Comparative Study of Three Culture Systems for Optimal Recovery of Mycobacteria from Different Clinical Specimens

A new broth-based nonradioactive culture system, MB-Redox (Heipha Diagnostika Biotest, Germany), was compared with the liquid radiometric Bactec 460 TB system and the solid Löwenstein-Jensen (L-J) medium for recovery rate and time to detection of mycobacteria. Of the 605 clinical specimens studied, 100 grew acid-fast bacilli (AFB). The isolation rate for all AFB was 84% for Bactec, 69% for MB-Redox, and 48% for L-J. Eighteen percent of the 100 isolates grew only in Bactec, 10% only in MB-Redox, and 5% only in L-J. The average times to detection of the 100 isolates were 13.2, 17.8, and 28.3 days, respectively, and the mean growth times for the 34 AFB detected by all three media were 13.7, 15.2, and 26.8 days, respectively. Mycobacterium tuberculosis was isolated from 15 clinical samples, with detection times of 16.8 days for Bactec, 18.3 for MB-Redox, and 22.8 days for L-J medium; 10 of these isolates grew in all media, with detection times of 14.2, 16.4, and 18.7 days, respectively. Seven were also positive on direct smear, with detection times of 12.4, 13.7, and 17.7 days. Two of the Mycobacterium tuberculosis isolates were recovered only in Bactec and another one only in L-J medium. Mycobacterium haemophilum grew only in the liquid systems, which provided the special growth factors this bacterium requires. Only the combination of all three systems yielded optimal recovery. MB-Redox gave reliable results, offering the advantages of ready-to-use tubes in which the antibiotic supplement is already incorporated and easy and immediate reading of the results. Since this system does not contain any radioactive substance, results can be confirmed with acid-fast staining and conventional and molecular tests.     

Fully Automated Liquid Culture System Compared with Löwenstein-Jensen Solid Medium for Rapid Recovery of Mycobacteria from Clinical Samples

The aim of this study was to compare the rate of recovery of mycobacteria and the time to detection in 5208 samples using the MB/BacT culture system (Organon Teknika, USA) and Löwenstein-Jensen medium. Mycobacteria were recovered from 301 (5.7%) samples. Two hundred fifty-seven (85.3%) isolates from 114 patients were Mycobacterium tuberculosis[135 (52.5%) smear-positive, 122 (47.4%) smear-negative], and 44 (14.6%) were potentially pathogenic environmental mycobacteria. The yield with the MB/BacT was higher than that with Löwenstein-Jensen [287 (95.3%) vs. 200 (66.4%), P<0.001] for both Mycobacterium tuberculosis[247 (96.1%) vs. 187 (72.7%), P<0.001] and potentially pathogenic environmental mycobacteria [40 (90.9%) vs. 13 (29.5%), P<0.001], mainly at the expense of the smear-negative samples. Moreover, 70 (27.2%) samples were positive only in the MB/BacT, whereas ten (3.8%) samples were positive only in Löwenstein-Jensen. The number of patients with tuberculosis detected by the MB/BacT was higher than that detected by Löwenstein-Jensen medium [111 (97.3%) vs. 89 (78%), P<0.001]. In 25 (21.9%) patients the diagnosis was established solely by means of the MB/BacT. In smear-positive and smear-negative samples, the mean times to detection of Mycobacterium tuberculosis were 16.7 and 26.3 days, respectively, with Löwenstein-Jensen and 11.5 and 19.3 days, respectively, with the MB/BacT. These results indicate that the MB/BacT is more efficient and faster than Löwenstein-Jensen for the recovery of mycobacteria.     

Comparison of the Automated Nonradiometric Bactec MGIT 960 System with Löwenstein-Jensen, Coletsos, and Middlebrook 7H11 Solid Media for Recovery of Mycobacteria

The aim of this study was to evaluate the sensitivity of as well as the time to detection of mycobacteria by three procedures: solid media with traditional reading, microscopy on solid media, and liquid culture using the automated nonradiometric Bactec MGIT 960 system. A total of 2832 respiratory specimens were tested, 315 of which were positive for mycobacteria. The most frequently isolated species was Mycobacterium tuberculosis (201 isolates). One hundred twenty mycobacteria other than tuberculosis were isolated, 72 of which were Mycobacterium xenopi strains. Sensitivity of each of the different media compared to all media combined for growth of Mycobacterium tuberculosis was 93%, 76.1%, 79.6%, and 75.1% for Bactec MGIT 960, Middlebrook 7H11 plates, Löwenstein-Jensen, and Coletsos, respectively. Sensitivity of the Bactec MGIT 960 for detection of all mycobacterial isolates was 75.1%. When this automated system was supplemented with visual inspection, the sensitivity increased to 89.4%. The sensitivity of Middlebrook 7H11 plates, Löwenstein-Jensen, and Coletsos was 50.8%, 60.7%, and 52.3%, respectively. Time to detection of Mycobacterium tuberculosis using the Bactec MGIT 960 system and Middlebrook 7H11 plates with microscopic reading was 12.7 and 13 days, respectively; using the traditional Löwenstein-Jensen and Coletsos media, time to detection was 22.8 and 22.7 days, respectively.     

