RESUMO
Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller-Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller-Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3-5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000-0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000-0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/efeitos dos fármacos , Humanos , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/microbiologia , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/crescimento & desenvolvimento , Micobactérias não Tuberculosas/isolamento & purificação , Fatores de TempoRESUMO
BACKGROUND: Although risk factors for HIV infection are known, it is important for blood centres to understand local epidemiology and disease transmission patterns. Current risk factors for HIV infection in blood donors in Brazil were assessed. METHODS: A case-control study was conducted at large public blood centres located in four major cities between April 2009 and March 2011. Cases were persons whose donations were confirmed positive by enzyme immunoassays followed by Western blot confirmation. Audio computer-assisted structured interviews (ACASI) were completed by all cases and controls. Multivariable logistic regression was used to estimate adjusted odds ratios (AORs) and associated 95% confidence intervals (CIs). RESULTS: There were 341 cases, including 47 with recently acquired infection, and 791 controls. Disclosed risk factors for both females and males were sex with an HIV-positive person AOR 11.3, 95% CI (4.1, 31.7) and being an IVDU or sexual partner of an IVDU [AOR 4.65 (1.8, 11.7)]. For female blood donors, additional risk factors were having male sex partners who also are MSM [AOR 13.5 (3.1, 59.8)] and having unprotected sex with multiple sexual partners [AOR 5.19 (2.1, 12.9)]. The primary risk factor for male blood donors was MSM activity [AOR 21.6 (8.8, 52.9)]. Behaviours associated with recently acquired HIV were being a MSM or sex partner of MSM [13.82, (4.7, 40.3)] and IVDU [11.47, (3.0, 43.2)]. CONCLUSION: Risk factors in blood donors parallel those in the general population in Brazil. Identified risk factors suggest that donor compliance with selection procedures at the participating blood centres is inadequate.
Assuntos
Doadores de Sangue , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , HIV-1 , Auditoria Médica , Adolescente , Brasil/epidemiologia , Estudos de Casos e Controles , Feminino , Infecções por HIV/prevenção & controle , Humanos , Masculino , Fatores de Risco , Assunção de Riscos , Sexo sem ProteçãoRESUMO
Low-colony-number counts on solid media are considered characteristic of cross-contamination, although they are normally observed in true-positive cultures from some groups of patients. The aim of this study was to evaluate low-yield growth cultures as a microbiological marker for cross-contamination. We evaluated 106 cultures with <15 colonies from 94 patients, and the proportions of false-positive cultures were 0.9% per sample and 1.1% per patient, which indicates that low-yield growth is not a reliable marker of cross-contamination.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Adulto , Idoso , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , Contagem de Colônia Microbiana , Impressões Digitais de DNA , DNA Bacteriano/genética , Reações Falso-Positivas , Feminino , Genótipo , Humanos , Masculino , Polimorfismo de Fragmento de Restrição , Sensibilidade e EspecificidadeRESUMO
SUMMARY: We investigated an outbreak caused by non-tuberculous mycobacteria (NTM) related to breast implant surgery in the city of Campinas, Brazil, by means of a retrospective cohort and molecular epidemiological study. A total of 492 records of individuals having breast surgery in 12 hospitals were evaluated. Twelve isolates were analysed using four different molecular typing methods. There were 14 confirmed cases, 14 possible cases and one probable case. One probable, nine possible and 12 confirmed cases were included in a cohort study; all occurred in eight of the hospitals and the confirmed cases in five. Univariate analysis showed that patients who had had breast reconstruction surgery in hospitals A and B were more likely to have NTM infections. No risk factor was independently associated with NTM infection in the multivariate model. The isolates obtained from patients at each hospital showed different molecular patterns, excluding isolates from hospital C that repeatedly showed the same genotype for approximately one year. In conclusion, this outbreak was caused by polyclonal strains at different institutions, and in one hospital a unique genotype caused most cases. No specific risk factors were found.
