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1.
Cytokine ; 43(2): 124-31, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18558493

RESUMO

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.


Assuntos
Tatus/metabolismo , Expressão Gênica , Interferon gama/metabolismo , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium leprae/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Tatus/genética , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , DNA Complementar/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/química , Interferon gama/genética , Macrófagos/citologia , Dados de Sequência Molecular , Nitritos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
2.
Clin Microbiol Rev ; 19(2): 338-81, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16614253

RESUMO

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.


Assuntos
Hanseníase , Mycobacterium leprae , Animais , Anti-Infecciosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Farmacorresistência Bacteriana , Genes Bacterianos/genética , Predisposição Genética para Doença , Genoma Bacteriano , Humanos , Imunidade Celular , Imunidade Inata/genética , Hansenostáticos/farmacologia , Hansenostáticos/uso terapêutico , Hanseníase/diagnóstico , Hanseníase/microbiologia , Hanseníase/terapia , Camundongos , Mycobacterium leprae/química , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/fisiologia , Nervos Periféricos/microbiologia , Doenças do Sistema Nervoso Periférico/microbiologia , Doenças do Sistema Nervoso Periférico/patologia , Reação em Cadeia da Polimerase , Células de Schwann/imunologia , Células de Schwann/microbiologia
3.
s.l; s.n; 2006. 44 p. ilus, tab.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLACERVO, SES-SP | ID: biblio-1241811

RESUMO

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.


Assuntos
Humanos , Animais , Camundongos , Anti-Infecciosos , Células de Schwann , Doenças do Sistema Nervoso Periférico , Farmacorresistência Bacteriana , Genes Bacterianos , Genoma Bacteriano , Hansenostáticos , Hanseníase , Imunidade Celular , Imunidade Inata , Modelos Animais de Doenças , Mycobacterium leprae , Nervos Periféricos , Predisposição Genética para Doença , Proteínas de Bactérias , Reação em Cadeia da Polimerase , Research Support, N.I.H., Extramural , Suscetibilidade a Doenças , Vacinas Bacterianas
4.
Cytokine ; 32(5): 219-25, 2005 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-16338142

RESUMO

The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type leprosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression. The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lymphoblasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of immunological tools will assist in advancing the armadillo as a translational model for leprosy.


Assuntos
Tatus/genética , Interleucina-2/genética , Sequência de Aminoácidos , Animais , Tatus/imunologia , Células Cultivadas , Escherichia coli/genética , Expressão Gênica , Genoma , Humanos , Interleucina-2/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência
5.
J Clin Microbiol ; 39(6): 2083-8, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11376039

RESUMO

Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the folP1 gene of Mycobacterium leprae, and definitive evidence linking these mutations with DDS resistance in M. leprae has been obtained. Based on these mutations, a heteroduplex DDS M. leprae (HD-DDS-ML) assay was developed for the simultaneous detection of M. leprae and of its susceptibility to DDS. The assay relies on the PCR amplification of an M. leprae-specific 231-bp fragment of folP1 containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis. M. leprae was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from M. leprae-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for M. leprae in a comparison with results obtained from 14 species of mycobacteria other than M. leprae and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100 M. leprae organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of M. leprae and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.


Assuntos
Dapsona/farmacologia , Análise Heteroduplex/métodos , Hansenostáticos/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/isolamento & purificação , Sequência de Bases , DNA Bacteriano/análise , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos , Humanos , Hanseníase/microbiologia , Dados de Sequência Molecular , Mycobacterium leprae/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA
6.
Antimicrob Agents Chemother ; 44(6): 1530-7, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10817704

RESUMO

Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli (C600DeltafolP::Km(r)). Expression of M. leprae folP1 in C600DeltafolP::Km(r) conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC(50)]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600DeltafolP::Km(r) knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC(50) 68-fold. These results prove that the folP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600DeltafolP::Km(r) knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.


