Cord formation in BACTEC 7H12 medium for rapid, presumptive identification of Mycobacterium tuberculosis complex.

We evaluated cord formation in BACTEC 7H12 medium as a criterion for rapid identification of Mycobacterium tuberculosis complex. Kinyoun-stained smears, prepared from 270 radiometrically positive BACTEC 7H12 bottles, were examined independently by three observers. Smears from 93.2, 88.6, and 83.0% of the M. tuberculosis complex cultures were read as cord positive, and smears from 97.3, 97.8, and 99.5% of the mycobacteria other than M. tuberculosis cultures were read as cord negative by the three observers, respectively. There was 93.3% agreement between the observers. The presence of cords in BACTEC 7H12 medium can be a reliable criterion for rapid, presumptive identification of M. tuberculosis complex.     

Reliability of cord formation in BACTEC media for presumptive identification of mycobacteria.

We evaluated cord formation in BACTEC media as a criterion for presumptive identification of Mycobacterium tuberculosis complex. Auramine-rhodamine smears from 673 radiometrically positive BACTEC vials were examined. The presence of cording had a sensitivity, a specificity, and positive and negative predictive values of 22.9, 98.5, 82.9, and 80.3%, respectively. Before cord formation is used to report presumptive identification of M. tuberculosis complex, workers should determine the reliability of this finding in their own laboratories.     

Modern laboratory diagnosis of mycobacterial infections

This review summarises recent advances made in microscopic techniques (fluorescence and peptide nucleic acids) and culture techniques (solid, liquid, radiometric, and non-radiometric systems) and in the development of rapid methods for the identification of mycobacterial cultures (high performance liquid chromatography, thin layer chromatography, RNA sequencing, and polymerase chain reaction restriction enzyme assays). The role of molecular amplification systems in identifying Mycobacterium tuberculosis is described. Most methods record high specificity and sensitivity for smear positive sputum but have variable sensitivity for sputum smear negative and extrapulmonary specimens. Specimen quality will affect the performance of these assays and organisational delays, such as the batching of specimens, can reduce the time saved. In house assays can be as effective as commercial systems as long as appropriate controls are used.     

Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex,M. avium,and M. intracellulare in sputum samples

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63 degrees C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63 degrees C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 micro l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 micro l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.     

Use of microscopic morphology in smears prepared from radiometric cultures for presumptive identification ofMycobacterium tuberculosis complex,Mycobacterium avium complex,Mycobacterium kansasii, andMycobacterium xenopi

The aim of this study was to assess the feasibility of a method for presumptive identification of mycobacteria, based on the morphology in smears prepared from radiometric Bactec-positive cultures (Becton Dickinson, USA) and to select the appropriate DNA probe (AccuProbe; Gen Probe, USA). The smear morphology of acid-fast bacilli was evaluated in 468 positive cultures from clinical samples: 313Mycobacterium tuberculosis complex, 67Mycobacterium avium complex, 32Mycobacterium kansasii, 49Mycobacterium xenopi, and sevenMycobacterium gordonae. The sensitivity and specificity for various morphological patterns were as follows: cord formation forMycobacterium tuberculosis complex 90% and 100%, respectively; striped bacilli forMycobacterium kansasii, 66% and 99%; sea urchin forMycobacterium xenopi, 96% and 99%; short bacilli forMycobacterium avium complex, 61% and 99%; fine-striped bacilli associated withMycobacterium avium complex from blood samples, 33% and 98%. This criterion was applied in the selection of a suitable DNA probe for the identification of 178 cultures. The correct probe was selected in 98%, 97%, and 72% of cultures, respectively, forMycobacterium tuberculosis complex,Mycobacterium avium complex, andMycobacterium kansasii. The observation of acid-fast bacilli morphology in radiometric cultures is a rapid and cost-efficient method for presumptive identification of common clinical isolates of mycobacteria.     

Algorithm for use of nucleic acid probes for identifying Mycobacterium tuberculosis from BACTEC 12B bottles.

