A nonphotochromogenic, rapidly growing Mycobacterium strain was isolated in pure culture from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia by using axenic media and an amoebal coculture system. Both isolates grew in less than 7 days at 24 to 37°C with an optimal growth temperature of 30°C. The isolates exhibited biochemical and antimicrobial susceptibility profiles overlapping those of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium immunogenum, indicating that they belonged to M. chelonae-M. abscessus group. They differed from M. abscessus in ␤-galactosidase, ␤-N-acetyl-␤-glucosaminidase, and ␤-glucuronidase activities and by the lack of nitrate reductase and indole production activities, as well as in their in vitro susceptibilities to minocycline and doxycycline. These isolates and M. abscessus differed from M. chelonae and M. immunogenum by exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that of M. abscessus and >99.6% similarity with those of M. chelonae and M. immunogenum. Further molecular investigations showed that partial hsp65 and sodA gene sequences differed from that of M. abscessus by five and three positions over 441 bp, respectively. Partial rpoB and recA gene sequence analyses showed 96 and 98% similarities with M. abscessus, respectively. Similarly, 16S-23S rRNA internal transcribed spacer sequence of the isolates differed from that of M. abscessus by a A3G substitution at position 60 and a C insertion at position 102. Phenotypic and genotypic features of these two isolates indicated that they were representative of a new mycobacterial species within the M. chelonae-M. abscessus group. Phylogenetic analysis suggested that these isolates were perhaps recently derived from M. abscessus. We propose the name of "Mycobacterium massiliense" for this new species. The type strain has been deposited in the Collection Institut Pasteur as CIP 108297 T and in Culture Collection of the University of Göteborg, Göteborg, Sweden, as CCUG 48898 T .During the last few years, the number of nontuberculous mycobacteria (NTM) reported in various clinical situations has greatly increased because of opportunistic infections in immunocompromised patients and improved culture and identification techniques (18). The 16S rRNA gene sequence analysis of NTM led to the description of 40 new species since 1992 and contributed to the description of new clinical forms (19,35,43,44). Particularly, mycobacteria of the Mycobacterium chelonaeMycobacterium abscessus group (M. chelonae, M. abscessus, and Mycobacterium immunogenum) emerged as opportunistic pathogens (45, 47-49, 51), causing hypersensitivity pneumonitis in automobile production workers and chronic lung disease in elderly women with bronchiectasis and in young adults with cystic fibrosis (4,9,16,36,49). Precise species identification in this group of mycobacteria remains difficult. Only two biochemical tests, i.e., sodium chloride tolerance and utili...

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Incidental blood agar-based recovery of Mycobacterium tuberculosis led us to further investigate this routine medium for primary isolation and culture of M. tuberculosis. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (P ‫؍‬ 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node M. tuberculosis isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (P < 0 0.001, ANOVA test). These results demonstrate that M. tuberculosis grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of Bartonella species, should consider the potential safety implications of encountering this highly infectious pathogen.Mycobacterium tuberculosis is a slow-growing bacterium that is the etiological agent of tuberculosis. Agar-based and eggbased media incorporating green malachite and Middlebrook broths or solid media are recommended as the "gold standard" for isolation, culture, and definite diagnosis of M. tuberculosis (6). Although there has been one anecdotal report of the isolation of M. tuberculosis on blood agar (M. Arvand, M. E. Mielke, T. Weinke, T. Regnath, and H. Hahn, Letter, Infection 26:254, 1998), microbiologists and medical students have been taught for decades that isolation of the organism requires a defined, egg-based medium, such as Lowenstein-Jensen medium (6).When trying to isolate Bartonella henselae from a lymph node by prolonged incubation on blood agar (5), we were surprised to isolate M. tuberculosis instead. The purpose of this preliminary work was to evaluate whether blood agar and prolonged incubation could support the growth of a variety of strains of M. tuberculosis obtained from different specimens. We now report that the current basic blood agar medium, used widely for nearly all primary isolations of bacteria, is at least as efficient as the widely recommended Lowenstein Jensen medium.Twenty three Ziehl-Neelsen-positive samples (15 respiratory tract specimens and 8 lymph node aspirates) were inoculated in parallel into tubes containing egg-based Coletsos medium, a local enriched formulation of Lowenstein-Jensen medium (bioMérieux, Marcy l'Etoile, France) and 5% sheep-blood agar (Bio Technologie Appliquée, Dinan, France). The respiratory tract specimens were digested using dithiothreitol and decontaminated using 2% NaOH before inoculation (6). Tubes were incubated at 37°C in a non-CO 2 atmosphere for 30 days, and they were examined every 2 days for the pre...