Comparison of solid and liquid culture media with the polymerase chain reaction for detection ofMycobacterium tuberculosis in clinical samples

The aim of this study was to determine the sensitivity of different methods — two commercial polymerase chain reaction (PCR) kits (a protocol of nested PCR and a protocol of amplification of the IS6110 insertion element), the radiometric Bactec system, the Septi-Chek AFB culture system, and culture in Löwenstein-Jensen (LJ) solid medium — for the detection ofMycobacterium tuberculosis. One hundred clinical samples from 51 patients with culture-positive tuberculosis (81 specimens) and 19 controls (19 specimens) were used. Eighty-nine percent of the samples were smear negative. In the 81 specimens obtained from patients with tuberculosis, the frequency of positivity was 66.6% for nested PCR, 63% for culture in liquid media, 38.3% for the IS6110 assay, and 28.4% for culture on LJ medium. In 18 samples obtained by invasive procedures in patients with tuberculosis, mycobacterial DNA was detected by nested PCR in 83.3% (including all samples positive by culture on liquid media), by culture in liquid media in 77.7%, by culture on LJ medium in 27.7%, and by the IS6110 assay in 11.1%. No false-positive results were obtained from the negative control specimens with any of the techniques tested. The sensitivity of the reamplification protocol appears to be superior to that of the IS6110 assay and similar to that of the Bactec system.     

Four-Year Experience of Use of the Cobas Amplicor System for Rapid Detection of<Emphasis Type="Italic"> Mycobacterium tuberculosis</Emphasis> Complex in Respiratory and Nonrespiratory Specimens in Greece

To evaluate the experience of a clinical microbiology laboratory with a DNA amplification assay for routine detection of Mycobacterium tuberculosis, the Cobas Amplicor Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) assay (Roche Diagnostics Systems, USA) was performed on 7,722 respiratory and 1,451 nonrespiratory specimens collected from 3,321 patients. The results were compared with those of culture in conventional Lowenstein-Jensen medium, culture in the MB/BacT system (Organon Teknika, France), and clinical investigations. A total of 240 of the 254 respiratory specimens culture positive for Mycobacterium tuberculosis were also positive in the PCR assay. Of the 7,300 culture-negative specimens, 45 (0.6%) were positive in the PCR. After detailed interpretation, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 84.5, 99.8, 94.1, and 99.4%, respectively, for respiratory specimens. The PCR assay was more sensitive for smear-positive respiratory specimens (97.1%) than for smear-negative respiratory specimens (48.6%). Of the 18 culture-positive (smear-negative) nonrespiratory specimens, 9 were positive in the PCR. None of the 1,384 culture-negative nonrespiratory specimens were positive in the PCR. The inhibition rates detected by the internal control of the test were 2.2% for respiratory specimens and 3.4% for nonrespiratory specimens. After resolving the discrepancies, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 82.5, 99.8, 94.3, and 99.4%, respectively, when compared to the results of diagnostic culture. In conclusion, the use of the Cobas Amplicor MTB-PCR assay might enable clinical microbiology laboratories with considerable previous experience in molecular biology testing to perform PCR and confirm tuberculosis infection immediately, leading to improved patient management.     

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium

Background: Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. Aim: We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Löwenstein-Jensen and TK-selective (SLC) medium. Materials and Methods: The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Löwenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. Results: The growth of mycobacterial strains was observed in 10–12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. Conclusion: With the additive effect of growth on TK-SLC medium in 10–12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.     

Comparison of the Septi-Chek AFB and BACTEC systems and conventional culture for recovery of mycobacteria.

