Métodos tintoriais utilizados na identificação do Mycobacterium leprae: revisão histológica

Foi feita revisão histórica sobre métodos tintoriais utilizados na identificação baciloscópica do Mycobacterium leprae. Ao lado da descrição de cada método, e suas variantes, é feita extensa revisão bibliográfica.

Isolamento de cepas de Mycobacterium avium em búfalos abatidos para consumo

Duas cepas micobacterianas, isoladas no parênquima pulmonar e linfonodo apical de búfalos abatidos para consumo, procedentes de criatórios localizados na Ilha de Marajó (PA) e submetidas à identificação segundo ensaios recomendados para o gênero Mycobacterium, foram identificadas como pertencentes ao complexo Mycobacterium avium. Apresentaram-se considerações relativas à associação desses organismos com a Aids -- e o papel dos alimentos nessa associação --, discutindo-se o impacto que a condição de germes oportunistas das espécies desse complexo têm na pandemia do HIV, assim como o risco potencial representado pelas infecções produzidas nos animais.

Outbreak of persistent cutaneous abscesses due to Mycobacterium chelonae after mesotherapy sessions, Lima, Peru

Outbreaks of rapidly growing mycobacteria have been occasionally described. The article reports an outbreak of cutaneous abscesses due to Mycobacterium chelonae following mesotherapy in Lima, Peru. From December 2004 through January 2005, 35 subjects who had participated in mesotherapy training sessions presented with persistent cutaneous abscesses. Thirteen (37%) of these suspected cases consented to underwent clinical examination. Skin punch-biopsies were collected from suspicious lesions and substances injected during mesotherapy were analyzed. Suspected cases were mainly young women and lesions included subcutaneous nodules, abscesses and ulcers. Mycobacterium chelonae was isolated from four patients and from a procaine vial. In conclusion, it is important to consider mesotherapy as a potential source of rapidly growing mycobacteria infections.

Efeito do Mycobacterium bovis BCG, lipopolissacarideo bacteriano e hidrocortisona no desenvolvimento de imunidade ao Plasmodium berghei em camundongos

Mycobacterium bovis (BCG) aumenta significantemente o desenvolvimento da imunidade nos camundongos CFW, C57BL/6, C57BL/l0ScN e BALB/c (Nu/+) para os estágios eritrocitos do Plasmodium berghei. Camundongos tratados com BCG requerem menos ciclos de infecção com P. berghei e cura pelo Fansidar (pirimetamina + sulfadoxina) para desenvolverem imunidade sólida a este parasita do que os controles. Contudo, os animais que receberam BCG 30 dias antes do início da imunização evidenciaram uma perda precoce da imunidade adquirida para o P. berghei, quando comparado com os animais que receberam BCG 14 dias antes ou que não receberam BCG. Assim, sendo, o BCG aumentada a indução na resposta imune do hospedeiro ao P. berghei no curso de infecções subseqüentes. O tratamento de camundongos CFW, BALB/c e C57BL/6 com lipopolissacarídeo bacteriano ou hidrocortisona faz com que os animais requeiram um número maior de ciclos de infecção e cura para tornarem-se imunes ao P. berghei que os controles. O tratamento dos camundongos C57BL/10ScN com hidrocortisona aboliu completamente a sua habilidade de sobrevida subseqüentes a ciclos de infecção com P. berghei e cura pelo Fansidar.

Radiometric studies of mycobacterium tuberculosis

An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C) acetate, (U-14C) glycerol, (1-14C) palmitic acid, 1-14C) lauric acid, (U-14C) L-malic acid, (U-14C) D-glucose, and (U-14C) D-glucose, but not (1-14C) L-glucose, (U-14C) glycine, or (U-14C) pyruvate to 14CO2. By using either 14C-for-mate, (1-14C) palmitic acid, or (1-14C) lauric acid, 10(7) organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

UltramicroELISA para la deteccion de anticuerpos IgM al Mycobacterium leprae utilizando muestras de sangre seca

En este trabajo se establecen las condiciones optimas para la detección de anticuerpos IgM al glicolipido fenólico-I (GF-I) en muestras de sangre en papel de filtro utilizando el UltramicroELISA HANSEN y la tecnologia SUMA. Se estudiaron 300 donantes de sangre y 58 pacientes leprosos. Para estas dos poblaciones se compararon los resultados de muestras de sangre seca colectadas en papel de filtro SS-2992 con los de suero, y se obtuvo una correlación de 0.919 para donantes de sangre, 0.969 para pacientes y 0.954 para el total de las dos poblaciones. Se obtuvo una coincidencia de 98% en pacientes y 96% en donantes. En la población de pacientes la sensibilidad fue de 93% y la especificidad de 100% para las muestras de sangre seca evaluadas por el UMELISA HANSEN, con respecto a las muestras de suero analizadas por esta misma tecnica.

