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.

Soft tissue abscess and lymphadenitis due to Mycobacterium avium Complex as an expression of immune reconstitution inflammatory syndrome after a second scheme of highly active antiretroviral therapy

Immune reconstitution inflammatory syndrome (IRIS) is an atypical and unexpected reaction related to highly active antiretroviral therapy (HAART) in human immunodeficiency virus (HIV) infected patients. IRIS includes an atypical response to an opportunistic pathogen (generally Mycobacterium tuberculosis, Mycobacterium avium complex, cytomegalovirus and herpes varicella-zoster), in patients responding to HAART with a reduction of plasma viral load and evidence of immune restoration based on increase of CD4+ T-cell count. We reported a case of a patient with AIDS which, after a first failure of HAART, developed a subcutaneous abscess and supraclavicular lymphadenitis as an expression of IRIS due to Mycobacterium avium complex after starting a second scheme of HAART.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

DIFFERENTIATION BETWEEN Nocardia spp. AND Mycobacterium spp.: CRITICAL ASPECTS FOR BACTERIOLOGICAL DIAGNOSIS

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

Identificação de micobactérias não tuberculosas através de métodos moleculares não comerciais

Embora Mycobacterium tuberculosis, o agente etiológico da tuberculose humana, seja a principal causa de micobacteriose no Homem, outras micobactérias não tuberculosas (MNT), podem também causar infecção em seres humanos, sendo cada vez mais frequentemente isoladas no laboratório de micobacteriologia. Dada a morosidade da identificação de MNT por métodos convencionais, torna-se essencial o desenvolvimento de métodos rápidos e fiáveis para a sua identificação. Neste estudo foram comparados três métodos moleculares para a identificação de MNT, baseados na análise de restrição enzimática após amplificação de: (i) região rRNA 16S-23S “Internal Transcribed Spacer” (ITS), (ii) gene hsp65, e (iii) zona ITS e regiões adjacentes, 16S e 23S rDNA. Para este fim, avaliamos 50 isolados, correspondendo a 47 isolados clínicos de MNT e três estirpes de referência, representando as 11 espécies de MNT mais frequentemente isoladas no laboratório de Micobactérias do Instituto de Higiene e Medicina Tropical (IHMT, UNL) e uma estirpe referência de M. tuberculosis, e identificadas anteriormente utilizando o sistema comercial GenoType® Mycobacterium CM/AS (Hain Lifescience). O PCR-RFLP do gene hsp65 proporcionou os melhores resultados, identificando correctamente 42 dos 49 isolados de MNT, pertencentes a M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. fortuitum, M. abscessus, M. szulgai, M. peregrinum e M. xenopi. O PCR-RFLP da região ITS identificou correctamente 28 dos 49 isolados testados, não distinguindo M. intracellulare/M. scrofulaceum, M. avium/M. bohemicum e M. kansasii/M. szulgai. Finalmente, o PCR-RFLP da região ITS e regiões adjacentes mostrou o pior desempenho, com apenas oito isolados correctamente identificados e falhando na amplificação de diversos isolados. Dos três métodos testados, o PCR-RFLP do gene hsp65 e da região ITS mostraram maior capacidade de identificação e reprodutibilidade. No entanto, a sua aplicação exige uma optimização cuidadosa das condições de análise e a sua aplicabilidade depende, em grande parte, da diversidade xi de MNT em cada laboratório. Verificaram-se também várias dificuldades a nível da interpretação dos resultados. Assim, apesar das vantagens referidas na literatura para estes três métodos, verificou-se que o grau de variabilidade e dificuldade de interpretação associados aos padrões obtidos, limitam a sua implementação no laboratório de diagnóstico de micobacteriologia.

Caracterização de estirpes Mycobacterium Tuberculosis da família Lisboa: análise de regiões de diferença e single nucleotide polymorphisms

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia