Mycobacteria In Clinical Specimens Research Articles

Review of methods for detection and characterization of non-tuberculous mycobacteria in aquatic organisms.

Mycobacteriosis is an emerging and often lethal disease of aquatic organisms caused by several non-tuberculous mycobacteria (NTM) species. Early diagnosis of mycobacteriosis in aquaculture and aquatic settings is critical; however, clinical diagnoses and laboratory detection are challenging, and the available literature is scarce. In an attempt to fill the gap, here we review the most relevant approaches to detect and characterize mycobacteria in clinical specimens of aquatic organisms. Emphasis is given to recent advances in molecular methods used to differentiate NTM species spanning from targeted gene sequencing to next-generation sequencing. Further, given that there are major gaps in our understanding of the prevalence of the different NTM species, partially because of their distinct requirements for in vitro growth, we also reviewed the most relevant NTM species reported to cause disease in aquatic organisms and their specific in vitro growth conditions. We also highlight that traditional bacterial culture continues to be relevant for NTM identification, particularly in non-automated laboratories. However, for NTM species discrimination, a high level of accuracy can be achieved with MALDI-TOF MS and molecular approaches, especially targeted gene sequencing applied from clinical specimens or from pure NTM isolates.

Factors Associated with the Performance of Direct PCR Detection of Mycobacteria in Clinical Specimens: Retrospective Real-world Data

Factors Associated with the Performance of Direct PCR Detection of Mycobacteria in Clinical Specimens: Retrospective Real-world Data

An 8-year study on the prevalence and drug resistance of mycobacteria in clinical specimens (2011–2018)

Non-tuberculous mycobacteria cause a wide range of clinical disorders. Isoniazid (INH) and rifampin (RIF) are the most effective first-line antibiotics against for Mycobacterium tuberculosis. The aim of this study was to investigate the prevalence of mycobacteria in clinical samples collected during 8 years (2011–2018) in the National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran and to determine the tuberculosis drug resistance to first-line antibiotics, INH and RIF. In this study, 15829 different clinical specimens were collected at the NRITLD National Tuberculosis Center. A multiplex allele specific polymerase chain reaction (MAS-PCR) was used to identify mutations related to RIF and INH resistance. The genes involved were katG315, inhA for INH and rpoB516, rpoB526 and rpoB531 for RIF. In total (7528/15829, 47.56%), mycobacterial isolates including 6937 MTBC (43.82%) and 591 NTM (3.73%) were obtained. The frequency of MTBC isolates decreased from 65.17% (2015/3092) in 2011 to 47.06% (1224/2601) in 2018. Among NTM isolates, M. simiae was the most prevalent with 55.33% (327/591). The average INH resistance ratio between 7528 MTB and NTM isolates was 21%, from 15.98% in 2011 to 18.76% in 2018. In the case of RIF, the same resistance trend has been gradually increasing from 12.45% in 2011 to 14.55% in 2018. The prevalence of MDR TB has increased during the study period, from 6.49% (134/2065) in 2011 to 12.58% (174/1354) in 2018. The results of this study indicate that early detection of mycobacterial strains and determination of their drug resistance are necessary.

Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR

AimsMycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control....

Isolation and Identification of Mycobacterium tuberculosis with Mixed Growth from Positive MGIT 960 Cultures by Re-Decontamination

Tuberculosis (TB) caused by Mycobacterium tuberculosis has been identified as a remerging infectious disease with public health importance globally. Exploitation of better diagnosis techniques for precise isolation of mycobacteria in clinical specimens is of great importance to improve the diagnosis as part of the global TB control efforts. Contamination of broth cultures of acid-fast bacilli (AFB) by bacterial species other than Mycobacterium species frequently occurs. Many of these contaminated cultures require re-decontamination and re-incubation before the appropriate tests can be performed for identification. This study aimed at the isolation/recovery of M. tuberculosis from contaminated MGIT tubes by making minor modification to the standard protocol and access whether the modification has any impact on recovery rate. Among all confirmed 451 mixed growth positive cultures, 89.57% were culture positive (positive for MTBC), 3.80% were culture negative, 4.65% were culture contaminated and 1.99% were found NTM.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Early Detection of Mycobacteria Using a Novel Hydrogel Culture Method

