RESUMO
Buruli ulcer (BU) is the third most common mycobacterial disease in immunocompetent hosts. BU is caused by Mycobacterium ulcerans, which produces skin ulcers and necrosis at the site of infection. The principal virulence factor of M. ulcerans is a polyketide-derived macrolide named mycolactone, which has cytotoxic and immunosuppressive activities. We determined the severity of inflammation, histopathology and bacillary loads in the subcutaneous footpad tissue of BALB/c mice infected with 11 different M. ulcerans isolates from diverse geographical areas. Strains from Africa (Benin, Ghana, Ivory Coast) induced the highest inflammation, necrosis and bacillary loads, whereas the strains collected from Australia, Asia (Japan, Malaysia, New Guinea), Europe (France) and America (Mexico) induced mild inflammation. Subsequently, animals were infected with the strain that exhibited the highest (Benin) or lowest (Mexico) level of virulence in order to analyse the local immune response generated. The Mexican strain, which does not produce mycolactone, induced a predominantly T helper type 1 (Th1) cytokine profile with constant high expression of the anti-microbial peptides beta defensins 3 and 4, in co-existence with low expression of the anti-inflammatory cytokines interleukin (IL)-10, IL-4 and transforming growth factor (TGF)-beta. The highly virulent strain from Benin which produces mycolactone A/B induced the opposite pattern. Thus, different local immune responses were found depending on the infecting M. ulcerans strain.
Assuntos
Úlcera de Buruli/imunologia , Mycobacterium ulcerans/patogenicidade , Animais , Austrália , Benin , Ensaio de Unidades Formadoras de Colônias , Congo , Côte d'Ivoire , Citocinas/análise , Citocinas/genética , Citocinas/imunologia , Expressão Gênica , Gana , Japão , Malásia , Masculino , México , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Infecções por Mycobacterium não Tuberculosas/imunologia , Papua Nova Guiné , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da Espécie , Trinidad e Tobago , Virulência/genética , beta-Defensinas/análise , beta-Defensinas/genéticaRESUMO
SETTINGS: Tuberculosis (TB) diagnostic laboratories in Latin America. OBJECTIVES: Evaluation of thin-layer agar (TLA) compared to Löwenstein-Jensen (LJ) culture for the diagnosis of TB. DESIGN: Phase II prospective study in six laboratories. Samples included sputum and extra-pulmonary specimens from patients with a clinical diagnosis of TB. Respiratory samples were decontaminated using NaOH/ NALC; all samples were centrifuged, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on LJ and TLA and identified according to recommended procedures. Sensitivity and likelihood ratios (LR), growth detection time and contamination rate were calculated for both media. RESULTS: A total of 1118 clinical specimens were studied. Cultures detected Mycobacterium tuberculosis in all AFB-positive samples, whereas for AFB-negative specimens LJ detected 3.2% and TLA 4.4%. Sensitivity was 92.6% (95%CI 87.9-95.9) and 84.7% (95%CI 78.8-89.0) for TLA and LJ, respectively. Positive and negative LRs were similar. Contamination was 5.1% for TLA and 3.0% for LJ. Median time to detection of a positive culture was 11.5 days (95%CI 9.3-15.0) for TLA and 30.5 days (95%CI 26.9-39.0) for LJ (P < 0.0001). CONCLUSION: Difference in the characteristics of the participating laboratories, the disease prevalence and the number and type of specimens processed did not affect the overall performance of TLA as compared to LJ, supporting the robustness of the method and its feasibility in different laboratory settings.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Ágar , Técnicas Bacteriológicas/métodos , Humanos , América Latina , Estudos Prospectivos , Fatores de TempoRESUMO
Buruli disease (BU) is a progressive necrotic and ulcerative disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU is considered the third most common mycobacterial disease after tuberculosis and leprosy. Three clinical stages of the cutaneous lesions have been described in BU: pre-ulcerative, ulcerative and healed lesions. In this study we used immunohistochemistry and automated morphometry to determine the percentage of macrophages and of CD4/CD8 lymphocytes and their expression of interferon (IFN)-gamma, interleukin (IL)-10, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta. Expression of these cytokines was correlated with the inflammatory response evaluated by histopathology. All the studied BU ulcerative cases showed extensive necrosis and chronic inflammation. The most important feature was the presence or absence of granulomas co-existing with a mixed pro-inflammatory/anti-inflammatory cytokine balance. When granulomas were present significantly higher expression of IFN-gamma was seen, whereas in ulcerative lesions without granulomas there was increased expression of IL-10 and significantly higher bacillary counts. These features correlated with the chronicity of the lesions; longer-lasting lesions showed granulomas. Thus, granulomas were absent from relatively early ulcerative lesions, which contained more bacilli and little IFN-gamma, suggesting that at this stage of the disease strong suppression of the protective cellular immune response facilitates proliferation of bacilli.
