 10, 11, 15).The effects of inoculum concentration, volume, and homogeneity, as well as the lack of reproducibility in BT960 PZA tests, have been cited (1, 5, 6, 9). However, we are not aware of a study where BT960-resistant strains were tested with the 460TB plus PZase and molecular testing.There are several differences in the inocula used for testing PZA in the two systems. First, in the 460TB, the ratio of inoculum to medium is 1:42, whereas in the BT960 system, the ratio is 1:16.6. The concentration of inoculum in the test medium in the BT960 is thus more than 2.5 times greater than that used in the 460TB. The volume of inoculum used in the BT960 is 0.5 ml, versus 0.1 ml of inoculum used in the 460TB. The higher concentration and volume of inoculum used in the BT960 has a higher likelihood of containing organisms resistant to PZA, as it is estimated that in M. tuberculosis, between 1 in 10⁷ and 1 in 10¹⁰ cells are resistant to any drug (9).Second, there is variability in the concentration of the inoculum used in the BT960 test according to the day of test setup. For days one and two after the culture flags positive, there is no dilution of the MGIT seed vial, but for days 3 to 5, the inoculum is diluted 1:5. This may lead to considerable variation in the amount of the organism in the inoculum and could cause the lack of reproducibility found during repeat testing.Third, the inoculation method differs between the two systems. For the 460TB, a fine-needle tuberculin syringe is used, and for the BT960, the inoculum is dispensed with a disposable pipette tip, which may result in uneven distribution of bacilli due to “clumping.”The sensitivity of the PZase assay is reported to vary between 79 and 96% (1, 5, 7). All of the discordant isolates that were tested showed the presence of PZase. Sequencing of the pncA gene that encodes PZase has shown that 74% to 97% of all PZA-resistant strains of M. tuberculosis carry a mutation in several different regions of the gene (7, 8, 12). None of the discordant strains were found to have pncA gene mutations.These results corroborate the 460TB results as PZA susceptible and the BT960 results as falsely resistant. For laboratories which perform a large number of susceptibility tests with the BT960 system, this discordance could lead to a significant number of false resistant and major error (3) results for PZA, as well as therapeutic issues in patient management.Due to the potential for false resistant results during PZA testing with the BT960, laboratories should consider retesting all PZA-resistant isolates with the 460TB reference method before reporting results. Since PZA is considered an essential component of first-line TB therapy, it is important that laboratories find a successful algorithm to provide rapid and accurate susceptibility results for PZA.     

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.     

Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium

We considered samples received for culture of mycobacteria using BACTEC MGIT 960 system over a period of 1 year. Tubes flagged positive by MGIT were evaluated for presence of serpentine cording. The cord formation was compared with isolates identified as Mycobacterium tuberculosis complex (MTC) based on p-nitrobenzoic acid (PNB) test. Cords were found in 591 isolates of which 584 (98.8%) were confirmed as MTC. The sensitivity and specificity of cord formation were found to be 99.7% and 89.9%, respectively.     

Improvement of tuberculosis diagnosis by the Mycobacteria Growth Indicator Tube (MGIT) in a developing country laboratory

In order to improve tuberculosis diagnosis in a developing country (Senegal), we evaluated a new liquid-based medium and nonradioactive system, Mycobacteria Growth Indicator Tube (MGIT), with individual clinical specimens collected in Dakar. The main purpose was to compare the time to detection and the rate of recovery of Mycobacterium tuberculosis complex and to determine its importance with respect to Lowenstein-Jensen (LJ), a liquid-based-medium for isolation of M. tuberculosis complex. 531 specimens were processed with Mycoprep kit containing NaOH-N-acetyl L-cystein and inoculated on both LJ and MGIT and incubated at 37 degrees C for 60 days. For each medium, the recovery rate and the time to detection were recorded. Among the 531 specimens, of which 121 smears were positive, 32.5% (173/531) grew the M. tuberculosis complex. Of these, 103 were smear positive (S+) and 70 smear negative (S-). LJ recovered 54.9% (95/173) and MGIT recovered 91.9% (159/173). Disagreements were observed with 92 isolates, LJ failed to recover 78 while MGIT failed to recover 14. The overall mean time to detection was 20.1 days for LJ and 10.5 days for MGIT. MGIT has shown a better sensitivity in isolation with significant reduction in reporting culture for M. tuberculosis complex. As a simple and a nonradiometric system, it could be used in conjunction with egg-based media in developing countries laboratories.