Assuntos
Implante Mamário/efeitos adversos , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Infecções por Mycobacterium/epidemiologia , Infecção da Ferida Cirúrgica/epidemiologia , Adolescente , Adulto , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Estudos de Coortes , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Epidemiologia Molecular , Análise Multivariada , Infecções por Mycobacterium/microbiologia , Estudos Retrospectivos , Fatores de Risco , Infecção da Ferida Cirúrgica/microbiologiaRESUMO
A cluster of cases of post-augmentation mammaplasty surgical site infections occurred between 2002 and 2004 in Campinas, in the southern region of Brazil. Rapidly growing mycobacteria were isolated from samples from 12 patients. Eleven isolates were identified as Mycobacterium fortuitum and one as Mycobacterium porcinum by PCR-restriction digestion of the hsp65 gene. These 12 isolates, plus six additional M. fortuitum isolates from non-related patients, were typed by pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: 16S-23S rRNA internal transcribed spacer (ITS) genotyping; randomly amplified polymorphic DNA (RAPD) PCR; and enterobacterial repetitive intergenic consensus (ERIC) PCR. Four novel M. fortuitum allelic variants were identified by restriction analysis of the ITS fragment. One major cluster, comprising six M. fortuitum isolates, and a second cluster of two isolates, were identified by the four methods. RAPD-PCR and ITS genotyping were less discriminative than ERIC-PCR. ERIC-PCR was comparable to PFGE as a valuable complementary tool for investigation of this type of outbreak.
Assuntos
Técnicas de Tipagem Bacteriana , Mamoplastia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium fortuitum/classificação , Mycobacterium fortuitum/isolamento & purificação , Infecção da Ferida Cirúrgica/microbiologia , Proteínas de Bactérias/genética , Brasil , Chaperonina 60 , Chaperoninas/genética , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Mycobacterium fortuitum/genética , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Técnica de Amplificação ao Acaso de DNA Polimórfico , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
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.
Assuntos
Genoma Bacteriano , Sequências Repetitivas Dispersas/genética , Repetições Minissatélites/genética , Complexo Mycobacterium avium/genética , Animais , Argentina , Sequência de Bases , Brasil , Cromossomos Bacterianos/genética , Humanos , Epidemiologia Molecular , Complexo Mycobacterium avium/classificação , Filogenia , Mapeamento Físico do Cromossomo , Polimorfismo de Fragmento de Restrição , Alinhamento de SequênciaRESUMO
How the immune system kills Mycobacterium tuberculosis is still a puzzle. The classical picture of killing due to phagocytosis by activated macrophages may be only partly correct. Based on recent evidence, we express here the view that cytotoxic T lymphocytes also make an important contribution and suggest that DNA vaccines might be a good way to enhance this.
Assuntos
Proteínas de Bactérias , Chaperoninas/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T Citotóxicos/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Chaperonina 60 , Chaperoninas/genética , Humanos , Ativação de Macrófagos/imunologia , Camundongos , Mycobacterium tuberculosis/genética , Ratos , Ratos Endogâmicos Lew , Tuberculose/imunologia , Tuberculose/microbiologia , Vacinas de DNA/administração & dosagemRESUMO
New diagnostic tests and vaccines for tuberculosis are being developed by means of a strategy based on the study of antigens exclusive to Mycobacterium tuberculosis. These antigens were initially identified by Western blots using sera from active pulmonary tuberculosis patients against sonic extracts from M. tuberculosis and M. bovis BCG. Several proteins present in the M. tuberculosis but absent in the Mycobacterium bovis BCG sonic extracts were selected and are currently under investigation. One of these, denoted MTP40, has been extensively studied. The nucleotide sequence of the mtp40 gene has been obtained; hybridization studies have shown that this DNA fragment is exclusive to M. tuberculosis. Using this genomic fragment, a polymerase chain reaction (PCR)-based diagnostic test which allows the specific identification of a minimum of 10 fg of M. tuberculosis DNA was developed. The diagnostic assay is now being tested on uncultured clinical samples in order to determine its usefulness in routine diagnosis. Peptides synthesized from the derived sequence for the MTP40 protein and also from other M. tuberculosis proteins are now being studied as possible candidates for a new generation of synthetic vaccines against tuberculosis.
