Detecção de Mycobacterium lepra por PCR em "SWAB" nasal e "SWAB" da linfa do lóbulo da orelha de pacientes hansenianos

Recentemente vários estudos têm usado a técnica Reação em Cadeia de Polimerase (PCR) para detecção do DNA do Mycobacterium leprae, em diversas amostras biológicas, demonstrando alta sensibilidade. O objetivo deste trabalho foi avaliar a sensibilidade da PCR na detecção de M. leprae em “swab” nasal e “swab” da linfa do lóbulo da orelha de pacientes hansenianos e comparar os resultados da PCR com a baciloscopia e histopatologia e formas multibacilares (MBs) e paucibacilares (PBs) da hanseníase. Foram coletadas amostras de secreção nasal e linfa do lóbulo da orelha de 24 pacientes hansenianos. Para amplificação do DNA foram testados três pares de primers: S13 e S62, R1 e R2, LP1 e LP2 que amplificam fragmentos de DNA de 531 pb, 372pb e 129pb, respectivamente. Os iniciadores LP1 e LP2 expressaram maior sensibilidade, independente das amostras clínicas. Os resultados da PCR foram altamente significativos para as amostras de secreção nasal (p<0.0000) e significativos para os espécimes de linfa do lóbulo da orelha (p=0.0000). Comparando os resultados da PCR, usando os primers LP1 e LP2 e conservante lise 1, com a baciloscopia e histopatologia, os estudos apontaram que a PCR, em amostras de secreção nasal, obteve maior sensibilidade para as formas MBs (41,67%), seguida da baciloscopia (25%) e histopatologia (8,33%). Nas formas PBs, a sensibilidade foi considerada a mesma entre a PCR e Histopatologia (8,33%). A baciloscopia não apresentou sensibilidade (0%). Nas amostras da linfa do lóbulo da orelha, a baciloscopia demonstrou maior sensibilidade para as formas MBs (25%), seguido da PCR (20,83%) e histopatologia (16,7%). Nas formas PBs, a PCR e Histopatologia apresentaram a mesma sensibilidade (4,17%). Não houve sensibilidade na baciloscopia (0%). A PCR, apesar de não demonstrar uma sensibilidade de 100% é uma ferramenta com perspectivas futuras para auxiliar no monitoramento do tratamento e cura dos pacientes hansenianos.

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.

Cultura cromogênica dum bacilo ácido-álcool resistente isolado de pus de lesão fechada de lepra humana

Last October 2nd the Author smeared nine tubes of Loewenstein medium with material obtained from closed pustulae of a seven years old boy, L2 case of leprosy. This material was very rich in Hansen bacilli in its different forms, inclusive globus, as is seen in the figures 2 and 3 of Plate 1. Part of this material obtained from pustulae opened by galvanocautery, was inoculated, at the same day, into white rats and guinea-pigs. November 26th a new biopsy gave more rich material, which was smeared again into Loewenstein fresh medium. December 15th three of the first and two of the second series of tubes of cultures showed germination of a yellow, dry and rough culture, covering almost the total surface of the medium. Microscopic examination of the culture showed that it was a pure culture of an acid-fast organism. Passages into glycerinated potatoes germinated well covering the surface of the same with a clear yellow granulated culture remaining the fluid (glycerinated water) quite limpid. The germination in glycerinated broth produced a yellow velum in the surface of the medium, as is seen in fig. 3 of Plate 2, without becoming turbid. The microorganism isolated twice from the same source of material was cocciforme (as Mycobacterium pulviforme of Marchoux), in the original culture, becoming more bacilliforme, always acid-fast, after passage into glycerinated media. The A. sent his culture to foreigner colleagues to study it and will inoculate it soon into laboratory animals.