BackgroundEarly laboratory detection of Mycobacterium tuberculosis is crucial for controlling tuberculosis. We developed a hydrogel mycobacterial culture method that retains the advantages of both solid and liquid methods in terms of speed, cost, and efficiency.MethodsMycobacterium bovis bacillus Calmette-Guérin (BCG) suspensions and 200 acid-fast bacilli (AFB)-positive clinical specimens were inoculated in Middlebrook 7H9 liquid media (Becton-Dickinson and Company, USA) and mixed with 75 µL of 9-fluorenylmethoxycarbonyl (Fmoc)-Phe-Phe-OH hydrogel stock solution in an Eppendorf tube just before culture incubation. The mixtures were cultured at 37℃ for as long as 14 days to monitor culture status.ResultsThe number of M. bovis BCG increased with time. For 200 AFB smear-positive specimens, 155 of 158 conventional culture-positive specimens and 4 culture-negative or contaminated specimens yielded positive cultures within 14 days. For 128 specimens positive with the liquid culture method, the time to positive culture using the hydrogel method (mean, 12.6 days; range, 7 to 14 days) was significantly shorter than that for conventional liquid culture (mean, 16.2 days; range, 6 to 31 days; P<0.0001).ConclusionsThe hydrogel scaffold culture system is useful for timely, economical, and efficient detection of mycobacteria in clinical specimens.

Evaluation of the effectiveness of BACTEC MGIT 960 for the detection of mycobacteria in Bangladesh

ObjectiveTuberculosis (TB) caused by Mycobacterium tuberculosis has been identified as a re-emerging infectious disease with public health importance globally. Exploitation of new laboratory techniques for precise identification of mycobacteria in clinical specimens is of great importance to improve the diagnosis as part of the global TB control efforts. MethodsThe current study was conducted for the evaluation of BACTEC MGIT 960 method in comparison with Lowenstein–Jensen (LJ) culture and light emitting diode (LED) fluorescence microscopy for isolation of mycobacteria among TB suspects from Bangladesh. A total of 421 specimens were tested with these methods. ResultsAmong the tested samples, 3.6% (n=15) were LED fluorescence microscopy positive; while 18 (4.2%) and 45 (10.6%) were recovered from LJ and MGIT 960 culture. The relative positivity found through MGIT 960 system were 60% and 66.7% higher than that of LJ culture and LED fluorescence microscopy, respectively. Recovery rate of Mycobacterium tuberculosis complex ([MTC], 21 by MGIT and 16 by LJ culture) and non-tubercular mycobacteria ([NTM], 24 by MGIT and 2 by LJ culture) by MGIT 960 was 24% and 96% greater, respectively than LJ culture. Moreover, MGIT 960 was found to be highly sensitive (100%), specific (93.3%), accurate (93.6%) and a more rapid method in detecting mycobacteria when compared with LJ culture. ConclusionExtended recovery of NTM and MTC through MGIT 960 urged frequent application of this method to detect mycobacteria more effectively and rapidly.

Comparison of two in-house real-time PCR assays with MTB Q-PCR Alert and GenoType MTBDRplus for the rapid detection of mycobacteria in clinical specimens

An in-house IS6110 real-time PCR (IH IS6110), MTB Q-PCR Alert (Q-PCR) and GenoType MTBDRplus (MTBDR; Hain Lifescience) were compared for the direct detection of Mycobacterium tuberculosis complex (MTBC) in 87 specimens following automated NucliSENS easyMAG DNA extraction. This included 82 first smear-positive specimens and three smear-negative specimens. Another in-house real-time PCR with a Mycobacterium genus-specific probe for the internal transcribed spacer (ITS) region (IH ITS) was used to allow a full comparison with culture results. The sensitivities of IH IS6110, Q-PCR, MTBDR and IH ITS for MTBC detection were 100, 92, 87 and 87 %, respectively, compared with culture. Both IS6110-based real-time PCRs (in-house and Q-PCR) were similar in performance, with 91.2 % concordant results for MTBC detection. Inhibition rates were low, with zero to three specimens producing uninterpretable results. However, the Q-PCR failed to detect MTBC in five samples that were smear negative or had few acid-fast bacilli (one to 10 bacilli in 10 microscopic fields) detected by IH IS6110. IH ITS was the least sensitive assay but may be useful when used in conjunction with IS6110 PCR results to determine the presence of non-tuberculous mycobacteria in smear-negative specimens. None of the real-time PCR assays tested provided drug-resistance data. It was concluded that an IH IS6110 assay could easily be incorporated into the workflow of a diagnostic laboratory for rapid and accurate identification of MTBC from clinical specimens. The inclusion of an internal control and amplification of an ITS target enhance the diagnostic utility of the test.