Assuntos
Antígenos de Bactérias/análise , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Animais , Antígenos de Bactérias/química , Sequência de Bases , Western Blotting , Camundongos , Dados de Sequência Molecular , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Reação em Cadeia da Polimerase , Coelhos , Tuberculose/prevenção & controle , Vacinas SintéticasRESUMO
The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity. beta-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Soros Imunes , Mycobacterium tuberculosis/enzimologia , Regiões Promotoras Genéticas/genética , Fosfolipases Tipo C/isolamento & purificação , Animais , Proteínas de Bactérias/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Fosfolipases Tipo C/genéticaRESUMO
In order to develop a new experimental animal model of infection with Mycobacterium chelonae in keratomileusis, we conducted a double-blind prospective study on 24 adult male New Zealand rabbits. One eye of each rabbit was submitted to automatic lamellar keratotomy with the automatic corneal shaper under general anesthesia. Eyes were immunosuppressed by a single local injection of methyl prednisolone. Twelve animals were inoculated into the keratomileusis interface with 1 microl of 10(6) heat-inactivated bacteria (heat-inactivated inoculum controls) and 12 with 1 microl of 10(6) live bacteria. Trimethoprim drops (0.1%, w/v) were used as prophylaxis for the surgical procedure every 4 h (50 microl, qid). Animals were examined by 2 observers under a slit lamp on the 1st, 3rd, 5th, 7th, 11th, 16th, and 23rd postoperative days. Slit lamp photographs were taken to document clinical signs. Animals were sacrificed when corneal disease was detected and corneal samples were taken for microbiological analysis. Eleven of 12 experimental rabbits developed corneal disease, and M. chelonae could be isolated from nine rabbits. Eleven of the 12 controls receiving a heat-inactivated inoculum did not develop corneal disease. M. chelonae was not isolated from any of the control rabbits receiving a heat-inactivated inoculum, or from the healthy cornea of control rabbits. Corneal infection by M. chelonae was successfully induced in rabbits submitted to keratomileusis. To our knowledge, this is the first animal model of M. chelonae infection following corneal flaps for refractive surgery to be described in the literature and can be used for the analysis of therapeutic responses.
Assuntos
Ceratite/microbiologia , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium chelonae , Animais , Modelos Animais de Doenças , Método Duplo-Cego , Masculino , Estudos Prospectivos , CoelhosRESUMO
More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.
Assuntos
Proteínas de Bactérias/genética , Chaperoninas/genética , Enzimas de Restrição do DNA/análise , Infecções por Mycobacterium/diagnóstico , Mycobacterium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição/métodos , Chaperonina 60 , Enzimas de Restrição do DNA/economia , Mycobacterium/química , Infecções por Mycobacterium/microbiologia , Sensibilidade e EspecificidadeRESUMO
As tuberculosis re-emerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabelled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Fragmentos de Peptídeos/imunologia , Tuberculose/imunologia , Sequência de Aminoácidos , Formação de Anticorpos , Antígenos de Bactérias/metabolismo , Linfócitos B/imunologia , Fibronectinas/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologiaRESUMO
Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.
Assuntos
Elementos de DNA Transponíveis , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Tuberculose Pulmonar/epidemiologia , Análise por Conglomerados , Cuba/epidemiologia , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologiaRESUMO
The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and 'Mycobacterium canettii'. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium tuberculosis/enzimologia , Polimorfismo Genético , Tuberculose Pulmonar/microbiologia , Fosfolipases Tipo C/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis , Deleção de Genes , Variação Genética , Humanos , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA , Fosfolipases Tipo C/metabolismoRESUMO
Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans.
Assuntos
Genes Bacterianos/genética , Proteínas Hemolisinas/genética , Mycobacterium tuberculosis/genética , Fosfolipases Tipo C/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette-Guérin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-gamma (IFN-gamma)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-gamma-producing T cells. When the cells were separated into CD4+CD8- and CD8+CD4- and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4- and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations.
