Caracterização dos perfis genéticos e de resistência a fármacos de isolados de mycobacterium tuberculosis associados com casos de tuberculose multirresistente na Bahia, Brasil / Characterization of genetic profiles and drug resistance of Mycobacterium tuberculosis isolates associated with cases of multidrug-resistant tuberculosis in Bahia, Brazil

A resistência aos fármacos utilizados no tratamento da tuberculose (TB) é um importante desafio no combate à doença. A rifampicina e a isoniazida são dois fármacos de primeira linha essenciais para a cura da doença, a qual tem como agente o M. tuberculosis. Pacientes com TB cujos isolados de M. tuberculosis apresentem resistência in vitro simultânea a estes dois fármacos desenvolvem a TB multirresistente (TBMR). A resistência do M. tuberculosis está relacionada com mutações em genes importantes para a sobrevivência do bacilo. O tratamento da TBMR é mais longo e utiliza fármacos anti-TB de segunda linha, os quais são de maior toxicidade, predispondo os pacientes à não adesão aos esquemas de tratamento. O paciente com TBMR, quando não devidamente tratado, pode selecionar cepas resistentes aos fármacos anti-TB de segunda linha, proporcionando o surgimento da TB extensivamente resistente (TBXDR). Por sua vez, estas cepas podem ser transmitidas em comunidades, constituindo um grave problema de saúde pública. Segundo a Organização Mundial de Saúde, a TBXDR tem sido documentada em alguns países, mas no Brasil estes dados são escassos. A caracterização genética de cepas de M. tuberculosis envolvidas com os casos TBMR/TBXDR pode facilitar a identificação de vias de transmissão. OBJETIVO: Pesquisar casos de TBXDR na Bahia e caracterizar perfis genéticos de isolados de M. tuberculosis de pacientes com TB multirresistente, associando o perfil genético encontrado com as características sócio-demográficas e clínicas dos pacientes envolvidos. MATERIAIS E MÉTODOS: Isolados de M. tuberculosis obtidos de pacientes com diagnóstico de TBMR entre 2008-2011 residentes no Estado da Bahia (Brasil) foram submetidos ao teste de sensibilidade utilizando fármacos anti-TB de primeira e segunda linha e genotipados pela técnica do Número Variável de Repetições em Tandem de Unidades Repetitivas Inter-espaçadas Micobacterianas (MIRU-VNTR) para obtenção de perfis genéticos que foram associados com perfis da base de dados internacional MIRU-VNTRplus. Isolados com perfis genéticos não associáveis a linhagens com o uso desta técnica foram adicionalmente genotipados por Spoligotyping e ambas as informações foram consideradas para assimilação de linhagens utilizando esta mesma base de dados. Informações clínico-epidemiológicas foram obtidas do banco de dados “Sistema TBMR” do Ministério da Saúde. RESULTADOS: Foram analisados 392 isolados. Destes, 35% foram excluídos por ausência de crescimento ou contaminação e 12% constituíam amostras em duplicata, resultando em 206 pacientes com TBMR no estudo. Comprovou-se a ocorrência da TBXDR em 7% (14/206) dos pacientes; destes, dois não possuíam registro anterior para qualquer tratamento anti-TB. Os pacientes estudados foram provenientes de 45 municípios do Estado. A capital, Salvador, concentrou 71% dos casos TBMR e 76% dos TBXDR. Dos casos TBXDR, 36% (5/14) apresentaram isolados resistentes a todos os fármacos testados. Observou-se associação de resistência combinada entre estreptomicina e etambutol (8/14, 57%) e o perfil TBXDR (RP 4,0; IC95% 1,2-13,8; P=0,01). Dos casos TBXDR, 71% (10/14) desenvolveram uma ou mais comorbidades (P=0,04), sendo o transtorno mental uma comorbidade significativa neste grupo (21%; 3/14; P=0,04). Encontrou-se 56 perfis genéticos, 38 únicos e 18 agrupados em clusters (contendo de 2 a 11 isolados). Quase a totalidade (92%) dos casos TBXDR esteve agrupada em clusters, diferindo dos casos não-TBXDR (P=0,049). Os perfis genéticos estiveram principalmente associados a seis famílias: LAM (70%), Cameroon (16%), Haarlem (10%) e as famílias X, S, Uganda I, que combinadas perfizeram 4%. Os casos TBXDR foram representados pelas famílias LAM (45%, ST’s 376, ST42, ST20), Cameroon (36%, ST61 único) e Haarlem (18%, ST50). CONCLUSÕES: A Bahia apresentou casos de TBXDR e as famílias de M.tuberculosis envolvidas com estes casos foram LAM, Cameroon e Haarlem. A genotipagem auxiliou na descoberta de casos epidemiologicamente relacionados.

Drogas antituberculose: interações medicamentosas, efeitos adversos e utilização em situações especiais - parte 1: fármacos de primeira linha / Antituberculosis drugs: drug interactions, adverse effects, and use in special situations - part 1: first-line drugs

Os objetivos principais do tratamento da tuberculose são curar o paciente e minimizar a possibilidade de transmissão do bacilo para indivíduos saudáveis. Reações adversas ou interações das drogas antituberculose entre si e com outros fármacos podem causar modificação ou descontinuação da terapêutica. Revisamos sucintamente o novo tratamento farmacológico da tuberculose introduzido pelo Ministério da Saúde do Brasil em 2009 e mostramos os mecanismos gerais de ação, absorção, metabolização e excreção dos medicamentos utilizados no esquema básico. Descrevemos as reações adversas e as interações (com medicamentos, alimentos e antiácidos) assim como a abordagem mais adequada para situações especiais, como gravidez, amamentação, insuficiência hepática e renal. Também descrevemos os mecanismos pelos quais as interações das drogas antituberculose do esquema básico podem causar hepatite medicamentosa e as possíveis alternativas nessa situação.