The performance of the Septi-Chek AFB system was compared with that of the BACTEC radiometric system and that of Lowenstein-Jensen agar slants (LJ) for detection of mycobacteria in clinical specimens. A total of 642 specimens were cultured; 61 (9.5%) yielded mycobacteria. Mycobacterium tuberculosis (34 isolates) and Mycobacterium avium complex (25 isolates) were the predominant species isolated. Of the 61 culture-positive specimens, 30 were smear positive and 31 were smear negative. Overall, 95% of the positive specimens were detected by Septi-Chek and BACTEC (100% of M. tuberculosis isolates) and 75% by LJ (82% of M. tuberculosis isolates). The mean times to detection were 15 days for BACTEC, 23 days for Septi-Chek, and 27 days for LJ. Of the 30 smear-positive specimens, 100% were recovered by Septi-Chek and BACTEC and 90% were recovered by LJ. Of the 31 smear-negative specimens, 90% were detected by Septi-Chek and BACTEC and 61% were detected by LJ. The Septi-Chek and BACTEC systems are superior to the conventional (LJ) mycobacterial culture method. Although Septi-Chek requires more time for the detection of mycobacteria than BACTEC, it is comparable in terms of overall recovery.     

Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.     

Clinical Evaluation of the Automated COBAS AMPLICOR MTB Assay for Testing Respiratory and Nonrespiratory Specimens

We evaluated the COBAS AMPLICOR PCR system (Roche Diagnostics) for the routine detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Diagnostic culture, considered as the reference method, was performed with BACTEC, Löwenstein-Jensen, Stonebrink, and Kirchner media. Occasionally MB-Redox, ESP, or MGIT medium was also used. A total of 643 respiratory and 506 nonrespiratory specimens collected from 807 patients were investigated. Of the 95 culture-positive specimens, 80 were COBAS AMPLICOR MTB positive, and of the 1,054 culture-negative specimens, 1,044 were COBAS AMPLICOR MTB negative. After resolving discrepancies by review of the medical history, the overall sensitivity, specificity, and positive and negative predictive values for the COBAS AMPLICOR MTB assay, respectively, were 83.5, 98.8, 86.7, and 98.6% compared to those of diagnostic culture. In smear-positive specimens, the sensitivity of the COBAS AMPLICOR MTB assay was 96%, versus 48% for smear-negative specimens. No significant differences in the test performance between respiratory and nonrespiratory specimens were observed. The overall inhibition rate was less than 2%, excluding stool specimens. The clear advantages of the COBAS AMPLICOR PCR system are standardized procedures and reagents for specimen processing as well as an internal control for reliable monitoring of PCR inhibitors. By simplifying the work flow through a completely automated amplification and amplicon detection procedure, the COBAS AMPLICOR PCR system proved itself as a very useful component for routine diagnostic procedures.     

Detection and Identification of Mycobacterium tuberculosis Directly from Sputum Sediments by Ligase Chain Reaction

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis [MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for M. tuberculosis, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations of M. tuberculosis. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.     

Evaluation of the Roche Septi-Chek AFB system for recovery of mycobacteria.

The Septi-Chek AFB system for the recovery of mycobacteria from clinical specimens was compared with a conventional approach using Lowenstein-Jensen and Middlebrook 7H11 agars. A total of 1,532 clinical specimens were analyzed; 132 yielded mycobacteria. Mycobacterium tuberculosis and Mycobacterium avium complexes were the predominant isolates. With the conventional combination of Lowenstein-Jensen and 7H11 agars, 75.8% of the isolates were recovered; the Septi-Chek AFB allowed recovery of 100% of the isolates. Septi-Chek AFB required less time for the detection of mycobacteria than did the conventional media.     

Evaluation of a New Commercial Assay for Diagnosis of Pulmonary and Nonpulmonary Tuberculosis

A new commercial assay for the diagnosis of tuberculosis, the BDProbeTec ET Direct Detection assay (Becton Dickinson, USA), was evaluated using 351 respiratory and 372 nonrespiratory specimens. The results were compared to detection of Mycobacterium tuberculosis complex (MTC) by conventional culture. Among the 351 respiratory specimens, MTC bacteria were identified in 150, of which 85 were positive by both microscopy and the assay. Sixty-five specimens culture positive for MTC were microscopy negative; of these, 39 were positive in the assay. All 26 specimens culture positive for nontuberculous mycobacteria (NTM) were negative by the assay. Of 175 specimens culture negative for MTC, 3 were falsely positive by the assay and 1 yielded inhibition. The overall sensitivity and specificity values were 82.7% and 98.5%, respectively. The sensitivity for microscopy-positive and -negative respiratory specimens was 100% and 60%, respectively. After correction for discrepancies, the specificity was 99% compared with notification data. The BDProbeTec ET assay detected 66 of 67 microscopy-positive and 50 of 125 microscopy-negative nonrespiratory specimens. The result for one specimen was inconclusive. All nine specimens containing NTM were negative by the assay. Of 171 specimens culture negative for MTC, 6 were falsely positive by the assay. The overall sensitivity and specificity values obtained with nonrespiratory specimens were 60.7% and 96.7%, respectively. After examining discrepancies by reviewing the patients' histories, the specificity was 98.9%. The sensitivity was 98.5% in microscopy-positive specimens and 40.3% in microscopy-negative specimens. The overall inhibition rate was 0.3%. The BDProbeTec ET assay is a fast, effective, and user-friendly system that can be used for rapid detection of MTC bacteria in respiratory and microscopy-positive nonrespiratory specimens as an important supplement to smear and culture. Electronic Publication     

Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis,Tuberculosis (TB) Drug Resistance,and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV⁺) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.     