Assuntos
Antibacterianos/farmacologia , Dapsona/farmacologia , Di-Hidropteroato Sintase/metabolismo , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Di-Hidropteroato Sintase/genética , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular
7.
Lepr Rev ; 71 Suppl: S91-5, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11201896

RESUMO

The folP1 gene of Mycobacterium leprae, which encodes dihydropteroate synthase (DHPS), was studied for the presence of mutations associated with resistance to dapsone (DDS). When the folP1 of several DDS-resistant clinical isolates of M. leprae were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting isoleucine for threonine in DHPS-1, and a second mutation occurred in codon 55, substituting arginine for proline. DNA sequencing of strains of M. leprae resistant to 0.01 g% DDS in the mouse diet revealed that 13 of 14 strains contained either the 53 or 55 folP1 mutation. None of the susceptible strains and only one of five strains resistant to 0.001 g% DDS revealed a mutation in folP1, suggesting that only high-level DDS resistance is associated with the mutations identified in folP1. Development and application of simple molecular tests to assess drug-related mutations in M. leprae could establish current levels of drug resistance in leprosy as a reference point for future monitoring of drug resistance at the global level.


Assuntos
Dapsona/farmacologia , Hansenostáticos/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Resistência Microbiana a Medicamentos , Humanos , Hanseníase/tratamento farmacológico , Testes de Sensibilidade Microbiana
8.
Indian J Lepr ; 71(1): 11-8, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10439322

RESUMO

Evidence suggests that resistance to dapsone (DDS) in Mycobacterium leprae is related to the enzyme dihydropteroate synthase (DHPS). Two M. leprae genes (folP-1 and folP-2) encoding DHPS-1 and DHPS-2, respectively, have been identified through the M. leprae genome project. We have studied DDS-susceptible and resistant strains of M. leprae to determine whether the DDS-resistant phenotype is associated with a mutation(s) in folP-2 and to establish the number of genomic copies of the gene encoding DHPS-2 (folP-2). RFLP analysis of genomic DNA from DDS-susceptible and resistant strains of M. leprae exhibited a unique 4.2 kb restriction fragment consistent with a single genomic copy of folP-2 in both phenotypes. DNA encoding folP-2 was amplified by PCR and sequenced from two susceptible and two resistant strains of M. leprae. The folP-2 sequences from these strains were identical indicating that resistance to DDS was not associated with mutation(s) in the gene encoding DHPS-2.


Assuntos
Dapsona/farmacologia , Di-Hidropteroato Sintase/genética , Hansenostáticos/farmacologia , Mutação , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Animais , DNA Bacteriano/análise , Resistência Microbiana a Medicamentos/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética
10.
Am J Clin Pathol ; 109(5): 642-6, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9576586

RESUMO

The differentiation of leprosy from other cutaneous granulomatous diseases is routinely based on characteristic histopathologic features and the demonstration of Mycobacterium leprae by acid-fast staining. Increased ascertainment of other mycobacterial infections in the skin has made this task more difficult, but the distinction remains fundamental for the selection of appropriate treatment. Experience with formalin-fixed, paraffin-embedded tissues, frozen tissues, and tissue lysates referred for detection of M. leprae DNA by a polymerase chain reaction (PCR) assay during the past 4 years was reviewed. This assay was done by using primers and probes previously developed in our laboratory to amplify a 360-base-pair fragment of the gene for an 18-kD protein of M. leprae. Among biopsy samples obtained from 37 patients, PCR results were positive for 10 of 20 samples diagnosed as leprosy by histopathologic criteria and in 0 of 17 not diagnosed as leprosy. The specificity of the assay was 100% in this clinical referral material; sensitivity ranged from 50% to 83%. The PCR assay also identified M. leprae in one third of samples in which acid-fast organisms were seen and the histopathologic features were consistent with but not definitive of leprosy. In a nonendemic population, the sensitivity and specificity of PCR assay recommend its use primarily to identify M. leprae when acid-fast organisms are discernible but atypical clinical or histopathologic features obscure the diagnosis. The assay is not highly informative when acid-fast bacilli are not detectable by light microscopy.


