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Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.

The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis complex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosis mc 2 6030 cells into distilled water and M. tuberculosis -negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Genedrive to that of the Xpert MTB/RIF assay using M. tuberculosis cultures as the reference standard. The Genedrive assay had an LOD of 1 pg/μl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10 4 CFU/ml and 2.5 × 10 5 CFU/ml for cells spiked into water and sputum, respectively. False-positive rpoB probe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus , Mycobacterium gordonae , or Mycobacterium thermoresistibile . In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Genedrive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Genedrive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

AccuProbe (Hologic, Marlborough, MA) is an FDA-approved commercial test used for the identification of Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium avium complex (MAC), Mycobacterium gordonae, and Mycobacterium kansasii from growth in solid or

In a previous study, we have evaluated genetic identification by using the rpoB gene, which was recently introduced by Kim et al. (J. Clin. Microbiol. 39:2102-2109, 2001; J. Clin. Microbiol. 37:1714-1720, 1999). In this process, we examined the rpoB gene heterogeneity of clinical isolates identified as Mycobacterium gordonae with the conventional biological and biochemical tests and/or a commercially available DNA probe kit. Sequencing of the rpoB gene of 34 clinical isolates revealed that M. gordonae clinical isolates were classified into four major clusters (A, B, C, and D). Interestingly, organisms belonging to cluster D (15 isolates) did not hybridize with M. gordonae ATCC 14470 and specifically possessed urease activity. Therefore, it could be considered to be a novel mycobacterium. The identification of M. gordonae is known to have ambiguous results sometimes. On the other hand, identification of clinical isolates seems to be inconvenient and unsuitable because of a more than 99% 16S rRNA gene similarity value between clusters. These findings suggest that the existence of M. gordonae -like mycobacteria that share similar biochemical and biological characteristics with the 16S rRNA gene of an M. gordonae type strain but less similarity at the genomic DNA level may have complicated the identification of M. gordonae in many laboratories. Furthermore, compared with hsp65 PCR restriction analysis (PRA), rpoB PRA would have the advantage of producing no ambiguous results because of the intracluster homogeneity of the rpoB gene. In this case, rpoB would provide clearer results than hsp65 , even if PRA analysis was used. We demonstrated that these M. gordonae -like mycobacteria were easily distinguished by PRA of the rpoB sequence. Additionally, the significance of this M. gordonae -like cluster may help to establish the comparison between the M. gordonae isolates from a clinical specimen and an infectious process in a given patient and to determine the true incidence of infection with this microorganism.

The performances of two continuously monitoring mycobacterial culture systems—ESP Culture System II (ESP II; Trek Diagnostics, Inc., Westlake, Ohio) and BACTEC MGIT 960 (BD Biosciences, Sparks, Md.)—were compared. In addition to both liquid media, all specimens were plated onto Middlebrook 7H11/7H11 selective agar. A total of 3,151 specimens of all types (56.3% were respiratory specimens) were cultured; 231 (7.3%) yielded mycobacteria. The most common species recovered were Mycobacterium avium complex (69 isolates) and Mycobacterium tuberculosis complex (MTBC; 65 isolates). The recovery rates for ESP II, BACTEC MGIT 960, and Middlebrook agar, respectively, were 71.2, 63.9, and 61.8% for all mycobacteria; 70.2, 72.6, and 66.3% for all mycobacteria except Mycobacterium gordonae ; and 73.8, 84.6, and 87.7% for MTBC. For liquid plus solid medium combinations, recovery rates for all mycobacteria and for MTBC, respectively, were 84.1 and 92.3% for ESP II plus Middlebrook agar and 81.5 and 98.5% for BACTEC MGIT 960 plus Middlebrook agar. The differences in recovery of all mycobacteria by ESP II and by BACTEC MGIT 960 were not significant; for the individual species, the only significant difference was recovery of more isolates of M. gordonae by ESP II. For those isolates recovered in both automated systems, mean times to detection of all mycobacteria and MTBC, respectively, were 15.8 and 17.4 days for ESP II and 12.5 and 11.9 days for BACTEC MGIT 960 ( P < 0.05). False-positive signals occurred with 23 (0.7%) BACTEC MGIT 960 cultures and 84 (2.7%) ESP II cultures ( P < 0.01). Overall contamination rates were 17.1% for BACTEC MGIT 960, 18.9% for ESP II, and 11.0% for Middlebrook agar. In summary, the ESP II and BACTEC MGIT 960 systems performed comparably with regard to growth and detection of mycobacteria, and the contamination rates were similar. However, with ESP II, times to detection of all mycobacteria and of MTBC were significantly longer, the recovery rate of M. gordonae was significantly higher, and the number of false-positive signals was greater than with BACTEC MGIT 960.

