Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli.

Participação da apolipoproteína-E na atividade microbicida “in vitro” contra o Mycobacterium tuberculosis

A parede celular de Mycobacterium tuberculosis (Mtb) é constituída por 60% de lipídios, impedindo a passagem de uma grande quantidade de substâncias, além de desempenhar um importante papel na imunopatogênese. A apresentação desses antígenos aos linfócitos se dá por meio de moléculas do tipo CD1.Por sua vez a Apolipoproteína-E (ApoE), glicoproteína amplamente distribuída nos tecidos, pode facilitar a apresentação de lipídios pelo CD1. A ApoE possui três principais alelos ApoE- 2, 3 e 4, que codificam três isoformas de proteínas, tipos 2, 3 e 4, que possuem diferentes estruturas e funções. A presença de determinadas isoformas da ApoE está associada a doenças infecciosas, como herpes labial, dano hepático severo causado pelo vírus da hepatite C, diarréia infantil e tuberculose pulmonar. Neste contexto, avaliamos a participação da ApoE na atividade microbicida in vitro frente ao Mtb. Para tanto, foram arrolados 13 indivíduos PPD-, 17 indivíduos PPD+ e 4 indivíduos com tuberculose pulmonar ativa. O uso de plasma humano depletado de ApoE nos experimentos de atividade microbicida in vitro mostraram um aumento significante (p=0,02) no número de micobactérias (431.5 ± 81.92 UFC) quando comparado ao grupo controle (313.0 ± 74.61 UFC). Esses resultados foram confirmados por um modelo experimental utilizando esplenócitos de camundongos de camundongos C57BL/6 (815.9 ± 76.32 UFC) e animais APOE nocaute (1133 ± 86.85 UFC) (p = 0.021). Quanto à produção de IL-10, no grupo PPD+, observamos que o grupo com depleção de ApoE (866.7 ± 447.8) apresentou uma produção menor desta citocina com relação ao controle infectado (1089 ± 481.3) (p=0,023). Já em relação ao IFN-, em ambos os grupos observou-se, após 72 horas, uma tendência à diminuição da produção dessa citocina no grupo com depleção, com relação ao grupo controle. Esses dados sugerem que a ApoE tem papel distinto na ativação da resposta imune e sua ausência pode prejudicar a resposta imune frente à tuberculose

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

Survey on tuberculosis teaching in Brazilian nursing schools, 2004

OBJECTIVE: To characterise tuberculosis (TB) teaching in Brazilian nursing schools by state and region and its theoretical and practical contents. METHODOLOGY: In an educational research survey in 2004, 347 nursing schools were identified. Questionnaires were posted to faculties providing training in TB. Data were compiled in a database with a view to descriptive result analysis. Replies to the questionnaire were received by 32% of the nursing schools contacted. RESULTS: Undergraduate TB teaching is heterogeneous. For training in theory, the principle teaching method is through classes in 102 (91.9%) nursing schools. Practical TB teaching is carried out at the primary care level (89.2%). Teachers update their knowledge through events and internet; little reference is made to manuals. The time devoted to practical TB teaching ranges from 10 to 20 hours, although this is not always included in student training. CONCLUSION: Teaching in TB should go beyond the traditional model that focuses only on biological aspects. It should introduce tools that lead to permanent behavioural change, such as a more human approach and social and psychological aspects, such as living conditions, habits and customs. It should involve new partners, such as families, communities and other health professionals, and identify obstacles within the university. © 2006 The Union.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Case report: Disseminated Mycobacterium tuberculosis infection in a dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

Tuberculose: conhecimentos, representações sociais e experiências da doença na visão do portador