Assuntos
Citocinas/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium ulcerans , Dermatopatias Bacterianas/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Feminino , Granuloma/imunologia , Humanos , Macrófagos/imunologia , Masculino , Infecções por Mycobacterium não Tuberculosas/patologia , Dermatopatias Bacterianas/patologia , Úlcera Cutânea/imunologia , Úlcera Cutânea/patologiaRESUMO
OBJECTIVE: To collect data on non-tuberculous mycobacteria (NTM) isolated from clinical laboratories in different countries to establish: 1) whether the isolation of NTM was increasing, 2) which species were increasing, and 3) whether there was any pattern of geographical distribution. DESIGN: In 1996, the Working Group of the Bacteriology and Immunology Section of the International Union Against Tuberculosis and Lung Disease contacted 50 laboratories in different countries for the necessary information. RESULTS: The number of patients reported with NTM was 36099 from 14 countries. Mycobacterium avium complex, M. gordonae, M. xenopi, M. kansasii and M. fortuitum were the five species most frequently isolated. There was a significant upward trend for M. avium complex and M. xenopi. Pigmented mycobacteria predominated in Belgium, the Czech Republic and the Mediterranean coast of Spain. Non-chromogenic mycobacteria were found to be predominant in the area of the Atlantic coast of Brazil and in Turkey, the United Kingdom, Finland and Denmark. CONCLUSIONS: There was an increase in the number of NTM isolated from clinical samples of patients. Isolation of the most frequent species is constantly changing in most of the geographical areas, and newer species are emerging due to better diagnostic techniques to detect and identify NTM.
Assuntos
Mycobacterium/isolamento & purificação , Brasil , Europa (Continente) , Irã (Geográfico) , Complexo Mycobacterium avium/isolamento & purificação , Mycobacterium fortuitum/isolamento & purificação , Mycobacterium kansasii/isolamento & purificação , Mycobacterium xenopi/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Estudos Retrospectivos , TurquiaRESUMO
INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n = 18) or M. intracellulare (n = 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.
Assuntos
Proteínas de Bactérias , DNA Espaçador Ribossômico/genética , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Kit de Reagentes para Diagnóstico , Chaperonina 60 , Chaperoninas/genética , Sondas de DNA , Humanos , Mycobacterium/genética , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Fatores de TempoRESUMO
The line probe assay (LiPA), a rapid molecular method for detecting rifampicin resistance (RMPr) in Mycobacterium tuberculosis, correctly identified all 145 rifampicin-sensitive (RMPs) and 262 (98.5%) of 266 RMPr strains among 411 isolates collected from diverse countries. If used as a marker of multidrug-resistant tuberculosis (MDR-TB), detection of RMPr by LiPA would have detected 236 of the 240 MDR strains in this study but would have wrongly suggested the presence of MDR in 26 RMP-monoresistant isolates (sensitivity 98.3%, specificity 84.8%). Hence, the reliability of using LiPA (or any other rapid RMPr-detection method) as a surrogate marker of MDR-TB largely depends on the prevalence of RMP-monoresistance in the study population. This approach must therefore be validated in each local situation.
Assuntos
Antibióticos Antituberculose/farmacologia , Resistência a Medicamentos , Técnicas de Sonda Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , África/epidemiologia , Ásia/epidemiologia , Europa (Continente)/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , América do Sul/epidemiologia , Especificidade da Espécie , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologiaRESUMO
A total of 624 respiratory specimens from 543 patients (418 Belgian, 110 Rwandan, and 15 Colombian patients) were tested for the presence of Mycobacterium tuberculosis by the Mycobacterium Tuberculosis Direct Test (MTDT, Gen-Probe). Compared to culture, the MTDT on 497 samples of sputum or broncho-alveolar lavage from Belgium had a sensitivity, specificity and positive and negative predictive value of 86.4%, 96.0%, 50.0% and 99.3% respectively. The pooled results for Rwanda (112 specimens) and Colombia (15 specimens) were 97.8%, 65.7%, 88.2%, 92% respectively. After resolution of discrepant results by taking into account the clinical data, the results for the Belgian patients were 86.9%, 96.2%, 52.6%, 99.3% respectively, and for the Rwandan-Colombian patients 98.1%, 100%, 100% and 92% respectively. Results could be improved by testing more than one specimen from each patient and the inclusion of an internal control to detect inhibitors of the reaction. Culture remains necessary for drug susceptibility tests and the isolation and identification of non-tuberculous mycobacteria.
