Discordant molecular characterization results in a Mycobacterium avium complex strain isolated from an AIDS patient.

This report describes an unusual strain of Mycobacterium avium complex isolated from the sputum of an immunocompromised AIDS patient, which did not react with the MAC probe of the BDProbe Tec system, but was identified as Mycobacterium intracellulare by 16S rRNA gene sequencing. Its PCR restriction-enzyme analysis pattern was compatible with an allelic variant of M. avium. It was scotochromogenic, slow-growing and phenotypically identified as Mycobacterium scrofulaceum. Its clinical significance is not certain.

Use of the INNO-LiPA-MYCOBACTERIA assay (version 2) for identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex isolates.

Using INNO-LiPA-MYCOBACTERIA (Lipav1; Innogenetics) and the AccuProbe (Gen-Probe Inc./bioMérieux) techniques, 35 Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAC/MAIS) complex strains were identified between January 2000 and December 2002. Thirty-four of 35 isolates were positive only for the MAIS complex probe by Lipav1 and were further analyzed by INNO-LiPA-MYCOBACTERIA version 2 (Lipav2), hsp65 PCR restriction pattern analysis (PRA), and ribosomal internal transcribed spacer (ITS), hsp65, and 16S rRNA sequences. Lipav2 identified 14 of 34 strains at the species level, including 11 isolates positive for the newly specific MAC sequevar Mac-A probe (MIN-2 probe). Ten of these 11 isolates corresponded to sequevar Mac-A, which was recently defined as Mycobacterium chimerae sp. nov. Among the last 20 of the 34 MAIS isolates, 17 (by hsp65 PRA) and 18 (by hsp65 sequence) were characterized as M. avium. Ten of the 20 were identified as Mac-U sequevar. All these 20 isolates were identified as M. intracellulare by 16S rRNA sequence except one isolate identified as Mycobacterium paraffinicum by 16S rRNA and ITS sequencing. One isolate out of 35 isolates that was positive for M. avium by AccuProbe and that was Mycobacterium genus probe positive and MAIS probe negative by Lipav1 and Lipav2 might be considered a new species. In conclusion, the new INNO-LiPA-MYCOBACTERIA allowed the identification of 40% of the previously unidentified MAIS isolates at the species level. The results of the Lipav2 assay on the MAIS isolates confirm the great heterogeneity of this group and suggest the use of hsp65 or ITS sequencing for precise identification of such isolates.

Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae.

A group of pigmented, slowly growing mycobacteria identified by 16S rRNA gene sequencing as 'MCRO 33' (GenBank accession no. AF152559) have been isolated from several clinical specimens in various laboratories across Canada. Genotypically, the organism is most closely related to Mycobacterium simiae. However, it presents with a similar phenotypic profile to Mycobacterium scrofulaceum. Several reference strains obtained from ATCC and TMC culture collections, previously identified as M. scrofulaceum or M. simiae, have also been found to possess the MCRO 33 16S rRNA gene sequence. Biochemical testing, susceptibility testing, HPLC, hsp65 gene and 16S-23S spacer (ITS1) sequencing were performed on clinical and reference strains to characterize further this unique species. Of the clinical strains, one was isolated from a cervix biopsy whereas all other clinical isolates were obtained from respiratory samples. In one patient, symptoms, imaging and repeat clinical specimens positive on culture for this organism were suggestive of active clinical disease. The description of this species, for which the name Mycobacterium parascrofulaceum sp. nov. is proposed, follows the present trend of a large number of novel Mycobacterium species identified due in great part to sequence-based methods. The type strain is HSC68T (= ATCC BAA-614T = DSM 44648T).

Evidence of the presence of IS1245 and IS1311 or closely related insertion elements in nontuberculous mycobacteria outside of the Mycobacterium avium complex.

A PCR assay based on the simultaneous detection of IS1245 and IS1311 was developed and used to determine the host range of these insertion elements. Specific PCR products were observed in Mycobacterium malmoense, Mycobacterium scrofulaceum, and Mycobacterium nonchromogenicum, indicating that IS1245 and IS1311 are not limited to the Mycobacterium avium complex.

Evaluation of the LiPA MYCOBACTERIA assay for identification of mycobacterial species from BACTEC 12B bottles.