Tuberculosis is considered one of the most ancient human diseases, cases were registered 3900 years before Christ, and it is currently regarded as a serious public health problem in the world due to several factors such as income mismanagement, precarious standard of life and some sort of prejudice comprised by the word tuberculosis. Taking this into consideration, it was developed a descriptive and exploratory study aiming at analyzing the social representations of tuberculosis made by its patient from the Unidades de Saúde da Família (Family Health Units a public health program) in Campina Grande City PB, in relation to the decentralization of the policies that administrate the disease. It was interviewed 34 tuberculosis patient that were being treated from 2007 to 2008. The age group of the interviewees varied from 10 to 60 years old, but most of them were between 36 and 60 years old (58,8%, n=20), some were young adult and adult (21 35 years old), with 11 (32,3%) respondents, and, less frequent, children and teenagers (11 20 years old), with 03 (8,8%) participants. Data was collected through semi-structured interview. The questions that guided the research were elaborated based on the operational recommendations of DOTS strategy; that is: access to laboratory examinations; medication guarantee; directly observed treatment. Besides that, the experiences of the patient were considered in their relation with the family and the different social groups. The analysis of the discursive material was submitted to the Analyse Lexicale par Context d un Ensemble de Segments de Texte software - ALCESTE 4.7. Data interpretation showed five categories for the social representations of the tuberculosis patient that participated in DOTS strategy: 1) the accessibility of the health assistance service; 2) the patient perspective of the disease; 3) the change in the operation of the productive life; 4) the signals and symptoms of the tuberculosis disease; 5) the rearrangement and mechanisms used to face the disease. The Central Nucleus reveals that tuberculosis is a transmissible disease that can be prevented by people through educational practices, health promotion, active search for symptomatic respiratory and control of the carriers communication; these mechanisms should be incorporated to the routine of all participants of the family health groups. The Intermediate Elements, based on quotidian life, as well as the individual experiences of the tuberculosis patient, reveals prejudiced attitude and beliefs that lead to isolation and restriction of interpersonal relationship. Peripheral Elements were constituted by themes that showed the patient feelings of indignation because of the social barriers they had to face in the Family Health Units during the treatment. These elements demonstrate a negative perspective of the representation concerning the accessibility, i.e. inadequate structure of the health service; long distance to the Health Centre, this factormakes it difficult for the patient to continue the treatment; scheduling delay; and limited service regarding other requests (doctor, dentist etc). One expects to contribute for the construction of a new perspective of the health question between the different agents who make the assistencial institutions and formation of professionals, either in central or local scope

Caracterização da população portadora de tuberculose do município de Américo Brasiliense/SP, no período de 1992 a 2002

Tuberculosis (TB) is a serious public health problem all over the world, which was recognized by the WHO in 1993 as a global emergency because of its very high incidence in the 22 countries that concentrate 80% of TB cases. One of these countries is Brazil, where TB occurrences are well-documented in major cities, but little is known of its spread in rural areas and small towns. Therefore, an epidemiological study was done on medical records of TB sufferers in the rural district and small town of Américo Brasiliense, São Paulo state, from 1992 to 2002, with the aim of improving TB prevention and treatment. The results showed that the incidence of TB peaked in the years of intense migration of rural workers, largely cane-cutters. Among these, the disease attacks mainly the men, in their productive years (20 to 40 years old). The predominant clinical form observed was pulmonary tuberculosis. The treatment abandonment rate was less than 1.8%, while the cure rate was around 90%. The disease detection rate by examination of sputum for acid-fast bacilli was around 60%.

Ocorrência de tuberculose em um hospital psiquiátrico do interior de Goiás

Objective: To investigate the prevalence of infection, disease and eventual institutional outbreak of tuberculosis in a psychiatric hospital using the PPD test, as well as testing for mycobacteria in material collected from the respiratory tree and using molecular tracking technique based on insertion sequence 6110 (IS6110). Methods: Between February and August of 2002, PPD tests were given to 74 inpatients and 31 staff members at a psychiatric hospital in the city of Rio Verde, located in the state of Goiás, Brazil. In addition, respiratory tree material collected from the inpatients was submitted to testing for Mycobacterium tuberculosis. Results: Among the patients analyzed, mycobacteria were isolated from five (6.8%): four identified as M. tuberculosis and one as M. chelonae. The M. tuberculosis isolates were sensitive to isoniazid and rifampicin, and, when submitted to the restriction fragment length polymorphism/IS6110 technique, presented unique genetic profiles, totally distinct from one another, suggesting that all of the tuberculosis cases were due to endogenous reactivation. It was not possible to characterize this group of cases as an institutional outbreak. Performing the two-step tuberculin test in the patients, the infection rates were 23% and 31%, compared with 42% among staff members, who were submitted to the one-step test. Conclusion: The results indicate a high incidence of tuberculosis infection among inpatients and hospital staff, as well as a high occurrence of the disease among inpatients.