Baths with 2% alkaline glutaraldehyde are often reused for 14 days to decontaminate flexible fiberoptic endoscopes (FFEs) between patients, but the effect of such reuse on the disinfectant's activity has not been known. Many busy endoscopy units also disinfect FFEs with contact times shorter than those recommended by the disinfectant manufacturer. We therefore collected samples of the disinfectant over the 14-day reuse period from two manual and one automatic bath used for bronchoscopes and gastroscopes at a local hospital. Control samples were also collected from a manual bath of 2% alkaline glutaraldehyde which did not receive any endoscopes. The germicidal activities of the samples were assessed in a carrier test against a mixture of hepatitis A virus, poliovirus 1 (Sabin), and Pseudomonas aeruginosa; the mixture also contained either Mycobacterium bovis or Mycobacterium gordonae. Bovine serum (5%) was the organic load. The criterion of efficacy was a minimum of a 3-log10-unit reduction in the infectivity titers of the organisms tested. The initial disinfectant concentration in all the baths was nearly 2.25%; it became about 1.8% in the control bath and fell to approximately 1% in the three test baths after 14 days. No protein was detected in the control bath, while its concentration rose gradually in the test baths to a maximum of 1,267 micrograms/ml after 14 days. With a contact time of 10 min at 20 +/- 2 degrees C, all the samples from the control bath were effective against all the test organisms and all the samples from all the test baths were also effective against P. aeruginosa. With a contact time of 10 or 20 min at 20+/-2 degrees C, the virucidal and mycobactericidal activities of the samples from the test baths showed broad-spectrum germicidal activity when the contact time was increased to 45 min and the temperature was raised to 25 degrees C. These findings emphasize the care needed in the disinfection of FFEs, especially in view of the increasing threat of AIDS and the resurgence of tuberculosis.

The performance and cost of the Capilia TB assay were evaluated for use in a resource-limited setting. The sensitivity and specificity were 99.6% and 99.5%, respectively. The incremental costs of the Capilia test were estimated to be $1.46 and $1.84 when the test was added to liquid and solid culture processes, respectively. These findings suggest that the Capilia TB assay represents a rapid, simple, and inexpensive Mycobacterium tuberculosis identification test that can be used in resource-limited settings.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.

The reliability of the Gen-Probe enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD) for identification of Mycobacterium tuberculosis complex (MTBC) in BACTEC 12B broth cultures of respiratory specimens was evaluated by testing aliquots from 268 bottles with a growth index of ≥50. MTD results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen-Probe, Inc.) testing sediment from broth samples or colonies on a solid medium. For the first 134 cultures, from which 68 mycobacterial isolates (including 27 MTBC isolates) were recovered, both fresh and frozen aliquots were tested. MTD results for the frozen aliquots agreed with the identification by usual protocol in all cases, whereas there was one false-negative MTD result with fresh aliquots. For the remaining 134 cultures, only frozen aliquots were tested. Of the total 268 broth cultures (from 210 patients) evaluated, 137 (51.1%) grew mycobacteria, including 60 MTBC isolates. All 60 isolates were MTD positive, as was one additional culture that grew Mycobacterium gordonae . The latter culture was from a patient who was diagnosed with tuberculosis a few months earlier and was on therapy; therefore, the MTD result was considered a true positive. Sensitivity, specificity, and positive and negative predictive values of MTD were 100%. The mean times from specimen receipt to identification of MTBC were 15 (±1) days (range, 4 to 27 days) for BACTEC plus MTD and 19 (±1) days (range, 6 to 36 days) for the usual protocol ( P < 0.001). These data indicate that the MTD is a rapid, reliable method for identification of MTBC in fresh or frozen aliquots of broth from positive BACTEC 12B cultures of respiratory specimens.