Assuntos
Vacina BCG/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Tuberculose/imunologia , Vacinas de DNA/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Chaperonina 60 , Chaperoninas/imunologia , Receptores de Hialuronatos/análise , Interferon gama/biossíntese , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. avium PCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in the HaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria.
Assuntos
Genoma Bacteriano , Mycobacterium avium/genética , Mycobacterium avium/isolamento & purificação , Suínos/microbiologia , Tuberculose/microbiologia , Tuberculose/veterinária , Alelos , Animais , Sequência de Bases , Brasil/epidemiologia , Proteínas de Choque Térmico/genética , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Polyclonal infection by Mycobacterium avium was detected by hsp65 PCR-restriction enzyme analysis (PRA) in a bone marrow isolate from an AIDS patient. Two M. avium strains, differing in colony morphology, PRA HaeIII digestion pattern, insertion element (IS) 1245 amplification, and restriction fragment length polymorphism fingerprints with IS1245 and IS1311 probes, were isolated.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , Medula Óssea/microbiologia , Complexo Mycobacterium avium/genética , Infecção por Mycobacterium avium-intracellulare/microbiologia , Reação em Cadeia da Polimerase , Adulto , Sequência de Bases , Enzimas de Restrição do DNA/farmacologia , Elementos de DNA Transponíveis , Humanos , Masculino , Dados de Sequência Molecular , Complexo Mycobacterium avium/isolamento & purificação , Polimorfismo de Fragmento de RestriçãoRESUMO
One-hundred eight Mycobacterium avium isolates from pigs, humans, birds, and bovines were typed by the IS1245-based restriction fragment length polymorphism (RFLP) method and PCR-restriction enzyme analysis (PRA) of hsp65. Nine clusters of isolates showing more than 80% similarity in their RFLP profiles were detected. The largest cluster (cluster B) included 32 of 79 pig isolates (40.5%), 3 of 25 human isolates (12%), and 1 of 2 bovine isolates, comprising 33% of all isolates. The second largest cluster (cluster A) included 18 pig isolates (22.8%) and 6 human isolates (24%). Six smaller clusters included six pig isolates (clusters C and D), four and two human isolates (clusters E and F, respectively), two pig isolates (cluster I), and two pig isolates plus one bovine isolate and the avian purified protein derivative strain (cluster H). Cluster G represented the "bird-type" profile and included the bird isolate in this series, one pig isolate, plus reference strain R13. PRA revealed four allelic variants. Seventy-seven isolates were identified as M. avium PRA variant I, 24 were identified as M. avium PRA variant II, 6 were identified as M. avium PRA variant III, and 1 was identified as M. avium PRA variant IV. Except for three isolates from cluster B, each of the RFLP clusters was associated with a single PRA pattern. Isolates with unique (nonclustered) RFLP profiles were distributed between PRA variants I and II, and there was one unique isolate of PRA variant IV. These observations are consistent with divergent evolution within M. avium, resulting in the emergence of distinct lineages with particular competence to infect animals and humans.
Assuntos
Proteínas de Bactérias , Chaperoninas/genética , Mycobacterium avium/isolamento & purificação , Animais , Aves , Bovinos , Chaperonina 60 , Chaperoninas/análise , DNA Bacteriano/análise , Genótipo , Humanos , Mycobacterium avium/classificação , Mycobacterium avium/genética , Polimorfismo de Fragmento de Restrição , Especificidade da Espécie , SuínosRESUMO
Mycobacterium avium has become a major human pathogen, primarily due to the emergence of the AIDS epidemic. Restriction fragment length polymorphism (RFLP) typing, using insertion sequence IS1245 as a probe, provides a powerful tool in the molecular epidemiology of M. avium-related infections and will facilitate well-founded studies into the sources of M. avium infections in animal and environmental reservoirs. The standardization of this technique allows computerization of IS1245 RFLP patterns for comparison on a local level and the establishment of M. avium DNA fingerprint databases for interlaboratory comparison. Moreover, by combining international DNA typing results of M. avium complex isolates from a broad spectrum of sources, long-lasting questions on the epidemiology of this major agent of mycobacterial infections will be answered.