Morfologia do Mycobacterium leprae hominis e do M. leprae muris: estudo baseado na microscopia electrônica e de contraste de fases

Hansen's Bacillus: By electron microscopy this bacillus shows membrane and halo, this being more visible when sorrounding the globi or bundles of bacilli; shows, also, free granules of various sizes which were before considered as dust of the dyes; shows external granules bound with the membrane and some times branching. By phases contrast microscopy examining leproma suspensions and subcataneous lymph at 400 x we saw many free granules with intense rotatory movement; granulated bacilli with screw, skip or stroke motion, producing slow progressive motion. All such elementes are surrounded by a halo, corresponding to the classical gloea. By a patient and delayed examination we were able to see that the internal granules are motile and help the progression of the bacilli, giving the impression that the cytoplasm is liquid. By a lasting observation we could see the larger granules form prolapse, like a pseudopode and abandon the bacilli and going in very rapid rotatory movement. There are branched bacilli; there are pedunculated fred granules like comets. The addition of a drop of formol at the preparation stops all movements. Stefansky's Bacillus: Repeated examination by RCA electron microscope, type EMU-25 of fresh suspensions of rat lepromas, led us to confirm the close relationship between human and murine leprosy agents. We examined also material from carabo (Lepra bubalorum) from Java, but due to fixation, the material was unsuitable for comparative studies. The Stefansky's bacilli showed also emmbranes and halos, internal or external granules (smaller than those of Hansen's bacillus). The bacilli shaded by chromium look thicker and shorter than those of Hansen. Due to electron bombardment both, Hansen's and Stefansky's baccilli suffer considerable alterations in their structure, showing black barrs of chromatin condensation at their extremities as also in their centers. By phase microscopy the Stefansky's bacilli showed elements with 1, 2 (bipolar), 3 or more internal small granules, developing identical movements as those of Hansen. The globi seem to be non-motile but the free bacilli appearing around the globi show intense movement. At 1000 x the examination is less satisfactory than at 400 x. The addition of formol solution in the preparation suppresses all movements, even the brownian, but the material becomes more suitable for the study of static morphology of the bacilli. CONCLUSION - The electron and phases contrast microscopy of leprous material from different types and phases of the disease may explain some of the unknown aspects of the biology and morphology of the bacilli.

Modelagem molecular da Chiquimato quinase de Mycobacterium leprae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Perfis de resistência a agentes antimicrobianos de 5 estirpes do género Mycobacterium isoladas de ambiente Hospitalar

As infecções nosocomiais têm aumentado ao longo dos anos, resultando num aumento do tempo de permanência do doente no hospital, e permanecem como elevada causa de elevada morbilidade e mortalidade. As micobactérias são organismos que se encontram amplamente distribuídos no meio ambiente (M. mucogenicum, M. obuense e M. gordonae), incluindo, habitats marinhos (Mycobacterium marinum), sendo muitos deles patogénicos de mamíferos, e causadores de diferentes patologias, como a Lepra e a Tuberculose. M. marinum causa uma doença sistémica tal como tuberculose em peixes e pode causar infecções da pele em seres humanos (Granuloma de Aquário) que se podem propagar para estruturas mais profundas como ossos (osteomielite). Enquanto que M. obuense é causador de infecções do tracto respiratório, M. mucogenicum e M. gordonae promovem bacteremias. Este estudo teve como principal objectivo a identificação das populações bacterianas e o seu isolamento, em particular micobactérias ambientais em dois hospitais, que sabe serem responsáveis, cada vez mais por infecções atípicas como bacteremias (M. mucogenicum e M. gordonae), infecções pulmonares (M. obuense) e infecções cutâneas (M. marinum). Pretendeu-se também avaliar a resistência aos antibióticos e desinfectantes comummente utilizados no tratamento de infecções causadas por micobactérias não tuberculosas (MNT) através do cálculo da Concentração Mínima Inibitória (CMI) para aferir os perfis de resistência. Os resultados deste estudo demonstram a identificação de 186 espécies de bactérias em dois hospitais amostrados das quais se identificaram 5 estirpes de micobactérias – “M. gardonae” (10AIII, 29AIII e 35AIII), “M. obuense” (22DIII) e “M. mucogenicum” (24AIII). Das 5 estirpes de micobactérias identificadas “M. gardonae” 10AIII apresenta perfil de resistência ao imipenemo (CMI = 16 mg/L); “M. gardonae” 29AIII apresenta perfil de resistência à claritromicina (CMI = 8 mg/L) e “M. gardonae” 35AIII apresenta, por sua vez, apenas perfil de susceptibilidade intermédia ao imipenem (CMI = 8 mg/L). M. obuense 22DIII apresenta perfil de resistência ao imipenem (CMI = 32 mg/L), à tobramicina (CMI=32 mg/L) e à ciprofloxacina (CMI = 8 mg/L). “M. mucogenicum” apresenta perfil de resistência ao sulfametoxazol (CMI > 128 mg/L), à doxiciclina (CMI>64 mg/L), à tobramicina (CMI=16 mg/L) e à ciprofloxacina (CMI=4 mg/L).Em conclusão pôde-se verificar que além da presença de um grande leque de bactérias capazes de causar infecções nosocomiais nos hospitais, MNT também existem na forma multirresistente, o que revela uma problemática a ter em atenção. Esta requer mais estudo dos mecanismos de resistência e da sua disseminação, e obtenção de novos medicamentos com novos alvos, mais eficazes para combater as estirpes multirresistentes que ao longo dos anos tem aumentado.