Detection ofMycobacterium tuberculosis complex in clinical specimens by a commercial polymerase chain reaction kit

A total of 722 respiratory and 86 nonrespiratory specimens obtained from 456 patients were tested for detection ofMycobacterium tuberculosis complex by a commercial polymerase chain reaction (PCR) kit (Amplicor, Roche Diagnostic Systems) and the results compared with those of microscopy and culture (solid and radiometric media). Respiratory and nonrespiratory specimens were analysed separately. Of the respiratory specimens, 54 were positive forMycobacterium tuberculosis complex both in the PCR and in culture, five were positive in the PCR but negative in culture, and eight were positive in culture but negative in the PCR. Four cultures were positive for mycobacteria other thanMycobacterium tuberculosis; none of these gave a positive result in the commercial test. Resolution of discrepant results was performed by analysis of patients' clinical data. For respiratory specimens the sensitivity of the commercial test was 87.6%, the specificity 99.6%, the positive predictive value 96.6%, and the negative predictive value 98.7%. For nonrespiratory specimens the sensitivity was 60%, whereas the specificity ranged as high as 98.6%. For this group the positive predictive value was 85.7% and the negative predictive value 94.9%. When respiratory specimens are used, the commercial PCR test for detection ofMycobacterium tuberculosis complex, with its high sensitivity and specificity, is a good complementary diagnostic tool for rapid diagnosis of bronchopulmonary tuberculosis in a routine mycobacterial laboratory.     

Detection of Mycobacterium tuberculosis Complex in Sputum Specimens by the Automated Roche Cobas Amplicor Mycobacterium Tuberculosis Test

Three hundred twenty-four sputum specimens from 151 patients with suspected active pulmonary tuberculosis were tested for the presence of the Mycobacterium tuberculosis complex with auramine fluorochrome stain and automated PCR assay (Roche Cobas Amplicor Mycobacterium Tuberculosis Test [MTB]). The results were compared with those of the conventional Löwenstein-Jensen tube culture and the BACTEC radiometer liquid culture. A total of 76 specimens from 32 patients were culture positive for M. tuberculosis. In addition, 37 specimens from 15 patients were smear and culture positive for other Mycobacterium species but negative by the present nucleic acid amplification method and thus were not included in the comparison. Compared with culture, the sensitivities, specificities, and positive and negative predictive values for acid-fast smear were 67, 98, 93, and 91% and those for the Cobas Amplicor MTB were 83, 99, 97, and 95%, respectively. When three consecutive sputum specimens per patient could be obtained, the sensitivity of the Cobas Amplicor MTB improved to 91%, whereas the sensitivity of the acid-fast smear remained unchanged.     

Comparative evaluation of the MB-check system for recovery of mycobacteria from clinical specimens

The performance of the MB-Check system (Roche) in the recovery of mycobacteria from clinical specimens was compared with that of the Bactec radiometric system and conventional culture on Löwenstein-Jensen medium. A total of 1,582 clinical specimens were used for the study; 74 yielded mycobacteria. Organisms of theMycobacterium tuberculosis andMycobacterium avium complexes accounted for the majority of isolates. The rate of recovery of all mycobacteria with the Bactec system was 91.9%, with the MB-Check system 79.7% and on Löwenstein-Jensen medium only 54.1%. Combination of the Bactec and MB-Check systems allowed recovery of 100% of isolates. The Bactec system showed a faster detection time than the other two methods.     

Comparison of fluorescent BACTEC 9000 MB system, Septi-Chek AFB system, and Lowenstein-Jensen medium for detection of mycobacteria.

