Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico.

BACKGROUND: Little information is available on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites in humans. This study used molecular methods to determine the Mycobacterium species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease. METHODS: Bacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the Mycobacterium species present. DNA samples positive for M. tuberculosis complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the rpoB gene associated with rifampicin resistance. RESULTS: Of the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for Mycobacterium species by PCR or line probe assay, respectively. In tissues, M. tuberculosis complex and M. genus were found in lymph nodes, and M. genus was found in brain and vagina specimens. In body fluids, M. tuberculosis complex was found in synovial fluid. M. gordonae, M. smegmatis, M. kansasii, M. genus, M. fortuitum/M. peregrinum complex and M. tuberculosis complex were found in urine. M. chelonae/M. abscessus was found in pericardial fluid and M. kansasii was found in gastric aspirate. Two of M. tuberculosis complex isolates were also PCR and LiPA positive for the rpoB gene. These two isolates were from lymph nodes and were sensitive to rifampicin. CONCLUSION: 1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found.

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR-restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S-23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory.

BACKGROUND: The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only. RESULTS: During 1998-2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern. CONCLUSIONS: In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay.

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.

Mycobacterium noviomagense sp. nov.; clinical relevance evaluated in 17 patients.

Eighteen isolates of a nonchromogenic, slowly growing, non-tuberculous species of the genus Mycobacterium were cultured from respiratory specimens obtained over the last eight years from 17 patients in the Netherlands. These isolates were grouped because they revealed a unique 16S rRNA gene sequence and were related to Mycobacterium xenopi. None of the 17 patients met the American Thoracic Society diagnostic criteria for non-tuberculous mycobacterial disease, which distinguishes the novel isolates from the related species, M. xenopi. A polyphasic taxonomic approach, including identification by biochemical and phenotypical analysis, hsp65 gene sequencing and PCR restriction enzyme pattern analysis, and sequence analyses of the rpoB gene and 16S-23S internal transcribed spacer supported the separate species status of the novel isolates. The name Mycobacterium noviomagense sp. nov. is proposed for the novel strains. The type strain is NLA000500338(T) (=DSM 45145(T)=CIP 109766(T)). A more distinctive taxonomy of NTM is a prerequisite for the assessment of their clinical relevance.

ESAT-6 and CFP-10 in clinical versus environmental isolates of Mycobacterium kansasii.

Mycobacterium kansasii consists of 5 genetically distinct groups, of which 2 are associated with human disease. Determinants of the differences in virulence are unknown. Potential genes of interest are esat-6 and cfp-10, which are associated with virulence of Mycobacterium tuberculosis and Mycobacterium bovis but are lacking in bacille Calmette-Guérin and in most environmental mycobacteria (M. kansasii is an exception). We investigated esat-6 and cfp-10 genes in 22 clinical and 14 environmental isolates of M. kansasii. Both were present in all isolates; each genetic group had its own characteristic Southern-blot pattern corresponding to a highly conserved fingerprint pattern. Nucleotide sequences of the genes differed 12.6% and 10.1%, respectively, from the M. tuberculosis homologues, but the deduced amino acid sequences were <5% different. In vitro, clinical and environmental genotypes of M. kansasii expressed CFP-10 and ESAT-6. Thus, virulence of M. kansasii is not directly related to esat-6 and cfp-10 genes or gene expression.

Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and 'M. avium subsp. hominissuis' for the human/porcine type of M. avium.

In an attempt to clarify the taxonomy of the Mycobacterium avium complex, the relationship between IS1245 RFLP, growth temperature, 16S rDNA signature sequences and the 16S-23S rDNA internally transcribed spacer (ITS) of 160 M. avium-complex isolates from different sources was investigated. All 70 isolates identified as M. avium by INNO-LiPA MYCOBACTERIA (Innogenetics, Belgium), a DNA probe test that targets the ITS, and by 16S rDNA analysis carried multiple copies of IS1245. Three isolates with multiple copies of IS1245 were identified by 16S rDNA analysis as Mycobacterium intracellulare and by LiPA as M. intracellulare (n = 1) and M. avium-intracellulare complex (n = 2). A dichotomy among the M. avium isolates was found on the basis of a C and a G signature nucleotide at position 228 of the 16S-23S rDNA spacer sequence, and this grouping was largely confirmed on the basis of similarities in IS1245 RFLPs. Strains with the characteristic three-band IS1245 'bird-type', as well as M. avium subsp. silvaticum or 'wood-pigeon' strains, invariably contained the C signature. A third characteristic that separated the M. avium bird-type isolates from M. avium isolates from humans and other mammals was growth-temperature tolerance: in contrast to bird isolates, human/porcine isolates grew at 24 and 45 degrees C. Based on differences in IS1245 RFLP, 16S-23S rDNA ITS and growth temperature, M. avium isolates originating from birds should be considered as a separate, evolutionarily conserved taxon. Because all M. avium isolates from birds are invariably of this type, the designation M. avium subsp. avium should be reserved for these bird-type strains. For clarity in the epidemiology of M. avium-related disease, isolates from humans and pigs with multibanded IS1245 RFLPs merit a separate designation. The designation 'M. avium subsp. hominissuis' is suggested for this group of bacteria.