Evaluation of a low-cost calorimetric approach for rapid detection of tuberculosis and other mycobacteria in culture

The objective of this study was to evaluate the effectiveness of microcalorimetry in rapid detection of mycobacterium species using an inexpensive Isothermal microcalorimetry (IMC) instrument. In addition, we compared microcalorimetry with conventional monitoring techniques. Isothermal microcalorimetry measures heat production rate and can provide rapid detection of living mycobacteria in clinical specimens. Using liquid medium showed that bacterial activity measured by IMC using a TAM Air® agreed with the triphenyl tetrazolium chloride (TTC) assay. Using solid medium to enhance growth, fast-growing mycobacteria detection was achieved between 26 and 53 h and slow-growing mycobacteria detection was achieved between 54 and 298 h. In addition, the calorimetric data were analysed to estimate the growth rate and generation time of the mycobacteria monitored. Infections caused by mycobacteria are severe and difficult to treat. With 9·27 million new cases of tuberculosis in 2007, developing countries experience severe health and economic consequences owing to the lack of an affordable, fast detection method. Research-grade IMC instruments are too expensive to use in developing countries. Our study demonstrates that less-expensive instruments such as the TAM air® are adequate for mycobacteria detection and therefore establishes a clear proof of concept.

Efficacy of PCR-microwell plate hybridization method (Amplicor Mycobacterium) for detection of M. tuberculosis, M. avium and/or M. intracellulare in clinical specimens

Recently, a new kit to detect and identify mycobacteria in clinical specimens was developed by Japan Roche Co. Limited. The new method is based on amplification of DNA of mycobacteria in clinical specimens by PCR and hybridization of amplified DNA by microwell plate hybridization method, which is the "Amplicor Mycobacteria, Roche, (AMP-M)". Cooperative study was organized with 15 tuberculosis hospitals and institutions throughout Japan, and 349 clinical specimens from newly admitted tuberculosis patients and/or suspects were collected during July and August, 1993. All the specimens were examined by smear microscopy (Ziehl-Neelsen's staining), culture on Ogawa egg media, culture on variant 7H9 liquid media and by AMP-M. Excluding 25 specimens which had failed to identify the species of mycobacteria because of contamination, disability to multiply on the transplanted solid media and so on, the results of the examinations in 324 specimens consisting of 167 specimens from previously untreated cases and those of 157 specimens from previously treated cases were analysed. Main results obtained were as follows; 1. Of 70 smear positive specimens from previously untreated cases, culture positive on Ogawa media and 7H9 media, and by AMP-M positive were 59 (84.3%), 61 (87.1%) and 66 (94.3%), respectively. Of 97 smear negative specimens, culture positive were 20 (20.6%), 22 (22.7%) and 27 (27.8%), respectively. The AMP-M showed the highest positive rate in both groups. 2. The sensitivity and the specificity of AMP-M in previously untreated cases were calculated by assuming that positive on Ogawa and/or variant 7H9 media is "positive". The sensitivity was 95.8% (68/71) and the specificity was 94.8% (91/96) for M. tuberculosis in previously untreated cases. The sensitivity and the specificity for M. avium and M. intracellulare were all 100%, although the numbers observed were small. 3. So-called false positive of the AMP-M were observed in 5 cases out of 96 culture negatives on both Ogawa and variant 7H9 media. However, all 5 cases were positive by repeated AMP-M, 3 become culture positive later, and another 2 showed clinical findings consistent with tuberculosis. Hence, the authors considered that the false positive rate of the AMP-M method is to be very low in previously untreated cases. 4. Of 86 smear positive cases with history of previous chemotherapy, the positive culture on Ogawa media, variant 7H9 media and that by AMP-M method were 64 (74.4%), 77 (89.5%) and 85 (98.8%), respectively. In the smear negative cases, culture positive was 10 out of 71 (14.1%), 13 (18.3%) and 24 (33.8%), respectively.