Mycobacterium avium complex (MAC): an unusual potential pathogen in cerebrospinal fluid of AIDS patients

Mycobacterium avium complex (MAC) is frequently isolated from patients with late complications of Acquired Immunodeficiency Syndrome (AIDS), especially in North America and Europe. However, its isolation from the central nervous system (CNS) has been seldom reported in these countries. MAC infections in AIDS patients in African and Latin American countries are believed to be uncommon. We report the isolation of MAC from cerebrospinal fluid (CSF) of 11 AIDS patients out of 1723 (0.63%) seen at "Centro de Referência e Treinamento - AIDS", São Paulo and discuss the significance of its isolation.

Mycobacterium avium complex (MAC) isolated from AIDS patients and the criteria required for its implication in disease

Before the AIDS pandemia, the Mycobacterium avium complex (MAC) was responsible in most cases for the pneumopathies that attack patients with basic chronic pulmonary diseases such as emphysema and chronic bronchitis36. In 1981, with the advent of the acquired immunodeficiency syndrome (AIDS), MAC started to represent one of the most frequent bacterial diseases among AIDS patients, with the disseminated form of the disease being the major clinical manifestation of the infection8. Between January 1989 and February 1991, the Section of Mycobacteria of the Adolfo Lutz Institute, São Paulo, isolated MAC from 103 patients by culturing different sterile and no-sterile processed specimens collected from 2304 patients seen at the AIDS Reference and Training Center and/or Emilio Ribas Infectology Institute. Disseminated disease was diagnosed in 29 of those patients on the basis of MAC isolation from blood and/or bone marrow aspirate. The other 74 patients were divided into categories highly (5), moderately (26) and little suggestive of disease (43) according to the criteria of DAVIDSON (1989)10. The various criteria for MAC isolation from sterile and non-sterile specimens are discussed.

Au-nanossondas aplicação na detecção de Mycobacterium tuberculosis e plasmodium berghei

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Biotecnologia

A case report of vascular catheter-associated bacteremia caused by Mycobacterium tuberculosis in a non-immunosuppressed patient

Mycobacterium tuberculosis was isolated from a central venous catheter in a non-immunosuppressed patient with systemic tuberculosis. This case report represents a very uncommon form of isolation of Mycobacterium tuberculosis. A total improvement was obtained after treatment.

Clinical and laboratory findings of disseminated Mycobacterium avium complex infection (DMAC) in a pair matched case-control study

A pair matched case/control study was conducted from January 1991 to 30 June 1992 in order to define clinical and laboratory findings associated with DMAC infection in AIDS patients. Since DMAC infection is usually associated with advanced immunodeficiency, and therefore also with other opportunistic illnesses, in addition to the number of CD4+ lymphocytes, cases and controls were matched using the following criteria: date of AIDS diagnosis and antiretroviral therapy, number and severity of associated opportunistic infections and, whenever possible, type of Pneumocystis carinii prophylaxis, age and gender, in this order of relevance. Cases (defined as patients presenting at least one positive culture for MAC at a normally sterile site) and controls presented CD4+ lymphocyte counts below 50 cel/mm3. A significantly higher prevalence of general, digestive and respiratory signs, increased LDH levels, low hemoglobin levels and CD4+ cell counts were recorded for cases when compared to controls. Increases in gGT and alkaline phosphatase levels seen in cases were also recorded for controls. In conclusion, the strategy we used for selecting controls allowed us to detect laboratory findings associated to DMAC infection not found in other advanced immunossupressed AIDS patients without DMAC.

Caracterização de estirpes de Mycobacterium bovis: estudos de crescimento e de interacção com macrófagos

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report

We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.