Evaluation of the rapid MGIT TBc identification test for culture confirmation of Mycobacterium tuberculosis complex strain detection

A culture confirmation test for the detection of Mycobacterium tuberculosis complex strains that uses a lateral-flow immunochromatographic assay to detect the MPB64 antigen, the MGIT TBc identification (TBc ID) test, has been developed. We evaluated the performance of the TBc ID test in the detection of the M. tuberculosis complex in 222 primary-positive liquid cultures. We compared these results to those of nucleic acid-based identification and conventional biochemical tests. The validity of the TBc ID test was determined, and all of the nontuberculous mycobacteria (NTM) and Nocardia species tested were found to be negative. The detection limit of the TBc ID test was 5 × 10(5) CFU/ml, and for IS6110 real-time PCR it was 5 CFU/ml. All of the M. tuberculosis and M. africanum cultures were found to be positive, while M. bovis and M. bovis BCG cultures were negative. With the exception of 1 contaminated culture, the 221 culture-positive isolates contained 171 (77.5%) M. tuberculosis isolates, 39 (17.6%) NTM species, and 11 (5.0%) unidentified species. Two culture-positive isolates harbored a 63-bp deletion at position 196 of the mpb64 gene. The sensitivity, specificity, positive predictive values, and negative predictive values of the TBc ID test were 98.8, 100, 100, and 95.1%, respectively. Furthermore, the approximate turnaround time for real-time PCR was 4 h (including buffer and sample preparation), while for the TBc ID test it was less than 1 h. We suggest an algorithm for the primary identification of M. tuberculosis in liquid culture using the TBc ID test as an alternative to conventional subculture followed by identification using biochemical methods.     

Improved detection of Mycobacterium spp. using the Bactec MGIT 960 system

Until 1987, the notification rate for mycobacterial infection was on the decline; however, it now appears to be increasing once more. The reason for this may be multifactoral and include improved reporting of diagnosed cases, increased infection of an ageing population, homelessness, immunosuppression (e.g. due to human immunodeficiency virus infection), and immigration of people from countries where tuberculosis is endemic. This rising incidence and the increasing importance of resistant organisms mean that rapid identification by the clinical microbiology laboratory is required, and this is where an automated detection system can be an advantage. Over a two-year period, 2743 clinical specimen were examined for Mycobacterium spp. using the Bactec MGIT 960, and 286 were positive. Time to detection ranged from three to 14 days (mean: 9.3 days), and M. tuberculosis was recovered from 214 (75.5%). Contamination rate was higher (8.6%) than with manual methods, however. On balance, the Bactec MGIT 960 system proved a valuable tool in the routine microbiology laboratory.     

Evaluation of the VersaTREK system compared to the Bactec MGIT 960 system for first-line drug susceptibility testing of Mycobacterium tuberculosis

The aim of this study was to evaluate the reliability of the VersaTREK system for Mycobacterium tuberculosis drug susceptibility testing compared with results obtained with the Bactec MGIT 960 system. A total of 67 strains were evaluated. Overall agreement was at 98.5%. Kappa indexes were 1.0 for isoniazid, rifampin, and ethambutol, 0.937 for pyrazinamide, and 0.907 for streptomycin. The VersaTREK system is validated for M. tuberculosis drug susceptibility testing.     

Evaluation of Bactec MGIT 960 fluorescent method in diagnosis of tuberculosis

We evaluated the fluorescent Bactec MGIT 960 the new system in Poland, which is a fully automated, non-invasive, system for growth and detection of Mycobacterium with a capacity to incubate and continuously monitor 960 of 7-ml culture tubes. This system is equipped with special oxygen-quenching fluorescent sensor, which permits to continuously monitoring of microbial growth. Processed specimens were inoculated into Bactec MGIT 960, Bactec 460 Tb and MB/BacT as well as on to Lowenstein-Jensen slants. The greatest number of isolates of M. tuberculosis complex was recovered by using Bactec MGIT 960 system (49/19.5%). Other systems detected M. tuberculosis complex as follows: Bactec 460 Tb (47/18.7%), MB/BacT (43/17/1%), L-J (38/15.1%). Detection mean time of mycobacterial growth in smear-positive specimens were 15.4 days for Bactec MGIT 960, 16.2 days for Bactec 460 Tb, 15.1 days for MB/BacT and 28.2 days for L-J medium The rates of contamination for each of the system were: 3.8% for Bactec MGIT 960 and Bactec 460 Tb, 3.6% for MB/BacT and 2.9% for L-J. In conclusion Bactec MGIT 960 system is a valuable alternative of radiometric, semi automated Bactec 460 Tb system.     

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 the MGIT TBc identification test and meta-analysis of MPT64 assays for identification of the Mycobacterium tuberculosis complex in liquid culture

Rapid MPT64-based immunochromatographic tests (MPT64 ICTs) have been developed to detect Mycobacterium tuberculosis complex (MTBC) in culture. We demonstrated the noninferiority of one commercial MTP64 ICT, the MGIT TBc identification (TBcID) test, to GenoType line probe assays for MTBC identification in positive MGIT cultures. Meta-analysis of MPT64 ICT performance for identification of MTBC in liquid culture confirmed similar very high sensitivities and specificities for all three commercial MPT64 assays for which sufficient data were available.     