Development of slide-method to distinguish alive and dead mycobacteria by fluorescent staining--a trial for solving the biohazard problem in TB laboratories

An acid-fast staining can detect mycobacteria in clinical specimens rapidly and specifically. It equally stains living and dead bacteria. It would be of more clinical use if the viability of mycobacteria in a sample was determined by the staining. In the present paper, the problems of FDA/EB staining, which detects live or dead bacteria, were solved by establishing a new technique, a slide-method. An air-dried smear of Mycobacterium bovis BCG (Tokyo 172) on a glass slide was covered by a filter paper fully soaked in the staining solution (500 micrograms FDA and 40 micrograms EB per ml PBS). This was kept in an incubator at 37 degrees C for 20 min. The filter paper was removed after incubation and the slide was examined using a fluorescent microscope with a blue filter. Live bacteria were stained greenish yellow taking the FDA stain in while dead bacteria were stained red with EB. This new slide technique eliminated the problems associated with FDA/EB staining. Moreover, stained smears appeared to be more stable compared with the conventional tube method. To overcome the biohazard problems in smear examination of tubercle bacilli, heating of the slides on a heat block at 100 degrees C for 20 min or passing air dried smears in a flame 5 to 30 times was tried to kill the bacteria. The heat-treated slides were stained with FDA/EB and the number of green and red bacteria were counted. Samples of the smeared bacteria were taken after heating and cultured on a solid medium to determine the presence of any colony-forming unit. It was found that no CFU was observed after heating and the morphology of the stained sample was the same to that before heating. These facts suggest that the above mentioned method is a simple, safe yet inexpensive diagnostic tool for mycobacterial clinical specimens.

Evaluation of a Rapid Fluorescent Staining Method for Detection of Mycobacteria in Clinical Specimens

Rapid detection of mycobacterial disease is essential. Using multiple specimen types and concentrations of mycobacteria, we compared two commercial auramine O stains. The more rapid stain permitted consistent acid-fast bacillus quantitation and exhibited less debris staining, and the staining procedure required less time ( approximately 2 min) to perform. These results suggest that the rapid stain may be more cost-effective and efficient for use in clinical laboratories.

Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.

With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

Development and evaluation of triplex PCR for direct detection of mycobacteria in respiratory specimens

To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium-intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty-two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan-mycobacterial and 133 bp species-specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan-mycobacterial bands of approx. 300-350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96.4% of smear-positive specimens and 70.2% of smear-negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2.0%). Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.

Diagnosis of Extrapulmonary Tuberculosis by Smear, Culture, and PCR Using Universal Sample Processing Technology

Definitive and rapid diagnosis of extrapulmonary tuberculosis is challenging since conventional techniques have limitations. We have developed a universal sample processing (USP) technology for detecting mycobacteria in clinical specimens. In this study, this technology was evaluated blindly on 99 extrapulmonary specimens collected from 87 patients. USP-processed specimens were submitted to smear microscopy for detection of acid-fast bacilli (AFB), culture, and two PCR tests targeting devR (Rv3133c) and IS6110 gene sequences. On the basis of clinical characteristics, histology and cytology, conventional microbiology results, and response to antitubercular therapy, 68 patients were diagnosed with tuberculosis. Although USP smear and culture were significantly superior to conventional microbiology, which was not optimized (P < 0.0001), these approaches fell short of PCR tests (P < 0.0001). The low yields by smear and culture are attributed to the paucibacillary load in the specimens. The highest sensitivity in PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity values were 83 and 93.8%, 87.5 and 100%, and 66.7 and 75%, respectively, in pleural fluid, needle-biopsied pleural tissue, and lymph node specimens. In conclusion, the application of USP technology, together with clinicopathological characteristics, promises to improve the accuracy and confidence of extrapulmonary tuberculosis diagnosis.

Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens

Mycobacteria comprise a diverse group of bacteria that are widespread in nature, some of which cause significant disease in humans. Members of the Mycobacterium tuberculosis complex (MTBC) are the most important human pathogens of the genus Mycobacterium. Traditional methods for detection and identification of mycobacteria include microscopy, culture and phenotypic tests. These methods either lack sensitivity, specificity, or are time consuming. Advances in the field of molecular biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting MTBC and drug resistance in cultures and directly in clinical specimens from weeks to days. This review discusses the molecular genetic techniques for detecting and identifying MTBC as well as drug resistance of mycobacteria in clinical specimens.