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacterium species and differentiates M. tuberculosis complex, M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonae group, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive for M. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for Mycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

[Clinical analysis of 96 cases with pulmonary disease caused by nontuberculous mycobacteria].

OBJECTIVE: To study the clinical characteristics of nontuberculous mycobacteria (NTM) pulmonary disease. METHODS: Ninety-six out of 173 cases with NTM pulmonary disease diagnosed through identification of mycobacterium strains isolated from 5,592 sputum acid-fast staining positive cases from 1981-1996 were selected, and a retrospective analysis was made. RESULTS: According to Runyon classification, there were 14 cases with M. kansasii and 1 with M. marinum in type I, 4 with M. scrofulsceum in type II, 23 with M. intracellulare or M. avium in type III, 24 with M. chelonae and 30 with M. fortuitumin in type IV. Cases whose courses of disease were more than 10 years accounted for 31%. Main clinical symptoms included cough (78%), expectoration (71%), haemoptysis (58%) and fever (26%). Fifty-seven percent cases in X-ray chest film were seen lesions bilaterally, 42% in one side, 27% in the right and 15% in the left. One percent showed no obvious lesion in X-ray chest film. Excluding one case with incomplete data, the total resistance rate of NTM was 96% in other 95 cases, 93% in type I, 50%, 100% and 100% in type II, III, IV respectively. After antituberculous chemotherapy, sputum negative conversion was seen in 14 of 15 cases with type I NTM, 6 of 23 with type III and 14 of 54 with type IV, while definite data in 4 cases with type II were not available. CONCLUSIONS: NTM pulmonary disease is characterized by long course of disease, nonspecific symptoms, high resistance rate and unsatisfactory therapeutic efficacy.

Identification and subspecific differentiation of Mycobacterium scrofulaceum by automated sequencing of a region of the gene (hsp65) encoding a 65-kilodalton heat shock protein.

Mycobacterium scrofulaceum is most commonly recovered from children with cervical lymphadenitis, although it also accounts for approximately 2% of the mycobacterial infections in AIDS patients. Species assignment of M. scrofulaceum isolated by conventional techniques can be difficult and time-consuming. To develop a strategy for rapid species assignment of these organisms, a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein in 37 isolates from diverse sources was sequenced. Eight hsp65 alleles were identified, and these sequences formed phylogenetic clusters and lineages largely distinct from other Mycobacterium species. There was incomplete correlation between serovar designation and hsp65 allele assignment. The hsp65 data correlated strongly with the results of sequence analysis of the gene coding for 16S rRNA. Automated DNA sequencing of a 360-bp region of the hsp65 gene provides a rapid and unambiguous method for species assignment of these acid-fast organisms for diagnostic purposes.

Serovar determination and molecular taxonomic correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: a cooperative study of the International Working Group on Mycobacterial Taxonomy.

A cooperative study was conducted by the International Working Group on Mycobacterial Taxonomy to correlate the agglutination serovar designations of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains with the species ascriptions of these organisms according to molecular criteria and cultural properties and to assess the reproducibility of serovar determinations for a set of 63 reference strains of these species. Among the molecular criteria, the level of agreement between results obtained with nucleic acid probes and T-catalase serology results was 94% for strains of M. avium and M. intracellulare. Nucleic acid probes were not available for M. scrofulaceum, but none of the 10 strains ascribed to this species on the basis of catalase serology data reacted with a nucleic acid probe for M. avium or M. intracellulare. Ascription to a species on the basis of mycolic acid high-performance liquid chromatography patterns was in agreement with catalase serology results in 86% of the cases examined. Most strains belonging to serovars 1 through 6 and 8 through 11 were identified by molecular criteria as M. avium, most strains belonging to serovars 7, 12 through 20, 23, and 25 were identified as M. intracellulare, and most strains belonging to serovars 41 through 43 were identified as M. scrofulaceum, in agreement with common current practice. Evidence for assigning serovar 27 to M. scrofulaceum was obtained. However, two strains of a given serovar may, on occasion, be placed in different species. The dominant species assignments for strains belonging to serovars 21, 24, 26, and 28 remain unresolved.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Mycobacterium avium complex strains belonging to serovars 21-28 by three commercial DNA probe tests.