Evaluation of the enhanced amplified Mycobacterium tuberculosis direct test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

OBJECTIVE: To evaluate the performance of the enhanced Mycobacterium Tuberculosis Direct Test (E-MTD), for the direct detection of M tuberculosis complex (MTBC) in respiratory specimens. DESIGN: Two hundred seventy-four respiratory specimens from 151 patients in respiratory isolation were tested with the E-MTD, and the results were compared with the results of mycobacterial smear, culture, and the earlier form of the test, MTD-1. RESULTS: Forty-one specimens were culture positive for mycobacteria (20 MTBC and 21 nontuberculous mycobacteria), 23 of which were smear positive (16 MTBC, 7 nontuberculous mycobacteria). Twenty-four specimens were positive by E-MTD, and 21 were positive by MTD-1. Of the 20 MTBC culture-positive specimens, 19 were positive by the E-MTD and 19 were positive by the MTD-1. The remaining specimens were MTBC negative by all methods. After resolution of discrepancies, the sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 100%, 99.6% for the MTD-1 and 95.2%, 98.8%, 87.0%, and 99.6%, for the E-MTD. For the E-MTD smear-positive and smear-negative specimens, these same values were 93.8%, 100%, 100%, and 87.5% and 100%, 98.8%, 62.5%, and 100%, respectively. CONCLUSION: The results suggest that the E-MTD is a reliable method for the direct detection of MTBC in smear-positive respiratory specimens.     

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.     

Growth detection failures by the nonradiometric Bactec MGIT 960 mycobacterial culture system

Mycobacterial growth in liquid culture can go undetected by automated, nonradiometric growth detection systems. In our laboratory, instrument-negative tubes from the Bactec MGIT 960 system are inspected visually for clumps suggestive of mycobacterial growth, which (if present) are examined by acid-fast smear analysis. A 3-year review demonstrated that ～1% of instrument-negative MGIT cultures contained mycobacterial growth and that 10% of all cultures yielding mycobacteria were instrument negative. Isolates from instrument-negative MGIT cultures included both tuberculous and nontuberculous mycobacteria.     

Evaluation of the Cobas TaqMan MTB test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

The Cobas TaqMan MTB test, based on real-time PCR technology, was evaluated for direct detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. A total of 1,093 samples from 446 patients, including 118 acid-fast smear-positive and 975 acid-fast smear-negative specimens, were investigated. Diagnostic cultures performed with 7H11 agar, Löwenstein-Jensen medium, and the Bactec MGIT 960 system were considered the reference methods. When discrepant results between the Cobas TaqMan MTB test and culture occurred, additional results from the BD MGIT TBc identification test and the GenoType Mycobacterium CM test performed on growth-positive and acid-fast-stain-positive MGIT tubes and review of the patient''s medical history were used for discrepancy analysis. The overall sensitivity, specificity, positive predictive value, and negative predictive value for the Cobas TaqMan MTB test were 91.5%, 98.7%, 91.5%, and 98.7%, respectively. In general, the performance of the new Cobas TaqMan MTB test was comparable to that of the replaced Cobas Amplicor MTB system. The most prominent feature of the new system was its extraordinarily high sensitivity (79.5%) for detecting MTBC in smear-negative specimens; out of 44 smear-negative but culture-positive specimens, 35 were positive by the new system. The Cobas TaqMan MTB assay, including DNA extraction, can be completed within 3 h.     

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 offers 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 substantial 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 specimens 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] protocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time savings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three different 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% concordance 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.     

Direct detection of Mycobacterium tuberculosis complex in respiratory specimens by a target-amplified test system.

A total of 938 respiratory specimens (633 sputa, 249 bronchial and tracheal aspirates, and 56 bronchoalveolar lavages) from 589 patients were tested for direct detection of Mycobacterium tuberculosis complex by the Gen-Probe amplified Mycobacterium tuberculosis direct test (MTD), and the results were compared with those of the conventional methods of fluorescence microscopy and cultivation (solid and radiometric media). One series of specimens (n = 515) was decontaminated with N-acetyl-L-cysteine (NALC)-NaOH: the other one (n = 423) was decontaminated with sodium dodecyl (lauryl) sulfate (SDS)-NaOH. Of the specimens decontaminated with NALC, 39 were MTD and culture positive, 455 were MTD and culture negative, 18 were MTD positive and culture negative, and 3 were MTD negative and culture positive, indicating a sensitivity of 92.9% and a specificity of 96.2% for the MTD. Of the specimens decontaminated with SDS, 35 were MTD and culture positive, 372 were MTD and culture negative, 15 were MTD positive and culture negative, and 1 was MTD negative and culture positive, indicating a sensitivity of 97.2% and a specificity of 96.1% for the MTD. After resolution of discrepant results by review of the patients' clinical data, the sensitivity of the MTD was 93.9%, the specificity was 97.6%, the positive predictive value was 80.7%, and the negative predictive value was 99.3% for the NALC series; the corresponding values were 97.4, 96.9, 76.0, and 99.7%, respectively, for the SDS series. In conclusion, the MTD is a highly sensitive and specific technique for detecting M. tuberculosis complex within hours in both smear-positive and smear-negative respiratory specimens.     