At Yale-New Haven Hospital, 46 specimens submitted for mycobacterial culture during an 8-week period in 1989 were positive for Mycobacterium gordonae, a nontuberculous acid-fast bacterium (AFB) of low pathogenicity. The specimens were submitted from 34 patients who came from various inpatient and outpatient services. Four patients were begun on antimycobacterial therapy on the basis of an AFB isolate which was later identified as M. gordonae. Isolation of M. gordonae was associated with use of the BACTEC TB system (BACTEC TB; Becton Dickinson Diagnostic Instrument Systems, Towson, Md.) and an antimicrobial solution, BACTEC PANTA PLUS (PANTA; Becton Dickinson Diagnostic Instrument Systems). The manufacturer reported that two lots (B9K1 and C9K1) of PANTA kits containing a single production lot (N8C1) of PANTA, which had been shipped to 173 laboratories, had been contaminated with M. gordonae. A survey of mycobacteriology laboratories in the United States revealed that, during April to July 1989, the M. gordonae isolation rate was 5.8/1,000 AFB specimens processed at laboratories that did not use BACTEC TB, 11.4/1,000 AFB specimens at laboratories that used BACTEC TB but not the implicated lot of PANTA, and 23.5/1,000 AFB specimens at laboratories that used BACTEC TB and the lot of implicated PANTA. Intrinsic contamination of PANTA was attributed to ineffective sterilization of water used in the manufacturing process and was not detected prior to product shipment because cultures for AFB were not part of the quality control regimen. This episode emphasizes that clinical laboratories can detect pseudoepidemics promptly if they are alert to abrupt increases in isolation rates, especially of unusual or generally nonpathogenic organisms.

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii , Mycobacterium xenopi , Mycobacterium gordonae , the species of the Mycobacterium avium complex (MAC), Mycobacterium scrofulaceum , and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium , correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae , while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum , and Mycobacterium malmoense , may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

We evaluated the ability of the AccuProbe (Gen-Probe, San Diego, Calif.) to detect Mycobacterium gordonae and Mycobacterium avium complex directly in liquid medium flagged positive by the MB/BacT (Organon Teknika Corp., Durham, N.C.). Seventy-one bottles from clinical specimens containing M. gordonae and 34 containing M. avium , confirmed by culture, were tested by direct AccuProbe assay for both organisms after additional incubation for ≥48 h and centrifugation at 4,500 × g for 15 min. Relative light unit (RLU) values were analyzed using the manufacturer's recommended cutoff of 30,000 RLU and a lower cutoff of 10,000 RLU. Using the 30,000 RLU cutoff, 55 of 71 (77.5%) specimens containing M. gordonae yielded positive results, whereas 28 of 34 (82.3%) M. avium complex specimens were correctly identified by direct probe. No specimens shown by culture to contain either M. gordonae or M. avium complex tested positive with the probe for the opposite organism (100% specificity). When the cutoff was lowered to 10,000 RLU, 67 of 71 M. gordonae (94.4%) and 32 of 34 M. avium complex (94.1%) specimens were correctly identified. This difference was significant for M. gordonae ( P = 0.004) but not for M. avium complex ( P = 0.26) compared to detection using the recommended RLU cutoff. Specificity was 100% for specimens containing M. gordonae that were tested with the M. avium complex probe using the 10,000 RLU cutoff, whereas specificity for specimens containing M. avium complex tested with the M. gordonae probe was 97%. Using a lower RLU cutoff for determining a positive result using the M. gordonae or M. avium complex probes when testing instrument-positive MB/BacT bottles directly will improve sensitivity without substantially compromising specificity.

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.

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.

We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic acid hybridization assay for the detection of Mycobacterium tuberculosis directly from spiked human sputum. Specimens were processed by either an N-acetyl-L-cysteine-NaOH or a 2% NaOH digestion-decontamination method and then washed to neutralize the pH of the cell pellet. The washed sputum pellets were heated at 100 degrees C to inactivate the M. tuberculosis organisms. The heat-inactivated samples were mechanically lysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrument in the presence of zirconium oxide beads and a buffer containing guanidine thiocyanate. The released nucleic acid was subjected to the GENE-TRAK Q-Beta replicase-amplified, dual-capture assay. The assay sensitivity was 10(3) purified rRNA targets or 1 CFU of M. tuberculosis spiked into M. tuberculosis-negative human sputum. There was a low level of noise because of the limitations of performing a signal amplification assay in an open system. High levels of other mycobacterial rRNA (approximately 10(7) organisms), including rRNAs of Mycobacterium avium and Mycobacterium gordonae, did not interfere with the sensitivity of the assay.

