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.

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

Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

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

Novas estratégias no combate à tuberculose: a NADH-MENAQUINONA desidrogenase do tipo II de Mycobacterium tuberculosis

Dissertação para obtenção do Grau de Mestre em Microbiologia Médica

Imunopatologia da hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae

A hanseníase, cujo agente etiológico Mycobacterium Ieprae, é doença de amplo espectro clínico e imunopatológico. Suas apresentações clínicas estão correlacionadas com padrões imunológicos distintos, variando de uma vigorosa resposta imune mediada por células ao M. Ieprae, com padrão Th1 no pólo tuberculóide, a uma ausência de resposta celular específica aos antígenos do M. Ieprae no pólo lepromatoso, com predomínio da resposta Th2 e exacerbação da resposta humoral. É provável que a suscetibilidade ao M. Ieprae é determinada por diferentes genes polimórficos. Estudos adicionais são necessários para esclarecer os mecanismos das interações complexas entre as citocinas e a participação da diversidade fenotípica da rede de células que contribuem para a defesa do hospedeiro. O entendimento de tais mecanismos poderá oferecer novas abordagens para identificar agonistas e/ou antagonistas para os efeitos pró- ou anti-inflamatórios e em quais circunstâncias sua utilização seria apropriada para intervenções imunológicas e/ou imunoterapeuticas.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.