A fatal case of pulmonary infection by Mycobacterium colombiense in Para State, Amazon Region, Brazil.

Mycobacterium avium complex (MAC) is a heterogeneous group of species found in several environmental sources and that exhibit variable degrees of pathogenicity. Among the MAC members, Mycobacterium colombiense has been related to pulmonary disease and disseminated infection in HIV-infected patients in Colombia. Lymphadenopathy cases have also been reported. We have described a fatal case of M. colombiense pulmonary disease in a Brazilian patient without evidence of HIV infection or other known causes of immunosuppression.

Description of a new Mycobacterium intracellulare pattern of PCR restriction enzyme analysis of hsp65 gene.

This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile.

Identification of potential biomarkers to distinguish Mycobacterium colombiense from other mycobacterial species.

Mycobacterium avium complex (MAC) consists of 9 species of slow-growing mycobacteria with differing degrees of pathogenicity, host preference and environmental distribution. Mycobacterium colombiense is a novel member of MAC that is responsible for disseminated infections in HIV-infected patients in Colombia and lymphadenopathy cases in Europe. At present, methods to easily differentiate novel members of MAC are lacking. In this study, we identified possible biomarkers that are potentially useful for the detection of M. colombiense by PCR or chromatography. The Randomly Amplified Polymorphic DNA (RAPD) technique was used to amplify genomic fragments of M. colombiense CECT 3035 that were subsequently used in the development of a direct colony-specific PCR assay using specific primers. The designed primers amplified a 634-bp fragment of DNA from M. colombiense, which included a 450-bp genomic region that encodes a hypothetical protein of 149 amino acids that is exclusive to M. colombiense. Bioinformatic analyses revealed that this hypothetical protein had no signal peptide, active sites or functional domains to aid its identification or classification. In addition, using thin-layer chromatography, we identified a different profile of mycolates for M. colombiense strains. The test developed in this study has potential applications in the routine identification of M. colombiense and in molecular assays designed for the surveillance of MAC strains.

Genome sequence of the Mycobacterium colombiense type strain, CECT 3035.

We report the first whole-genome sequence of the Mycobacterium colombiense type strain, CECT 3035, which was initially isolated from Colombian HIV-positive patients and causes respiratory and disseminated infections. Preliminary comparative analyses indicate that the M. colombiense lineage has experienced a substantial genome expansion, possibly contributing to its distinct pathogenic capacity.

Mycobacterium avium complex infection in HIV/AIDS patients.

Disseminated Mycobacterium avium complex (MAC) infection is a severe complication of advanced HIV/AIDS disease. Disseminated infection due to MAC appeared later in the natural history of HIV disease and was an independent predictor of mortality in patients before the extended use of highly active antiretroviral therapy (HAART). The use of combination schemes, including three or four antimicrobial agents followed by secondary prophylaxis and HAARTs, improved the survival and reduced mortality rates. However, subjects who ignore their serological status for HIV, or who are not receiving or do not tolerate HAART, are at high risk of developing disseminated MAC disease. In addition, patients who show a good immunological and virological response to HAART can develop episodes of immune reconstitution inflammatory syndrome associated with MAC, including supurative lymphadenitis and subcutaneous or soft-tissue abscesses. In this article, we describe the epidemiological, clinical, immunological, therapeutic and preventive aspects of MAC infection in HIV-seropositive patients in the pre- and post-HAART era.

Molecular features of Mycobacterium avium human isolates carrying a single copy of IS1245 and IS1311 per genome.

Human clinical isolates of the Mycobacterium avium complex, from hospitals in Bogotá, were studied using a wide range of molecular tests including PCR restriction-enzyme analysis (PRA) of the hsp65 gene. Up to 21 of the isolates were identified as M. avium PRA variant III (Mav III), a variant obtained only from isolates on the American continent. In contrast to previous reports, restriction fragment length polymorphism analysis using IS1245 and IS1311 showed a single copy for each insertion sequence (IS) in the majority (19/21) of the Colombian Mav III isolates under study. In order to analyse whether the ISs were inserted in a relevant genomic region, experimental conditions were established to determine the insertion loci of each single copy of both ISs in the genome. Analysis of genomic insertion loci indicated that both IS1245 and IS1311 were present in areas containing putatively truncated integrases and/or transposases, which may have an influence on the mobility of the inserted IS. In addition, a conserved genomic region was identified for the insertion of IS1311; this region could be part of the IS1311 itself.

Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant.

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B(T) (=CIP 108962(T)=CECT 3035(T)).

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America.

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

IS1245 restriction fragment length polymorphism typing of Mycobacterium avium from patients admitted to a reference hospital in Campinas, Brazil.

Mycobacterium avium is an important pathogen among immunodeficient patients, especially patients with AIDS. The natural history of this disease is unclear. Several environmental sources have been implicated as the origin of this infection. Polyclonal infection with this species is observed, challenging the understanding of its pathogenesis and treatment. In the present study 45 M. avium strains were recovered from 39 patients admitted to a reference hospital between 1996 and 1998. Species identification was performed using a species-specific nucleic acid hybridization test (AccuProbe) from Gen-Probe. Strains were genotyped using IS1245 restriction fragment length polymorphism typing. Blood was the main source of the organism. In one patient with disseminated disease, M. avium could be recovered more than once from potentially sterile sites. Strains isolated from this patient had different genotypes, indicating that the infection was polyclonal. Four patient clones were characterized in this population, the largest clone being detected in eight patients. This finding points to a common-source transmission of the organism.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria.

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

[Mycobacterium avium-intracellulare complex: phenotypic and genotypic markers and the molecular basis for interspecies transmission]. / Le complexe Mycobacterium avium-intracellulare: marqueurs phénotypiques et génotypiques et les bases moléculaires de la transmission inter-espèces.

The Mycobacterium avium complex (MAC) comprises a heterogeneous group of slowly-growing mycobacteria that are pathogenic for both humans and animals. Two genetically distinct species within MAC are M. avium, which tends to infect HIV-infected patients, and M. intracellulare more common among immunocompetent individuals. Contrary to M. intracellulare which relates to a single species, M. avium is separated into three subspecies; M. avium subsp. avium, a major opportunistic pathogen leading to a disseminated disease among terminal AIDS patients; M. avium subsp. paratuberculosis, causing Johne's disease among ruminants and implicated in Crohn's disease among humans; and M. avium subsp. silvaticum, a pathogen affecting birds that may cause chronic enteritis among calves but has not yet been associated with human disease. With the exception of mycobactin-dependent growth of M. paratuberculosis, most of the biochemical and cultural tests cannot discriminate among the three subspecies of M. avium. However, recently developed molecular methods and fingerprinting of strains using insertion sequences allows not only to distinguish among them but also further to explore the polymorphism of human and animal isolates. Numerous studies have underlined the probable role of various ecological niches (water, dust, soil, pigs, poultry and ruminants etc.) as a possible source of contamination for AIDS patients. This paper reviews the phenotypic and genotypic markers and epidemiology of M. avium complex organisms and current knowledge of the molecular basis of of inter-species transmission.

Experiencia de una década en la tipificación y resistencia a fármacos antituberculosos en micobacterias aisladas de pacientes VIH+/SIDA / Ten years experience in practicing identification and susceptibility tests on mycobacteria isolated from HIV+/AIDS patients

El análisis de las pruebas de tipificación y de la resistencia a fármacos antituberculosos de micobacterias aisladas de pacientes seropositivos al VIH, en la década de los noventas, mostró una proporción de micobacterias no tuberculosas de 23,6 por ciento y una resistencia primaria y adquirida a los fármacos antituberculosos de primera línea de 17,5 por ciento y 43,9 por ciento respectivamente; estas cifras son significativamente más elevadas que las observadas en enfermos tuberculosos VIH(-). Estos hallazgos confirman la conveniencia de continuar la práctica sistemática de las pruebas mencionadas en todo aislamiento de micobacterias proveniente de pacientes VIH+, para adecuar oportunamente y de acuerdo a sus resultados, los esquemas terapéuticos a emplear

Comparison among three methods for mycobacteria identification.