Antimycobacterial activity of some Brazilian indigenous medicinal drinks

Tuberculosis (TB) is a very serious problem worldwide and the increasing number of multiple drugs resistant TB cases makes the search for new anti-TB drugs an urgent need. Indigenous knowledge about the use of native plants to treat illnesses has contributed to the discovery of new medicines. In this study, the antimycobacterial activity of seven medicinal drinks was assessed: Ananas sativus (hydroalcoholic fruit extract), Aristolochia triangularis (aqueous and hydroalcoholic leaf, root and stem extracts), Bromelia antiacantha (hydroalcoholic fruit extract), Stryphnodendron adstringens (hydroalcoholic bark extract), Tabebuia ovellanedae (hydroalcoholic bark extract), Vernonia polyanthes (hydroalcoholic root extract), all used by the Vanuíre indigenous community in the treatment of respiratory diseases. The activity was evaluated by using a time-to-kill assay, in which Mycobacterium tuberculosis H37Rv was cultured on Löwenstein-Jensen medium, after thirty minutes, one, three, six, twelve and twenty-four hours contact of the bacteria with each drink. Within half to one hour contact, the hydroalcoholic drinks of A. triangularis, S. adstringens, T. ovellanedae and V. polyanthes reduced the bacterial growth by 2 orders of magnitude in CFU/mL, and all bacterial growth was absent after three hours contact. In contrast, no mycobactericidal effect was detected in the aqueous extract of A. triangularis or in the hydroalcoholic beverages of A. sativus and B. antiacantha, even after twenty-four hours contact.

Efeitos da terapia fotodinâmica sobre cepas de Candida isoladas de pacientes submetidos à antibioticoterapia prolongada

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Diagnóstico nutricional de pacientes internados por tuberculose em Belém do Pará

Doenças infecciosas crônicas, como a tuberculose (Tb), são causas de distúrbios metabólicos que interferem na utilização e mobilização de nutrientes corporais, afetando o estado nutricional. A função imunológica de doentes desnutridos é alterada, o que, provavelmente, implica maior incidência de infecções. Neste estudo objetivou-se diagnosticar o estado nutricional de pacientes recém-internados por tuberculose no Hospital Universitário João de Barros Barreto, em Belém, aplicando uma técnica de avaliação subjetiva global (ASG) e verificando sua concordância com um método objetivo, o índice de massa corporal (IMC). Foi descritivo e parte dele descritivo-analítico, transversal, tendo como população de referência pacientes de tuberculose encaminhados para hospitalização, excluindo aqueles com meningite tuberculosa. As variáveis controladas foram peso, altura, sexo, formas de Tb, alterações de peso, de gordura subcutânea, de massa muscular, da ingesta alimentar e da capacidade funcional, a presença de sintomas gastrointestinais, sinais de edema e ascite. O perfil nutricional observado foi de 77,8% de desnutridos, sendo, destes, 83,78% moderados e 16,22% graves, com um IMC médio de 18,36 ± 3,86 Kg/m². A maioria relatou perda ponderal nos últimos seis meses antes da internação, em níveis de 5% a 10% do seu peso habitual; 73,82% relataram modificação para menos na ingesta alimentar, e presença de sintomas gastrointestinais em 64,58% deles, sendo a anorexia o mais freqüente, seguida de náuseas, vômitos e diarréia. A perda da capacidade funcional foi referida em 100% (nutridos e desnutridos). Ao exame físico foi possível perceber que 79,16% apresentavam perda de gordura subcutânea e massa muscular, conjuntamente, e nenhum caso apresentou edema ou ascite. A Tb pulmonar foi a mais incidente, sendo encontrados três casos de Tb ganglionar e um de Tb óssea, associada à SIDA. O IMC, em relação à ASG, apresentou um coeficiente de Kappa igual a 0,47, validade sensível de 67% e especificidade de 100%. A ASG se mostrou uma técnica de boa aplicabilidade, demonstrando na prática dados inerentes não só ao estado nutricional, mas também ao modo de vida dos indivíduos, oferecendo subsídios relevantes para a terapêutica no nível hospitalar. O diagnóstico nutricional mostrou uma prevalência elevada de desnutridos, tanto pelo método subjetivo, como pelo objetivo e que a preocupação com o estado nutricional precoce propicia uma adequada intervenção nutricional. Os resultados recomendam a continuidade deste estudo, avaliando a situação nutricional quando da internação e acompanhando, periodicamente, cada caso, de modo a subsidiar melhor assistência por parte de toda a equipe de saúde a este grupo de doentes.