Nucleic acid probes (Gen-Probe, San Diego, Calif.) can be used to identify mycobacteria in BACTEC 12B broth cultures prior to detection of growth on solid media. We developed an algorithm that can be used to make an initial choice of a probe (either Mycobacterium tuberculosis complex [MTB] or M. avium complex [MAC]) for use in testing respiratory specimens. The algorithm was based on both the fluorochrome smear result of the concentrated specimen and the time from inoculation until the BACTEC 12B broth culture is flagged (growth index 10) as presumptively positive. The MTB probe is used first for all 4+ smear specimens, 3+ smear specimens positive in 5 days, 2+ and 1+ smear specimens positive in 7 days, and smear-negative specimens positive in 11 days. The MAC probe is used for all other specimens. The algorithm is used when other information about the culture (e.g., previous positive cultures and colonial morphology of growth on solid media) is unknown. Use of the algorithm to probe 102 respiratory BACTEC 12B broth cultures (35 with MTB; 1 with MTB, MAC, and M. gordonae; 47 with MAC; and 19 with other mycobacterial species) from 1 September through 30 November 1992 resulted in the initial use of the MTB probe for 35 (97%) of the cultures positive for MTB and the use of the MAC probe for 35 (73%) of the cultures positive for MAC. Use of the algorithm aided in the efficient use of laboratory resources without delaying the time to identification of MTB isolates.     

Identifying sputum specimens of high priority for examination by enhanced mycobacterial detection, identification, and susceptibility systems (EMDISS) to promote the rapid diagnosis of infectious pulmonary tuberculosis

AIMS: To compare clinical information and sputum microscopy as methods for the selection of samples for enhanced mycobacterial detection, identification, and susceptibility systems (EMDISS) to promote the rapid diagnosis of patients with infectious pulmonary tuberculosis. METHODS: Two thousand, two hundred and sixty four specimen request forms were examined for clinical details, which were then used to identify specimens likely to yield Mycobacterium tuberculosis on culture. These results were compared with the results of sputum microscopy for acid fast bacilli (AFB). Both methods were assessed against the results of culture using a combination of continuous automated mycobacterial liquid culture (CAMLiC) and conventional solid culture. RESULTS: Classification based on clinical details was an inefficient method of identifying high priority specimens for EMDISS. Although, when given, clinical details were often consistent, a substantial proportion of specimens arrived with no details. This approach would result in the referral of at least 16% of the workload but lead to the detection by culture of only 46% of the M tuberculosis present within it. In contrast, microscopy for AFB defined a much smaller number of specimens (4.8% of the total), which contained 90% of the M tuberculosis isolates. CONCLUSIONS: Microscopy for AFB is the most efficient method for defining sputum specimens suitable for referral for enhanced mycobacteriological techniques. However, it is essential that the methods used for smear preparation and microscopy are of the highest possible standard, otherwise some patients with infectious pulmonary tuberculosis will be denied, unnecessarily, the benefits of important advances in mycobacteriology.     

Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex,Mycobacterium avium complex and Mycobacterium kansasii

Mycobacterium species has a specific morphology when grown in liquid medium. Mycobacterium tuberculosis complex (MTB) often exhibits serpentine cording, which is different from the dot and cross-barring morphology observed in Mycobacterium avium complex (MAC) and Mycobacterium kansasii (MK), respectively. These characteristic morphologies can be used as a cost-effective method for rapid, presumptive identification of mycobacterial isolates cultured from the MGIT 960 system. By using Kinyoun acid-fast stain, serpentine cording was found in 840 of 904 (92.1%) samples positive for MTB; dot or loose aggregation was observed in 112 of 136 (82.3%) samples positive for MAC; and the cross-barring, ladder-like, morphology was observed in 45 of 56 (80.5%) samples positive for MK. The sensitivity and specificity were 92.9% and 96.4% for MTB; 82.4% and 94.5% for MAC; and 80.4% and 94.6% for MK, respectively. Using growth rate selection to exclude rapid growers, the positive and negative predictive values were 98% and 87.6% for MTB; 78.3% and 98% for MAC; and 78.9% and 99.1% for MK, respectively. Twenty-eight (93.3%) of 30 strains with ball morphology were rapid growers. Microscopic morphology can be used for rapid, presumptive identification of M. tuberculosis complex, M. kansasii, and M. avium complex and act as a guide for appropriate selection of initial probes to reduce costs.     

Assessment of morphology for rapid presumptive identification of Mycobacterium tuberculosis and Mycobacterium kansasii

Mycobacterium tuberculosis often exhibits serpentine cording when grown in liquid medium, whereas Mycobacterium kansasii can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for M. tuberculosis (64%) and cross-barring was recognized within 63 of 76 (83%) of the specimens positive for M. kansasii, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for M. tuberculosis and 83, 95, 59, and 98%, respectively, for M. kansasii. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of Mycobacterium avium complex from M. tuberculosis and the long beaded forms of Mycobacterium gordonae from M. kansasii. Mycobacterium asiaticum and Mycobacterium szulgai, which rarely occur, are genetically related to M. kansasii and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of M. tuberculosis and M. kansasii, respectively, and as guides for initial probe selection to reduce costs.     