Clinical evaluation of PCR for rapid diagnosis of pulmonary tuberculosis in patients with respiratory specimens that were smear positive but culture negative for Mycobacterium tuberculosis

in clinical samples is becoming more and more important in the control of tuberculosis both for the clinical treatment of infected patients and for the identification of exposed individuals. Micro- scopic examination of sputum with Ziehl-Neelsen smears is insensitive, detecting acid-fast bacilli only when there are 2104 mycobacteria/mL. Culture on solid medium is labor-intensive and takes a minimum of 2 weeks and often longer for microscopy-negative samples. PCR can improve the rapid diagnosis of tuber- culosis by allowing the direct detection of mycobacteria in clinical specimens. Several PCR-based tests and other amplification assays specific for mycobacteria have been reported [l-31; however, a recent multi- center study involving PCR methodology using in- house primers reported an unexpectedly high variation in sensitivity and called into question the reliability of such an approach for rapid diagnosis of tuberculosis [4]. PCR appeared to be very useful in hagnosing or excluding tuberculosis in a subset of patients with smear-positive sputum but culture negative for

A promising cultural-biomolecular method for rapid isolation and identification of mycobacteria

Early detection and identification of mycobacteria in clinical specimens is of great importance for the control of infection and timely choice of drug therapy. Although direct detection of mycobacteria by polymerase chain reaction (PCR)-based methods is able to provide information within 24-48 h [1-5], conventional culture remains the most reliable diagnostic tool and permits subsequent identification of the isolated mycobacteria and susceptibility testing. It is therefore important to develop a system or medium for the culture of mycobacteria that is sensitive and provides specific identification by molecular methods in the shortest time possible. A preliminary study evaluating a new nonradiometric cultural method for the detection of mycobacteria has been reported [6]. This test, MB Redox (Biotest, Dreiech, Germany), is a manual colorimetric cultural system which includes a modified serum-supplemented Kirchner medium containing the antibiotic mixture PACT (polymixin B, amphotericin B, carbenicillin and trimethoprim) and specific growth additives. The purpose of this investigation was to evaluate the sensitivity and specificity of a coupled cultural-biomolecular method (MB Redox-multiplex PCR) for rapid isolation of mycobacteria and identification of the genus Mycobacierium, and the individual species M. tuberculosis, M. avium and M. intracellulare. The present study describes initial trial experiments with the method in which multiplex PCR was performed on cultures growing in the MB Redox system. The time taken to obtain a positive result was then compared with the time taken for culture alone. The mycobacterial strains used in this work were:

Rapid detection of mycobacteria in clinical specimens by using the automated BACTEC 9000 MB system and comparison with radiometric and solid-culture systems

Recovery rates of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens were determined by using the fluorescent BACTEC 9000 MB system. Data were compared to those assessed by the radiometric BACTEC 460 system and by cultivation on solid media. A total of 3,095 specimens were processed with N-acetyl-L-cysteine-NaOH by two laboratories. The contamination rates for the BACTEC 9000 MB system were 6.8% (center 1) and 9.8% (center 2). A total of 451 mycobacterial isolates were detected (Mycobacterium tuberculosis complex, n = 296; nontuberculous mycobacteria [NTM], n = 155). These isolates originated from 94 (20.8%) smear-positive and 357 (79.2%) smear-negative specimens. The BACTEC 9000 MB system was significantly better than solid media (P < 0.05) in detecting AFB, but it was less efficient than the radiometric system (P < 0.01). The BACTEC 9000 MB system plus solid media (combination A) recovered 393 (87.1%) of the isolates, while the BACTEC 460 system plus solid media (combination B) detected 430 (95.3%) of all AFB isolates. Between combination A and B there was no statistically significant difference for the detection of isolates from smear-positive specimens (P > 0.05), in contrast to the recovery of AFB from smear-negative specimens for M. tuberculosis complex, P < 0.05; for NTM, P < 0.01). The mean time to detection of M. tuberculosis complex was 12.2 days for smear-positive specimens and 18.1 days for smear-negative specimens with the BACTEC 9000 MB system; 9.3 and 15.6 days, respectively, with the BACTEC 460 system; and 21.2 and 28.4 days, respectively, with solid media. For NTM, the average detection times were 15.1, 17.3, and 31.3 days by the three methods, respectively. In conclusion, the BACTEC 9000 MB system is a rapid, less labor-intensive detection system which allows for higher levels of recovery of AFB than solid media. There is no risk of cross contamination, which is known to be the case for the BACTEC 460 system, and data management is greatly facilitated. As a whole, however, the BACTEC 9000 MB system should only be used in conjunction with solid media.