Because of the rising incidence of clinical mycobacterial infections and the difficulty in identification and characterization of mycobacteria at the subspecies and serovar levels, a technique for differentiation that could be performed quickly and with relatively little equipment and expense was developed. Lysis and fractionation of mycobacteria by matrix solid-phase dispersion followed by thin-layer chromatography were used to produce chemotype profiles of the lipid and glycolipid components of each isolate. Organisms tested included Mycobacterium scrofulaceum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium flavescens, Mycobacterium kansasii, Mycobacterium bovis, 11 isolates of Mycobacterium gordonae, 10 serovars of Mycobacterium avium, and four strains of Mycobacterium paratuberculosis. A relative retention (Rx) value was established for each visible band on the chromatographs by comparison with a band common to all organisms tested. The chemotype profiles produced were different for every separate species and serovar sampled. These findings suggest that matrix solid-phase dispersion and subsequent thin-layer chromatography may have the sensitivity and flexibility to characterize and identify mycobacteria at the species and subspecies levels and potentially to identify clinical isolates of mycobacteria that have been difficult to identify by standard means. The technique has certain advantages over current biochemical, immunologic, and genetic methods because it is relatively simple to perform, is inexpensive, and requires a small amount of bacterial sample.

The Mycobacterium gordonae Rapid Diagnostic System (Gen-Probe, Inc., San Diego, Calif.) was evaluated for sensitivity and specificity as well as for its application in the mycobacteriology laboratory. An 125I-labeled cDNA probe complementary to rRNA was employed. Hybridization of greater than or equal to 10% was considered positive. A total of 218 mycobacterial isolates, including 159 isolates of M. gordonae, were tested. Under optimum conditions, the specificity and sensitivity of the probe were 100 and 98.7%, respectively. A number of discrepancies were observed between the probe and conventional biochemical results in one laboratory. Further studies, designed to resolve these discrepancies, revealed a number of potential technical pitfalls. Hybridization incubation temperatures that varied from the manufacturer's recommended optimum, culture suspensions below the density of a no. 1 McFarland nephelometer standard, and extended storage times of culture suspension all adversely affected the final hybridization values. Additionally, it was determined that in one laboratory incorrect functioning of the sonicator caused false-negative hybridization values. The manufacturer's recommendations should be strictly followed, and the performance of the sonicator should be checked on a scheduled basis. Results show that the probe will allow fast and accurate identification of M. gordonae, thus eliminating time-consuming biochemical testing of this organism.

Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified.

Isolation techniques with membrane-filtered potable water samples resulted in the isolation of potentially pathogenic high-catalase strains of Mycobacterium kansasii from 8 of 19 representative outlets in a small central Texas town. Mycobacterium gordonae was isolated from all samples, and Mycobacterium fortuitum was isolated from two samples. Data on chlorine levels are presented along with a possible explanation for the unusually high numbers of mycobacteria in these potable water samples. Findings suggest that water is a source of M. kansasii and may be an important link in the epidemiological picture of the disease.

Cross-absorbed antisera have been prepared against catalase from reference strains of Mycobacterium asiaticum, Mycobacterium gordonae, Mycobacterium scrofulaceum, Mycobacterium simiae, and Mycobacterium szulgai. A total of 61 strains of mycobacteria were grown in small volumes of liquid medium and disrupted in sealed tubes in a cup horn sonicator, and the extracts were tested by a seroprecipitation technique against each of the reference antibody preparations. All 35 strains that belonged to one of the reference species reacted with the corresponding antibody, and none of the 61 extracts gave a significant cross-reaction with the absorbed antibody to a species to which it did not belong. This method appears to provide a rapid and accurate means of identifying mycobacterial cultures in the diagnostic laboratory.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.

A new crystalline reagent for nitrate reductase tests was compared with standard liquid reagents on 437 strains of mycobacteria. The results for isolates of Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae, Mycobacterium scrofulaceum, Mycobacterium fortuitum, and Mycobacterium chelonei agreed 100% with the expected results. Of the 177 Mycobacterium tuberculosis isolates, 4 were negative by the conventional method. Two of these four isolates were positive with the new reagent. Of the positive nitrate tests carried out with liquid reagents, 42% flashed instantly or faded in color; none of the tests carried out with the new crystalline reagent flashed or faded. A stronger color reaction was seen for 28% of the positive tests with the new reagent.

The first evidence of the potential pathogenicity of Mycobacterium thermoresistibile is presented. This mycobacterium, initially identified as Mycobacterium gordonae, was isolated repeatedly from sputum, a bronchoscopy specimen. and later, an open lung biopsy. The distinctive characteristics are described, including the unique ability of the organism to grow at 52 degrees C.

A simple procedure for detecting a few cells of Mycobacterium gordonae in laboratory water that yielded spurious smear results is described.