Assuntos
Técnicas Genéticas , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/microbiologia , Bélgica , Colômbia/etnologia , Humanos , Valor Preditivo dos Testes , Ruanda/etnologia , Sensibilidade e Especificidade , Tuberculose Pulmonar/etnologiaRESUMO
In order to study polymorphisms of the DNA insertion sequence 6110 (IS6110) in Mycobacterium tuberculosis strains isolated from Colombian patients, together with resistance to antituberculous medications in the Department of Quindío, Colombia, a prospective study was conducted using a consecutive sample of 59 patients with symptomatic pulmonary tuberculosis whose cases had been confirmed by bacilloscopy, both with and without a history of treatment. The patients, who were participating in the Tuberculosis Control Program of the Regional Health Institute of Quindío in Armenia, included all individuals attending local health centers and hospitals between March and July 1993 who were referred to the regional institute. Sputum specimens from each patient were cultured and subjected to drug sensitivity tests. Subsequently, restriction fragment length polymorphisms (RFLP) of IS6110 from 27 patients were analyzed. The patients' treatment histories were used to classify their cases according to WHO criteria. Forty-five cultures were found positive, 44 for M. tuberculosis and 1 for M. africanum. Initial drug resistance was observed in 4 of 42 new cases, or 9.5% (95% CI: 0.6, 18), 2 showing resistance to isoniazid (INH) and 2 to isoniazid plus streptomycin (INH-SM). Acquired resistance was observed in 2 of the 3 chronic cases and relapses, the bacteria being resistant to isoniazid, rifampicin, and streptomycin (INH-RM-SM) in one case and to isoniazid, ethambutol, rifampicin, and streptomycin (INH-EMB-RM-SM) in the other. In those 27 strains subjected to RFLP analysis, the number of copies of IS6110 ranged from 6 to 17. Similarity coefficients revealed five distinct groups of strains. Overall, the RFLP analysis permitted most of the strains to be distinguished from one another, implying that the polymorphisms involved are sufficient to permit effective employment of this technique, which appears to have considerable potential for use in epidemiologic studies and in work designed to provide a basis for tuberculosis control program decision-making.
Assuntos
DNA Bacteriano/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Fragmento de Restrição , Tuberculose Pulmonar/microbiologia , Mapeamento Cromossômico/métodos , Colômbia/epidemiologia , Humanos , Estudos Prospectivos , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Pulmonar/epidemiologiaRESUMO
The purpose of this study was to determine the polymorphism of insertion segment 6110 (IS6110) in strains of Mycobacterium tuberculosis isolated from Colombian patients as well as the current status of resistance to antituberculosis drugs in the department of Quindío, Colombia. To this end, a prospective study was performed with a consecutive sample of 59 patients who sought care at local health centers and hospitals in rural and urban areas of Quindío from March to July 1993. The patients in the sample had symptomatic pulmonary tuberculosis confirmed by bacteriologic inspection of sputum, with and without a history of treatment, and were participants in the Tuberculosis Control Program of the Sectional Health Institute of Quindío in Armenia, Colombia. Sputum cultures and drug sensitivity tests were done. Later, restriction fragment length polymorphisms (RFLP) of IS6110 were analyzed in accordance with the protocols of van Soolingen et al. (1992). Cases were classified by treatment history, applying the criteria of WHO (1991). The results showed 44 cultures positive for M. tuberculosis and one positive for M. africanum. Primary drug resistance was found in 4 of 42 cultures, or 9.5% (CI 95%: 0.6 to 18); 4.8% were resistant to isoniazid (INH) and 4.8% to isoniazid and streptomycin (INH-SM). Acquired resistance was found in two of three cultures, or 66% (to isoniazid, rifampicin, and streptomycin [INH-RM-SM] and to isoniazid, ethambutol, rifampicin, and streptomycin [INH-EMB-RM-SM]). In 27 strains submitted to RFLP analysis, the number of copies of IS6110 varied from 6 to 17. Similarity coefficients revealed five distinct groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mycobacterium tuberculosis/genética , Polimorfismo de Fragmento de Restrição , Tuberculose Pulmonar/epidemiologia , Antituberculosos/uso terapêutico , Técnicas Bacteriológicas , Colômbia/epidemiologia , Resistência Microbiana a Medicamentos , Etambutol/farmacologia , Humanos , Isoniazida/farmacologia , Modelos Genéticos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estudos Prospectivos , Rifampina/farmacologia , População Rural , Estreptomicina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , População UrbanaRESUMO
Oligonucleotide primers have been used to amplify DNA regions of the M. leprae genome by the polymerase chain reaction. A first set of primers, PLp1 and PLp2, identifies a specific 386 bp DNA fragment located in the gene coding for the 65 kDa antigen of M. leprae. A second pair of primers, targetted to the same gene, leads to the amplification of a 154 bp DNA piece conserved in mycobacteria. Primers PLp1 and PLp2 discriminate the pathogenic species from other mycobacteria, detect down to 40 bacilli, and constitute potentially useful tools for the identification of M. leprae in clinical specimens.