OBJECTIVE: To compare three methods: Biochemical tests, high-performance liquid chromatography (HPLC) and polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP), for the identification of mycobacteria, and to perform a cost-benefit analysis to define an optimum identification algorithm. MATERIAL AND METHODS: One-hundred-and-seven mycobacteria isolates were identified by the three methods at Instituto de Diagnóstico y Referencia Epidemiológicos, between February of 1999 and January of 2000 and the results were compared with those of a reference laboratory using the Q-Cochran statistical test. RESULTS: PCR-RFLP was the most rapid and specific procedure but also the most expensive; biochemical tests excelled for identification of Mycobacterium tuberculosis, but were lengthy and expensive for other mycobacteria; HPLC ranked in the middle for price, speed and specificity. CONCLUSIONS: Considering the expected proportion of M. tuberculosis, the following algorithm was proposed: Initially, biochemical tests should be performed; if the results indicate a non-tuberculous mycobacteria, the isolate should be analyzed with HPLC; if results are unclear, the isolate should be analyzed using PCR-RFLP. Isolates showing a previously undescribed PCR-RFLP pattern should be characterized by DNA sequencing.

IS1245 genotypic analysis of Mycobacterium avium isolates from patients in Brazil.

OBJECTIVE: Disseminated Mycobacterium avium infection is an emerging opportunistic disease among patients with acquired immunodeficiency syndrome (AIDS) in Brazil. The mode of transmission of M. avium in a developing country setting needs to be better characterized. METHODS: Mycobacterium avium strain collections in São Paulo and Rio de Janeiro were analyzed according to the strains' IS1245 DNA gel electrophoretic migration patterns. Medical records of the patients from whom M. avium isolates were available were reviewed, and their demographic characteristics were stratified according to the isolates' IS1245 DNA fingerprint patterns. RESULTS: Of 105 patients, 33 (31%) with M. avium isolated between 1990 and 1994 had strains having IS1245 patterns identical in patterns seen in isolates from two or more patients (designated as cluster pattern strains). Cluster pattern strains were isolated from 21 (39%) of 54 patients with disseminated infection (defined as infection due to M. avium isolated from a sterile site in an adult patient). Six of the cluster pattern strains were isolated only from sterile sites. In São Paulo, cluster pattern strains were significantly more likely to be isolated from patients with disseminated disease. CONCLUSIONS: These preliminary observations suggest that in large cities of Brazil, a high proportion (at least 39%) of disseminated M. avium infections in patients with AIDS results from a recent transmission. Some strains of M. avium may be more likely to cause disseminated disease than others after an infection.

Molecular epidemiology of Mycobacterium avium complex isolated from patients with and without AIDS in Brazil and England.

Mycobacterium avium complex (MAC) is ubiquitous throughout the world. It is an opportunistic pathogen in AIDS patients but the number of cases in HIV negative patients is also increasing. The aim of this study was to determine whether patients were being infected with different MAC strains or whether one strain was dominant. DNA obtained from isolates in Brazil and England were compared using pulsed field gel electrophoresis (PFGE). Strains from 22 Brazilian patients clustered into 7 groups but 68/90 patients had a unique strain. In all patients, Brazilian and English, the same strain was isolated repeatedly over time, some over several years. This study shows that it is most likely that Man is infected from the environment and that one strain can survive without change for many years both in the environment and in Man.

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwise have been missed by the AccuProbe system in a routine clinical microbiology laboratory.

Analysis of Mycobacterium avium complex serovars isolated from AIDS patients from southeast Brazil.

The purpose of this study was to assess the distribution of Mycobacterium avium serovars isolated from AIDS patients in São Paulo and Rio de Janeiro. Ninety single site or multiple site isolates from 75 patients were examined. The most frequent serovars found were 8 (39.2%), 4 (21.4%) and 1 (10.7%). The frequency of mixed infections with serovar 8 or 4 was 37.8%. Among the 90 strains examined, M. intracellulare serovars (7 strains) and M. scrofulaceum (4 strains) were found in 11 isolates (12%) indicating that M. avium (88%) was the major opportunistic species in the M. avium complex isolates in Brazilian AIDS patients.