Tuberculosis determined by Mycobacterium bovis in captive waterbucks (Kobus ellipsiprymnus) in São Paulo, Brazil

Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus.

Comparison of three decontamination methods for Mycobacterium bovis isolation

Sixty samples of tissue fragments with lesions suggestive of tuberculosis from bovine abattoirs, kept in saturated solution of sodium borate, were subjected to four treatments: 4% NaOH (Petroff Method), 12 % H2SO4 and 1.5% HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3%) when compared to control (88%, p<0.001), NaOH (33%, p<0.001) and H2SO4 (21.7%, p<0.002). Regarding the isolation success, the HPC method was better (40%) than the control (3%, p<0.001), NaOH (13%, p=0.001) and H2SO4 (1.7%, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method.

Detection of Mycobacterium avium in pet birds

The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential.

Isolation of Mycobacterium tuberculosis from Captive Ateles paniscus

An adult female red-faced black spider monkey (Ateles paniscus), housed for 2 years in the Parque Estoril Zoo in Sao Paulo, Brazil, showed apathy. Clinical examination revealed discrete emaciation, swelling and induration of lymph nodes, and presence of a mass in the abdominal cavity. Therapies with enrofloxacin, azithromycin, and ceftiofur were ineffective. The animal died after 6 months. Necropsy and histopathology confirmed granulommas in lymph nodes, parietal and visceral pleura, lungs, liver, spleen, and kidneys. Acid-fast bacilli were isolated and identified as Mycobacterium tuberculosis by polymerase chain reaction restriction analysis and Spoligotyping techniques. The zoo personnel and other animals that had had contact with the infected primate were negative to tuberculosis diagnostic procedures, such as sputum exam (baciloscopy) and thorax radiography. It was impossible to determine whether the infection occurred before or after the arrival of the animal to the Parque Estoril Zoo. This is the first report of M. tuberculosis infection in Ateles paniscus, a neotropical primate.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Sulfonyl-hydrazones of cyclic imides derivatives as potent inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase B (PtpB)

Searching lead compounds for new antituberculosis drugs, the activity of synthetic sulfonamides and sulfonyl-hydrazones were assayed for their potential inhibitory activity towards a protein tyrosine phosphatase from Mycobacterium tuberculosis - PtpB. Four sulfonyl-hydrazones N-phenylmaleimide derivatives were active (compounds 14, 15, 19 and 21), and the inhibition of PtpB was found to be competitive with respect to the substrate p-nitrophenyl phosphate. Structure-based molecular docking simulations were performed and indicated that the new inhibitor candidates showed similar binding modes, filling the hydrophobic pocket of the protein by the establishment of van der Waals contacts, thereby contributing significantly to the complex stability.