The newly developed fluorescent BACTEC 9000 MB system for automated culture of mycobacteria was compared with the Septi-Chek AFB system and Lowenstein-Jensen medium (LJ). A total of 2,005 clinical specimens were included in the study. Mycobacteria were isolated from 202 (10.1%) specimens, including 155 Mycobacterium tuberculosis complex isolates and 47 Mycobacteria other than M. tuberculosis isolates. Of 131 isolates detected by the BACTEC system, the Septi-Chek AFB system, or both, 120 (91.6%) were detected by the BACTEC system and 105 (80.2%) were detected by the Septi-Chek AFB system (P < 0.02). The recovery rate in the BACTEC system compared with that in the Septi-Chek AFB system was significantly higher for M. tuberculosis complex isolates (P < 0.005) and for isolates from acid-fast smear-negative specimens (P < 0.01). Of 148 isolates detected by the BACTEC system, LJ, or both, 142 (95.9%) were detected by the BACTEC system and 118 (79.9%) were detected by LJ (P < 0.001). The recovery rate in the BACTEC system compared with that on LJ was significantly higher for M. tuberculosis complex isolates (P < 0.001). The BACTEC system detected more mycobacteria from both smear-positive and smear-negative specimens than LJ. The mean times to detection of mycobacteria were 17.6 days for the BACTEC system, 26.0 days for the Septi-Chek AFB system, and 29.4 days for LJ. The BACTEC fluorescent 9000 MB system is a rapid, sensitive, and efficient method for the isolation of mycobacteria.     

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD₆₆₀) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.     

Comparative Evaluation of Initial and New Versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Direct Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

We evaluated the initial version of the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe) (AMTDT 1) and the new version of AMTDT (AMTDT 2) for the detection of Mycobacterium tuberculosis directly from respiratory and nonrespiratory samples and compared the results with those of culture and staining methods. The assays were applied to 410 respiratory and 272 nonrespiratory samples collected from 515 patients. The combination of the culture results and clinical diagnosis was considered to be the “gold standard.” Ninety-five respiratory specimens were collected from 67 patients with a diagnosis of pulmonary tuberculosis (TB) and 68 nonrespiratory specimens were collected from 61 patients with a diagnosis of extrapulmonary TB. With respiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 96%, respectively, for AMTDT 1 and 94.7, 100, 100, and 98.4%, respectively, for AMTDT 2. With nonrespiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 94%, respectively, for AMTDT 1 and 86.8, 100, 100, and 98.4%, respectively, for AMTDT 2. The overall results of AMTDT 1 and AMTDT 2 were concordant for 97% (661 of 682) of the samples. Statistically significant differences in sensitivities were found between AMTDT 1 and AMTDT 2 with respiratory specimens. It was concluded that although both nucleic acid amplification methods are rapid, sensitive, and specific for the detection of M. tuberculosis complex in all types of clinical samples, AMTDT 2 appeared to be more sensitive than AMTDT 1 when applied to smear-negative specimens. In contrast AMTDT 2 is more susceptible than AMTDT 1 to inhibitory substances in the amplification reaction. The turnaround time of AMTDT 2 is shorter (3.5 h) than that for AMTDT 1 (5 h).     

Mycobacterium growth indicator tube testing in conjunction with the AccuProbe or the AMPLICOR-PCR assay for detecting and identifying mycobacteria from sputum samples.

We have compared the ability of the Mycobacterium Growth Indicator Tube (MGIT) system, a new culture method with an oxygen-sensitive fluorescent sensor, to recover mycobacteria from sputum samples with the abilities of egg-based medium and the Septi-Chek AFB system. We have also assessed the clinical utility of the AccuProbe or the AMPLICOR-PCR assay to directly identify Mycobacterium tuberculosis complex and M. avium-M. intracellulare complex (MAC) from positive MGITs. From 382 sputum samples, 99 isolates of M. tuberculosis complex and 20 isolates of MAC were recovered. The MGIT system had the highest recovery rates for M. tuberculosis complex (97.0%) and MAC (100%), compared to recovery rates of 51.5 and 65.0%, respectively, with the egg-based medium and 81.8 and 85.0%, respectively, with the Septi-Chek AFB system. The shortest recovery times were also achieved with the MGIT system: 16.6 days for M. tuberculosis complex and 12.0 days for MAC, compared to 27.1 and 20.1 days, respectively, with the egg-based medium and 21.4 and 13.2 days, respectively, with the Septi-Chek AFB system. The AccuProbe identified 74 (77.1%) of the 96 M. tuberculosis complex-positive MGITs and 17 (85.0%) of the 20 MAC-positive vials. The AMPLICOR system correctly identified 94 (97.9%) of the 96 M. tuberculosis complex-positive MGITs and all 20 MAC-positive vials. Therefore, the MGIT system used in conjunction with the AMPLICOR system is a rapid and sensitive method for detecting and identifying M. tuberculosis complex and MAC isolates from sputum samples.