Various reference strains of Mycobacterium avium complex (MAC) belonging to serovars 21-28 were identified by three DNA probe tests, i.e., Gen-Probe, AccuProbe and SNAP tests. All of these DNA probe tests were in agreement for strains identified as M. avium or M. intracellulare. The tested serovar strains involved M. avium, M. intracellulare, MAC reactive only with Probe X of SNAP test ('Probe X-reactive MAC'), M. scrofulaceum reactive with Probe X of SNAP test ('Probe X-reactive M. scrofulaceum'), and typical M. scrofulaceum which did not react with any of the probes. Both reference strains belonging to serovar 21 were M. avium, and none of the other serovars included this species. On the contrary, M. intracellulare was found in serovars 22, 25, 26, and 28. 'Probe X-reactive MAC' were also widely found in serovars 23, 24, 26, 27, and 28, while 'Probe X-reactive M. scrofulaceum' was seen only in serovar 22. These results confirm the usefulness of SNAP test to identify the MAC showing no reactivity to Gen-Probe and AccuProbe.

A simple and rapid method for the detection and identification of mycobacteria using mycobactin.

A system was developed for the identification of mycobacteria such as Mycobacterium tuberculosis and M. avium, by thin layer chromatography of 55Fe-labelled mycobactin. Approximately 2 x 10(3) mycobacteria were detected within 24 h and little operator time or skill was required. M. avium, M. intracellulare and M. scrofulaceum were found to have lower requirements for iron than other mycobacteria and this may influence their growth in host organisms.

Analysis of cellular fatty acids and proteins by capillary gas chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis to differentiate Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) complex species.

Infections due to atypical mycobacteria have increased during the past 30 years. Species of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum are among the most common non-tuberculous mycobacteria isolated from patients with AIDS or immunosuppressed. These three organisms are taxonomically closely related and identification, according to cultural characteristics and biochemical tests, is not always evident, so some of these related strains are grouped in a "MAIS" complex. Analysis of cellular constituents is an aid to identification. Gas chromatography was used to study mycolic acids and a secondary alcohol was found which is a discriminating constituent between M. scrofulaceum and the other two species. The lipidic analysis was not able to separate M. avium and M. intracellulare, so cell proteins were considered. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins reflects genetic relatedness between strains; the different patterns obtained from these three species are described and it is shown that this method is very useful in classification and epidemiology.

Epidemiology of infection by nontuberculous mycobacteria IX. Evidence for two DNA homology groups among small plasmids in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.

A 12.9 kb plasmid, pVT2, from a clinical Mycobacterium avium isolate, MD1, was cloned and radiolabeled for use as a DNA probe to examine the relatedness of plasmids in M. avium complex. That probe hybridized with plasmids isolated from M. avium complex strains from the environment (7 of 16) and from non-acquired immunodeficiency syndrome (AIDS) (10 of 17) and AIDS (5 of 6) clinical isolates. The similarity of plasmids from the environment with those from patients supports the hypothesis that the environment is a source of human M. avium complex infection. More striking was the observation that pVT2 hybridized with every plasmid (13 of 13 clinical and 5 of 5 environmental isolates) of 13.5 kb or smaller. A second probe, consisting of a 15.3 kb plasmid (pLR7) from another clinical isolate of the M. avium complex, hybridized with plasmids of 15.3 to 25 kb from environmental and clinical (AIDS and non-AIDS) isolates. There was no hybridization between pVT2 and pLR7. Thus, these two probes define two different groups of small mycobacterial plasmids.

[Serotyping of strains of non-tuberculous mycobacteria]. / Serotipaje de cepas de micobacterias no tuberculosas.

Forty strains of mycobacteria, belonging to Mycobacterium-avium-intracellulare-scrofulaceum complex, isolated from symptomatic respiratory patients, were studied. For such study, agglutination-adsorption technique was applied, using specific antisera elaborated at the "Pedro Kouri" Institute of Tropical Medicine, National Reference Institute, with titers ranging close to 1:320. The results obtained demonstrated that the prevailing types were those of the species Mycobacterium intracellulare (31 strains), prevailing serotypes 9 (Darden), 8 (Davis), 12 (Haweel) and serotype 26 followed by the species M. avium (3 strains) and M. scrofulaceum (2 strains).