Assuntos
DNA Bacteriano/análise , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Biópsia , Humanos , Hanseníase/microbiologia , Mycobacterium leprae/genética , Sensibilidade e Especificidade , Pele/microbiologia , Estados Unidos
11.
Int J Lepr Other Mycobact Dis ; 65(4): 461-4, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9465155

RESUMO

For 39 patients suspected of early leprosy, skin biopsies of the lesions were done and bisected. One piece was used for histopathologic examination and the other for polymerase chain reaction (PCR) studies to detect Mycobacterium leprae. The diagnosis of early leprosy was made clinically in 14 patients and by histopathologic study in 26 patients. Acid-fast bacilli were seen in the histopathologic sections of only two patients, and M. leprae were detected using PCR techniques in 11 patients. In one patient the diagnosis of leprosy was made only because of the detection of M. leprae in the PCR study. Since even in endemic countries the profile of leprosy is changing, detection of leprosy lesions in their early stages has become increasingly important. Since the finding of M. leprae is crucial in the confirmatory diagnosis of early leprosy, it is suggested that PCR studies to detect M. leprae be done wherever possible in conjunction with histopathologic examination. It is also recommended that the feasibility and the cost-effectiveness of both of these methods to find M. leprae be evaluated.


Assuntos
Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Biópsia , Criança , Feminino , Humanos , Hanseníase Dimorfa/diagnóstico , Hanseníase Tuberculoide/diagnóstico , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Pele/microbiologia , Pele/patologia
12.
Clin Infect Dis ; 20(4): 776-80, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7795073

RESUMO

A case of ocular leprosy as the manifestation of persistent or relapsed Mycobacterium leprae infection approximately 20 years following treatment is reported. The clinical and pathological features of this case are described, and the molecular methods needed to arrive at the definitive diagnosis are examined. If blindness is to be averted, clinicians must have a high index of suspicion for the diagnosis of ocular leprosy when anterior segment changes are noted during ophthalmologic examination of a patient from an area in which M. leprae is endemic. The indolent nature of ocular leprosy may require lifelong surveillance and therapy to insure sight preservation.


Assuntos
Infecções Oculares Bacterianas/microbiologia , Hanseníase/complicações , Mycobacterium leprae , Adulto , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológico , Humanos , Masculino , Recidiva
13.
Antimicrob Agents Chemother ; 38(10): 2380-6, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7840574

RESUMO

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.


Assuntos
Resistência Microbiana a Medicamentos/genética , Mycobacterium/efeitos dos fármacos , Rifampina/farmacologia , Sequência de Aminoácidos , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Mycobacterium/genética , Reação em Cadeia da Polimerase
14.
Antimicrob Agents Chemother ; 38(7): 1651-4, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7979302

RESUMO

Fusidic acid was assessed for antileprosy activity in nine lepromatous leprosy patients. Patients received fusidic acid at either 500 mg/day for 12 weeks or 750 mg/day for 4 weeks followed by 500 mg/day for 8 weeks. All patients showed time-dependent clinical improvement and decreases in bacillary morphological index, radiorespirometric activity and PCR signal, and in serum phenolic glycolipid I. Fusidic acid appears to be a weakly bactericidal antileprosy agent which may have a role in the multidrug treatment of leprosy pending an evaluation of lepra-reaction-suppressive activity.


Assuntos
Antígenos de Bactérias , Ácido Fusídico/uso terapêutico , Hanseníase Virchowiana/tratamento farmacológico , Adolescente , Adulto , Animais , Criança , Feminino , Pé/microbiologia , Pé/patologia , Glicolipídeos/sangue , Humanos , Hanseníase Virchowiana/microbiologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/metabolismo , Reação em Cadeia da Polimerase , Pele/microbiologia , Espirometria , Fatores de Tempo
15.
Infect Immun ; 61(4): 1509-15, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8454357

RESUMO

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.


Assuntos
Antígenos de Bactérias/química , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Sequência de Bases , Reações Cruzadas , Genes Bacterianos , Dados de Sequência Molecular , Peso Molecular , Mycobacterium/genética , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , Alinhamento de Sequência
16.
Microbios ; 76(309): 251-61, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8302203

RESUMO

Mycobacterium tuberculosis and Mycobacterium leprae develop resistance against the drugs used to treat tuberculosis and leprosy, respectively. Now multidrug-resistant tuberculosis is spreading in many countries, especially with the emergence of AIDS. Multidrug treatment is being promoted at present to eradicate leprosy. Since M. leprae may also become multidrug-resistant, new approaches have to be adopted for controlling mycobacterial diseases. Mycobacteria usually synthesize beta-lactamase and are insensitive to beta-lactam antibiotics. M. tuberculosis contains a constitutive beta-lactamase; de-repression of beta-lactamase has been reported in M. leprae. Three different beta-lactam/beta-lactamase-inhibitor combinations (ampicillin/sulbactam, amoxicillin/clavulanate and piperacillin/tazobactam) were used to suppress the growth of several strains of mycobacteria (including M. tuberculosis H37Rv) in vitro. Ampicillin/sulbactam is a potent bactericidal agent against M. leprae multiplying in mouse foot pads. In the present work, ampicillin/sulbactam showed higher activity than the other drug combinations. The beta-lactam/beta-lactamase inhibitors are likely to be effective as rational therapeutic agents against mycobacterial infections.