Inhalable antitubercular therapy mediated by locust bean gum microparticles

Tuberculosis remains a major global health problem and alternative therapeutic approaches are needed. Considering the high prevalence of lung tuberculosis (80% of cases), the pulmonary delivery of antitubercular drugs in a carrier system capable of reaching the alveoli, being recognised and phagocytosed by alveolar macrophages (mycobacterium hosts), would be a significant improvement to current oral drug regimens. Locust bean gum (LBG) is a polysaccharide composed of galactose and mannose residues, which may favour specific recognition by macrophages and potentiate phagocytosis. LBG microparticles produced by spray-drying are reported herein for the first time, incorporating either isoniazid or rifabutin, first-line antitubercular drugs (association efficiencies >82%). Microparticles have adequate theoretical properties for deep lung delivery (aerodynamic diameters between 1.15 and 1.67 μm). The cytotoxic evaluation in lung epithelial cells (A549 cells) and macrophages (THP-1 cells) revealed a toxic effect from rifabutin-loaded microparticles at the highest concentrations, but we may consider that these were very high comparing with in vivo conditions. LBG microparticles further evidenced strong ability to be captured by macrophages (percentage of phagocytosis >94%). Overall, the obtained data indicated the potential of the proposed system for tuberculosis therapy.

Histamine Plays an Essential Regulatory Role in Lung Inflammation and Protective Immunity in the Acute Phase of Mycobacterium tuberculosis Infection

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Recent data suggest a regulatory role of histamine not only in the innate but also in the adaptive immune response. We used a model of pulmonary Mycobacterium tuberculosis infection in histamine-deficient mice lacking histidine decarboxylase (HDC(-/-)), the histamine-synthesizing enzyme. To confirm that mycobacterial infection induced histamine production, we exposed mice to M. tuberculosis and compared responses in C57BL/6 (wild-type) and HDC(-/-) mice. Histamine levels increased around fivefold above baseline in infected C57BL/6 mice at day 28 of infection, whereas only small amounts were detected in the lungs of infected HDC(-/-) mice. Blocking histamine production decreased both neutrophil influx into lung tissue and the release of proinflammatory mediators, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), in the acute phase of infection. However, the accumulation and activation of CD4(+) T cells were augmented in the lungs of infected HDC(-/-) mice and correlated with a distinct granuloma formation that contained abundant lymphocytic infiltration and reduced numbers of mycobacteria 28 days after infection. Furthermore, the production of IL-12, gamma interferon, and nitric oxide, as well as CD11c(+) cell influx into the lungs of infected HDC(-/-) mice, was increased. These findings indicate that histamine produced after M. tuberculosis infection may play a regulatory role not only by enhancing the pulmonary neutrophilia and production of IL-6 and TNF-alpha but also by impairing the protective Th1 response, which ultimately restricts mycobacterial growth.