PIP: Mycobacterium avium complex (MAC) organisms have been associated with severe opportunistic infections in patients with AIDS in the US. The present study analyzed the distribution of 90 MAC serovars isolated from 75 AIDS patients from Sao Paulo and Rio de Janeiro, Brazil, in 1990-94. 56 isolates (62.2%) showed a single serovar--predominantly serovars 8 (39.2%), 4 (21.4%), and 1 (10.7%). In the 34 isolates (37.8%) in which more than one serovar was identified, 19 (55.9%) were from a single site and 15 (44.1%) were isolated from different sites or from the same site but at different times. The most common serovars in mixed infections from both single and different sites were 8 (34.2% and 37%, respectively)) and 4 (21.1% and 25.9%, respectively). Only 11 isolates (12%) were M. intracellulare or M. scrofulaceum strains, indicating that M. avium was the opportunistic species in 88% of the MAC isolates in these Brazilian AIDS patients. Although the serovars detected in this series are similar to those found in US AIDS patients, the occurrence of mixed serovars was substantially higher in Brazilian AIDS patients. The clinical implications of polyclonal infections in Brazilian AIDS patients require further investigation, especially since serovars from distinct sites may have distinct drug resistance patterns.

Computer-assisted analysis of Mycobacterium avium fingerprints using insertion elements IS1245 and IS1311 in a Caribbean setting.

A total of 33 clinical isolates of the Mycobacterium avium complex from 25 patients, identified by means of biochemical and cultural characteristics, the Accuprobe system and DT1/DT6 PCR, were further analysed using novel insertion elements IS1245 and IS1311 in a French Caribbean setting. PvuII-cleaved DNA and non-radioactive Southern hybridization and detection systems were used for fingerprinting with both IS elements. The data confirmed the specificity of the two probes for M. avium in our setting and highlighted a significant proportion of M. intracellulare-infected patients in this region. Two distinct groups composed of 2-3 bands and 6-27 bands were found among M. avium isolates, and were composed of the same isolates both with IS1245 and IS1311. The computer analysis of polymorphic banding patterns identified two prevalent genotypes: one contained 4 isolates from 3 patients while a second 2-banded cluster was composed of 6 isolates from 4 patients; all the patients were from the same hospital in Guadeloupe. A single isolate from Martinique was falsely included in the 2-banded cluster initially upon IS1245 fingerprinting, but could be discriminated from other isolates on the basis of IS1311 fingerprinting of PvuII-cleaved DNA. These results were also confirmed upon IS1245 fingerprinting of PstI-digested DNA, as well as DT6 fingerprinting. A single case of polyclonal infection was also discovered in a patient at a 75-day interval. This is the first study comparing the two IS elements and constitutes a first description of disseminated M. avium complex disease from the Caribbean. We conclude that both elements possess a similar discriminatory potential for M. avium isolates. Coupled with computer analysis, this methodology would appear to be particularly suitable for larger epidemiological studies.

[Taxonomic study of species of the Mycobacterium genus isolated in Cuba]. / Estudio taxométrico de especies del género Mycobacterium aisladas en Cuba.

40 strains of the Mycobacterium genus corresponding to 12 species, which were subjected to 62 microbiological and biochemical tests, were studied. Each one was considered as a character. As a result of the similitude coefficient and their grouping, 9 phenomes represented by: Phenome I (Mycobacterium fortuitum), Phenome II (MAI Complex), Phenome III (Mycobacterium phlei), Phenome IV (Mycobacterium triviale), Phenome V (Mycobacterium smegmatis), Phenome VI (Mycobacterium gordonae), Phenome VII (Mycobacterium szulgai), Phenome VIII (MAI Complex), and Phenome IX (Mycobacterium scrofulaceum), were obtained. The strain identification work was consistent with grouping from the phenotypic point of view.