This investigation was undertaken to assess the effectiveness of an enzyme-linked immunosorbent assay (ELISA) using A60 antigen in ascertaining diagnosis in hospitalized patients suspected to have pulmonary tuberculosis (TB) but with negative sputum stains. Cultures were performed to confirm active or inactive disease. IgG and IgM antibody activity was determined by adding a 1:100 dilution of serum to plates coated with A60 antigen. After addition of peroxidase-conjugated antihuman IgG or IgM and color development, optical density (OD) was determined. A total of 83 patients was studied, taking into account their current disease status and prior history. Using as a cutoff value the mean value +/- 2 SD measured in the negative culture, no TB history group, that is, OD = 0.50 for IgG measurements and 0.43 for IgM measurements, the sensitivity, specificity, and positive predictive value of IgG measurements were equal to 48, 71, and 50%, respectively. Using IgM measurements, these parameters were equal to 76, 98, and 95%, respectively. Combining the results of IgG and IgM measurements, sensitivity, specificity, and positive predictive value were equal to 68, 100, and 100%, respectively. Thus, the ELISA described here can greatly facilitate the diagnosis of TB in patients with negative smears.

Two ELISA tests (IgG and IgM) for the serodiagnosis of tuberculosis, both based on antigen 60 (A60) of M. bovis BCG, were applied to 1,644 controls and patients to analyse the immune response in different forms of this infectious disease. Out of 200 healthy individuals, 148 being tuberculin--positive BCG-vaccinated adults, only 10 contacts--nurses of the pneumology department and laboratory technicians of the mycobacterial laboratory--were found positive for anti-A60 IgG. One quarter of hospitalized patients affected by non-tuberculous pneumopathies (194 in total) were found weakly positive for anti-A60 IgG. We suppose that these positive cases have suffered from inapperant infections and are in a "persistent state". Out of 344 cases of primary pulmonary tuberculosis, 88% were positive for anti-A60 IgG and 75% for the corresponding IgM. Among 97 cases of primary extra-pulmonary tuberculosis, 94% were found IgG positive and 33% IgM positive. The difference between active and inactive post-primary (chronic) tuberculosis was striking: about 100% of both pulmonary and extra-pulmonary cases (367 altogether) had high titers of anti-A60 IgG but IgM positivity was observed in only 15% of the cases, whereas in inactive and quiescent noncavitary tuberculosis (442 cases), 57% of the patients were weakly positive for anti-A60 IgG and none were positive for IgM. Kinetics of synthesis of anti-A60 IgG and IgM were analysed in primary and post-primary (chronic) active tuberculosis. The IgM tracing immune response to A60 was shorter and lower during primary tuberculosis as compared to post-primary tuberculosis. Our findings point to the high prognostic value of the A60- ELISA test for tuberculosis. Anti-A60 IgM mark initial stages of the disease or reactivation processes whereas anti-A60 IgG last longer than IgM and provide an evaluation of the intensity of the infectious process. Repeated serological tests allow monitoring of the course of the infection and the efficacy of therapy. The test is negative in healthy BCG-vaccinated persons (tuberculin-positive) and healed tuberculous infection cases. The combined use of both IgG and IgM tests helps in the correct diagnosis of "false positive" cases.

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The radiometric BACTEC 460-TB methodology has filled an increased need in the screening of a wide range of antimicrobial agents against Mycobacterium avium (MAC) isolates on a patient-to-patient basis. In this context, a multicenter study involving eight test sites across France was performed to determine the MICs of 10 antimicrobial agents for MAC organisms. The aim of the investigation was to compare the in vitro activities of D-cycloserine, ethambutol, ethionamide, rifampin, amikacin, streptomycin, ciprofloxacin, sparfloxacin, clofazimine, and clarithromycin against MAC isolates. All of the test sites received the same clinical isolates of MAC, and the MICs were determined by a common protocol. The overall interlaboratory reproducibility of the MICs within ؎1 dilution of the modal MICs varied from 79.70 to 100% (mean, 95.2% ؎ 2.1%), whereas overall agreement of the MICs among the test sites varied from a mean of 91% ؎ 4.1% to a mean of 98 ؎ 1.3%. We confirmed that the proposed methodology is easy, accurate, and sufficiently reproducible to be used routinely in a clinical laboratory. Despite variations in the MICs of the same drug among strains, no link between the origin of MAC isolates (from human immunodeficiency virus-positive or -negative patients) and their drug susceptibilities was established. On the basis of the MICs that inhibited 50 and 90% of isolates tested for the drugs used, clarithromycin, clofazimine, ethambutol, and streptomycin were the most uniformly active against MAC; this was followed by amikacin, rifampin, and sparfloxacin. On the other hand, ciprofloxacin, Dcycloserine, and ethionamide showed only marginal in vitro activities.