Cord formation in MB/BacT medium is a reliable criterion for presumptive identification of Mycobacterium tuberculosis complex in laboratories with high prevalence of M. tuberculosis

We evaluated cord formation in MB/BacT broth as a rapid method for presumptive identification of the Mycobacterium tuberculosis complex. Kinyoun acid-fast-stained smears from 370 positive MB/BacT bottles were examined for the presence of serpentine cording. The smears were examined independently by two observers. Observer 1 (the supervisor of the mycobacteriology laboratory) examined all of the smears while observer 2 (a clinical microbiologist not familiar with acid-fast bacillus [AFB] microscopy) examined 148 randomly chosen smears that were read by observer 1 without knowledge of which smear was which. The sensitivity, specificity, and positive and negative predictive values of cording for the presumptive identification of M. tuberculosis read by observer 1 were 88.2, 97.4, 99.2, and 69.7%, respectively. These values were reported at 90.6, 52.3, 82.8, and 69. 7%, respectively, by observer 2. Our laboratory prevalence of M. tuberculosis among positive cultures was 78% during the time this study was conducted. At the time of positive signal of the MB/BacT bottles, the broth of the bottles had sufficient cell mass to allow for observation of the presence or absence of serpentine cording. The presence of cords in MB/BacT broth is a reliable criterion for rapid, predictive identification of the M. tuberculosis complex for laboratories with a high proportion of the M. tuberculosis complex when the smears are examined by a microbiologist who has experience with AFB staining.     

Determination of mycobacterial antigens in sputum by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex.     

Clinical Evaluation of the Gen-Probe amplified mycobacterium tuberculosis direct test for rapid detection of Mycobacterium tuberculosis in select nonrespiratory specimens

The performance of the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of extrapulmonary tuberculosis was evaluated by testing 178 nonrespiratory specimens from 158 patients. Criteria for specimen inclusion were (i) a positive smear for acid-fast bacilli (n = 54) and (ii) the source if the smear was negative (tissue biopsies and aspirates and abscess material were tested; n = 124). Results were compared to those of mycobacterial culture; clinical history was reviewed when MTD and culture results disagreed. Forty-eight specimens (27.0%) were positive for mycobacteria, including 23 Mycobacterium tuberculosis complex specimens; of which 21 were smear positive. Twenty-five specimens were MTD positive; 20 of these grew M. tuberculosis complex. All of the five MTD-positive, M. tuberculosis complex culture-negative specimens were considered truly positive, based on review of the medical record. Of the three MTD-negative, M. tuberculosis complex culture-positive specimens, two contained inhibitory substances; one of the two was smear positive. Excluding the latter specimen from analysis, after chart review, the sensitivity, specificity, and positive and negative predictive values of the MTD were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient. Given the few smear-negative samples from patients with extrapulmonary tuberculosis in our study, additional similar studies that include more smear-negative, M. tuberculosis complex culture-positive specimens to confirm our data are desirable.     

Cord Formation in BACTEC Medium Is a Reliable, Rapid Method for Presumptive Identification of Mycobacterium tuberculosis Complex

Serpentine cord formation in BACTEC 12B medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex. Kinyoun acid-fast stained smears were prepared from 666 positive BACTEC 12B bottles and examined for the presence or absence of serpentine cording. Cord formation had a sensitivity, specificity, positive predictive value, and negative predictive value of 89.2, 99.2, 98.5, and 94.2%, respectively. The evaluation of the presence of cord formation in BACTEC 12B medium is reliable and permits the rapid presumptive reporting of M. tuberculosis.     

Genotype MTBDRplus for direct detection of Mycobacterium tuberculosis and drug resistance in strains from gold miners in South Africa

GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis and drug resistance. Assay performance as applied directly to consecutive unselected sputum samples has not been established. The objective of this study was to determine the accuracy of the MTBDRplus test for direct detection of M. tuberculosis (in sputum) and for drug resistance in consecutively submitted sputum samples. In this cross-sectional study in South Africa, one sputum specimen from each person suspected of having pulmonary tuberculosis was tested by smear microscopy, direct MTBDRplus, and Mycobacterial Growth Indicator Tube (MGIT) culture with MGIT drug susceptibility testing. MGIT results were the reference standard. We tested 2,510 sputum samples, and 529 (21.1%) were positive for M. tuberculosis by MGIT. Direct MTBDRplus identified M. tuberculosis in 256 of 529 specimens (sensitivity, 48.4%; 95% confidence interval [CI], 44.1, 52.7). The sensitivity of MTBDRplus for M. tuberculosis detection by sputum smear status was as follows: smear negative, 13.7% (95% CI, 9.8, 18.4); smear scanty, 46.2% (95% CI, 19.2, 74.9); smear 1+, 69.1% (95% CI, 55.2, 80.9); smear 2+, 86.3% (95% CI, 73.7, 94.3); smear 3+, 89.8% (95% CI, 83.7, 94.2). Direct MTBDRplus testing was negative for 1,594/1,612 sputum samples that were culture negative for M. tuberculosis (specificity, 98.9%; 95% CI, 98.2, 99.3). For specimens positive for M. tuberculosis by MTBDRplus, this assay's sensitivity and specificity for rifampin resistance were 85.7% (95% CI, 57.2, 98.2) and 96.6% (95% CI, 93.2, 98.6) and for isoniazid resistance they were 62.1% (95% CI, 42.3, 79.3) and 97.9% (95% CI, 94.8, 99.4). For sputum testing, the sensitivity of MTBDRplus is directly related to the specimen's bacillary burden. Our results support recommendations that the MTBDRplus test not be used for direct testing of smear-negative or paucibacillary sputum samples.     

Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.     

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.     

Direct detection of Mycobacterium tuberculosis complex DNA and rifampin resistance in clinical specimens from tuberculosis patients by line probe assay

The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n=160) and previously treated (n=260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB core region was amplified by nested PCR. The results were analyzed in conjunction with those obtained by Ziehl-Neelsen (ZN) microscopy and by culture on solid media. The LiPA test was positive for M. tuberculosis complex DNA in 389 (92.9%) specimens, including 92.0% (286 of 311) ZN-positive and 94.5% (103 of 109) ZN-negative specimens. Of these, 30.6% were RMP resistant. In contrast, 74.3% of the specimens were positive for M. tuberculosis by culture, and 30.8% of them were RMP resistant. LiPA detected M. tuberculosis complex DNA in 92.4% (110 of 119) of the culture-positive and 100.0% (41 of 41) of the culture-negative specimens from untreated patients. There was a 99.6% concordance between the RMP resistance as determined by culture and by the LiPA test. With an optimal DNA extraction method, LiPA allows rapid detection of M. tuberculosis complex DNA and RMP resistance directly from sputum specimens. LiPA can still provide useful information when culture fails for various reasons. The rapid availability of this information is necessary to adjust patient treatment and avoid the risk of amplification of drug resistance.     

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.     

Molecular methods in the detection and identification of mycobacterial infections.

Nucleic acid amplification (NAA) tests for direct detection of Mycobacterium tuberculosis complex in respiratory specimens have the potential to provide a more rapid diagnosis of pulmonary tuberculosis (TB) than is currently possible by conventional stain, culture, and identification tests. Currently, 2 NAA tests-enhanced Amplified Mycobacterium Tuberculosis Direct (MTD) Test (Gen-Probe, Inc) and Amplicor Mycobacterium tuberculosis Test (Roche Molecular Systems, Inc)-have been approved by the Food and Drug Administration for testing respiratory specimens that are smear positive for acid-fast bacilli (AFB). This restriction to AFB smear-positive specimens was based on data from the initial clinical trials conducted to evaluate these products that showed low sensitivity (ie, 48%-53%) and less-than-optimal specificity (ie, 96%-99%) in AFB smear-negative specimens. Data from the clinical trial for the enhanced MTD test and from 2 subsequent studies, however, suggest that this version of the MTD test is a reliable tool for rapid diagnosis of pulmonary TB, regardless of the AFB smear result. Both NAA tests have been evaluated for diagnosis of extrapulmonary TB, and results were comparable to the results of tests performed with respiratory specimens. The NAA tests also appear to be reliable for rapid identification of M tuberculosis complex in positive broth cultures of all specimen types except blood. The impact of the NAA tests on patient outcome varies based on the AFB smear result. With smear-positive results, public health and hospital infection control resources are predominantly affected. With smear-negative results, however, the potential for affecting patient outcome is much greater. In patients with smear-negative results, the NAA test can result in earlier diagnosis of TB and subsequent initiation of therapy. Use of these tests also may eliminate the need for invasive diagnostic procedures, which are costly and pose an added risk to the patient, and they may allow earlier discharge of hospitalized patients.