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.     

Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures

Tuberculosis (TB) is a disease of major public health concern worldwide, especially in developing countries. In addition, the human immunodeficiency virus (HIV) epidemic has increased the incidence of infection with nontuberculous mycobacteria (NTM). Rapid, accurate, and simple methods for differentiation of Mycobacterium tuberculosis complex (MTBC) isolates from NTM is greatly needed for successful control of the TB epidemic. This study was done to evaluate the clinical performance of the BD MGIT TBc identification test (TBc ID) for rapid identification of MTBC in samples from broth cultures. A total of 229 Ziehl-Neelsen (ZN) stain-positive MGIT cultures were tested using the TBc ID test, and the results were compared with those of the AccuProbe MTBC identification test (GenProbe, San Diego, CA). The agreement between the TBc ID test and the AccuProbe assay was 96% (kappa = 0.92; confidence interval [CI] = 0.869 to 0.971). The sensitivity, specificity, and negative and positive predictive values of the TBc ID test compared to the AccuProbe assay were 100%, 92.4%, 100%, and 92.2%, respectively. After additional molecular testing, the agreement between the two methods increased to 97.8% (kappa = 0.96; CI = 0.917 to 0.994), and the specificity and positive predictive value increased to 95.6% and 95.7%, respectively. The TBc ID test is a simple, sensitive, and specific test for identification of MTBC in samples from acid-fast bacillus (AFB) smear-positive cultures. The TBc ID test could be a good alternative to the AccuProbe test in TB diagnostic laboratories.     

Comparison of enhanced Mycobacterium tuberculosis amplified direct test with COBAS AMPLICOR Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens

The new Roche COBAS AMPLICOR Mycobacterium tuberculosis Assay was compared to the Gen-Probe enhanced Mycobacterium tuberculosis Amplified Direct Test (AMTDII). A total of 486 specimens (296 respiratory and 190 extrapulmonary) collected from 323 patients were tested in parallel with both assays. Results were compared with those of acid-fast staining and culture, setting the combination of culture and clinical diagnosis as the "gold standard." After resolution of discrepant results, the sensitivity, specificity, and positive and negative predictive values for AMTDII were 85.7, 100, 100, and 90.4% for respiratory specimens and 82.9, 100, 100, and 95. 5% for extrapulmonary specimens, respectively. The corresponding values for AMPLICOR were 94.2, 100, 100, and 96.6% for respiratory specimens and 85, 100, 100, and 96.1% for extrapulmonary specimens, respectively. No significant differences were observed between the results of both assays or, within each one, between respiratory and extrapulmonary specimens. The difference between AMTDII and AMPLICOR sensitivities was related to the presence of inhibitory samples, which the former assay, lacking an internal amplification control (IAC), could not detect. The overall inhibition rate for the AMPLICOR assay was 3.9% (19 specimens). It is concluded that, although both amplification assays proved to be rapid and specific for the detection of M. tuberculosis complex in clinical samples, AMPLICOR, by a completely automated amplification and detection procedure, was shown to be particularly feasible for a routine laboratory setting. Finally, AMTDII is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay.     

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.     

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.     

Contribution of PCR for detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens

AIM OF THE STUDY: To evaluate the sensitivity of PCR versus culture of complex tuberculosis mycobacteria and to determine the delay between PCR results and identification of mycobacteria in culture. MATERIALS AND METHODS: Ninety-nine pulmonary and 66 extrapulmonary specimens were analyzed. Samples were inoculated on liquid (MGIT, Bactec) and solid media (Coletsos) and respectively incubated 6 and 12 weeks. Identification was performed by reverse hybridization of PCR products to their complementary probes immobilized on membrane strips (Genotype MTBC, HAIN). Specimens DNA detection was realized by PCR (Cobas Amplicor Mycobacterium tuberculosis test, Roche). RESULTS: Sensitivity of PCR for acid fast bacilli smear positive pulmonary (50/50) and extrapulmonary (7/7) specimens was 100%. Delay between PCR result and identification was 11 days for pulmonary specimens and 8 days for extrapulmonary specimens. Sensitivity of PCR for smear negative samples was, respectively, of 78.7% (37/47) and 51.8% (29/56) for pulmonary and extrapulmonary specimens. In case of PCR positive result of a smear negative sample, a gap of respectively 13 and 12 days was obtained for pulmonary and extrapulmonary specimens compared to identification. CONCLUSION: Positive PCR result for respiratory specimens allows a gap of 11 to 13 days in diagnosis in comparison with identification of mycobacteria in culture.