Assuntos
Antibacterianos/farmacologia , Quimioterapia Combinada/farmacologia , Mycobacterium/efeitos dos fármacos , Inibidores de beta-Lactamases , Amoxicilina/farmacologia , Combinação Amoxicilina e Clavulanato de Potássio , Ampicilina/farmacologia , Animais , Ácidos Clavulânicos/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium/enzimologia , Mycobacterium/crescimento & desenvolvimento , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Piperacilina/farmacologia , Combinação Piperacilina e Tazobactam , Sulbactam/farmacologia
17.
J Clin Microbiol ; 30(12): 3095-8, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1452690

RESUMO

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.


Assuntos
Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Estudos de Avaliação como Assunto , Fixadores , Humanos , Hanseníase/diagnóstico , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade
18.
Mol Cell Probes ; 6(5): 401-10, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1474978

RESUMO

An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for detecting M. leprae in routine skin biopsies taken from leprosy patients throughout the clinical spectrum. Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or DNA hybridization to detect the amplified product. Similar analysis of paucibacillary patients, in which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74%. No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis. Evaluation of leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were either greatly diminished or absent after 2 months of continuous antibiotic therapy. PCR was also able to detect the presence of M. leprae in tissues of patients receiving antibacterial therapy when patients were suspected of harbouring drug-resistant M. leprae.


Assuntos
Clofazimina/uso terapêutico , Dapsona/uso terapêutico , Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Rifampina/uso terapêutico , Pele/microbiologia , Antibacterianos , Biópsia , Sondas de DNA , DNA Bacteriano/genética , Quimioterapia Combinada/uso terapêutico , Eletroforese em Gel de Ágar , Estudos de Viabilidade , Humanos , Hanseníase/tratamento farmacológico , Mycobacterium leprae/genética , Hibridização de Ácido Nucleico , Sensibilidade e Especificidade , Pele/patologia
19.
FEMS Microbiol Immunol ; 4(3): 165-74, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1349489

RESUMO

DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess DNA sequences homologous to the 18-kDa protein gene of M. leprae. RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.


Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/análise , Mycobacterium/genética , Sequência de Bases , Southern Blotting , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição
20.
Lepr Rev ; 62(4): 362-73, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1784151

RESUMO

Thirty, nine-banded armadillos weighing between 3 and 5 kilograms trapped from an area endemic for armadillo leprosy were collected at random; killed, autopsied and examined histopathologically. Also, one of the right inguinal lymph nodes was removed under sterile precautions and examined using PCR, direct smear examination, mouse footpad study, culture in laboratory media and histopathology with a view to detecting Mycobacterium leprae. Blood was collected at death and tested for IgM antibodies to PGL-1. According to the PCR study of the inguinal lymph nodes 16 of 30 armadillos (53.3%) had evidence of M. leprae. Significant levels of IgM antibodies to PGL-1 and identifiable lepromatous granuloma in inguinal lymph nodes were found in 2 animals (6.7%) with advanced disseminated disease. The prevalence of generalized leprosy according to autopsy study was 13.3% and according to histopathological examination of ear tissue 3.3%. The presence of M. leprae in the tissues evoked no special tissue reaction in the early stages. The pattern of spread of the disease in 2 animals closely resembled that found in experimental animals infected intracutaneously. Initiation of infection by inoculation of M. leprae through thorn pricks remains a distinct possibility.


Assuntos
Antígenos de Bactérias , Tatus/microbiologia , Hanseníase/veterinária , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Animais , Anticorpos Antibacterianos/análise , Técnicas Bacteriológicas , Ensaio de Imunoadsorção Enzimática , Glicolipídeos/imunologia , Imunoglobulina M/análise , Hanseníase/diagnóstico , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/imunologia
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