The main objectives of tuberculosis therapy are to cure the patients and to minimize the possibility of transmission of the bacillus to healthy subjects. Adverse effects of antituberculosis drugs or drug interactions (among antituberculosis drugs or between antituberculosis drugs and other drugs) can make it necessary to modify or discontinue treatment. We briefly review the new guidelines for the pharmacological treatment of tuberculosis, introduced by the Brazilian National Ministry of Health in 2009, and describe the general mechanism of action, absorption, metabolization, and excretion of the first-line drugs used in the basic regimen. We describe adverse drug reactions and interactions (with other drugs, food, and antacids), as well as the most appropriate approach to special situations, such as pregnancy, breastfeeding, liver failure, and kidney failure. We also describe the mechanisms by which the interactions among the antituberculosis drugs used in the basic regimen can cause druginduced hepatitis, and we discuss the alternatives in this situation.

Immunophenotypic and intracellular cytokine profile of Indian patients with tuberculosis with and without human immunodeficiency virus co-infection.

Background. Tuberculosis (TB) occurs in more than 50% of human immunodeficiency virus (HIV) infected Indian patients. This study was carried out to determine the immunophenotypic and intracellular cytokine profile of patients with HIV-TB co-infection. Patients and Methods. Fifteen patients with HIV-TB co-infection and 15 each with TB alone and healthy individuals were studied. Immunophenotypic analysis and intracellular cytokines were measured using appropriate antibodies on a flowcytometer. Results. Percentage of CD3+ did not differ significantly in the three groups. The ratio of CD4+ : CD8+ was reversed among patients with TB and HIV-TB. CD19+ and CD25+ were present on fewer cells of healthy individuals but this was not statistically significant. Significantly higher percentage of cells of patients with TB and HIV-TB were CD69 positive. Interferongamma (INF-g ) and tumour necrosis factor-alpha (TNF-a) levels are significantly reduced in the CD4+ cells of patients with HIV-TB when compared with those with TB and healthy individuals. In CD8+ cells of patients with HIV-TB, levels of TNF-a are higher when compared with the other two groups. Interleukin-2 (IL-2) producing cells were not significantly different in any of the above subsets. Monocytes in individuals with HIV-TB had significantly higher interleukin-6 (IL-6) and TNF-a. Conclusions. T-helper cells among patients with HIV-TB have significantly lower cytokine production. T-suppressor cells and monocytes produce more TNF-a. These findings may be significant in view of recent attempts to treat HIV-TB coinfected patients with anti-TNF therapy.

Relationship of nitric oxide and protein carbonyl in tuberculosis.

BACKGROUND: Tuberculosis (TB) is a highly contagious disease caused by Mycobacterium tuberculosis. The bacilli replicate within the macrophages and can remain dormant for years; activated macrophages show immunity against these bacilli. MATERIAL AND METHODS: A prospective study was carried out in newly diagnosed TB patients (n = 70) before their antituberculosis treatment and in normal control subjects (n = 35). Serum level of nitric oxide was estimated by Moshage method, 1995 and Bories and Bories method, 1995 and protein carbonyl by Levine method, 1990. Pearson's correlation (r) and Fisher's 'z' test was performed on the obtained results. RESULTS: In our study, serum nitric oxide and protein carbonyl levels were significantly increased (p < 0.001) in TB patients as compared to normal control group. Positive correlation was seen in pulmonary TB (r = 0.8892, p < 0.001) [Fisher's 'z' transformed = 0.7901 to 0.9430] and extra-pulmonary TB (r = 0.8330, p < 0.001) [Fisher's 'z' transformed = 0.6918 to 0.9128]; 'r' and Fisher's 'Z' was significantly different from zero (two sided p < 0.001). CONCLUSION: The mean serum nitric oxide and protein carbonyl levels were concomitantly increased and positively correlated with each other in patients with pulmonary TB and extra-pulmonary TB. The changes in the level of nitric oxide and protein carbonyl are a reflection of increased defence mechanism and free radical activity in tuberculosis.

Measurement of 24 h energy expenditure in male tuberculosis patients.

BACKGROUND & OBJECTIVES: The total daily energy expenditure in patients with infectious disease is presumed to be high because of an increase in the basal metabolic rate (BMR), a reason for the weight loss observed in these patients. A reduction in daily physical activity, which may reduce the total daily energy expenditure. The aim of this study was to measure the free living total daily energy expenditure and physical activity of newly diagnosed hospitalized patients with tuberculosis using the labelled bicarbonate method. METHODS: In 6 healthy volunteers and 6 patients with newly diagnosed tuberculosis, 13C labelled bicarbonate method was used to measure free living total daily energy expenditure and physical activity. The 13C sodium bicarbonate (NaH13CO3) tracer was infused intravenously over a 48 h period and breath samples collected at regular intervals to estimate expired 13CO2. RESULTS: The patients had a 14 per cent increase in their BMR although they were not febrile at the time of measurement. However, their total daily energy expenditure was lower than that of the controls (mean value of 8.3 and 10.3 mJ/day respectively) and their physical activity level was also lower (mean 1.4 and 1.6 units respectively). INTERPRETATION & CONCLUSION: The total daily energy expenditure of afebrile patients with newly diagnosed tuberculosis is not higher than that of sedentary controls, despite an increased basal metabolic rate. It is possible that the observed weight loss in patients with tuberculosis is due to a reduced energy intake linked to anorexia associated with the disease. These findings may have relevance in nutritional treatment of chronic infections.

Fingernails as biological indices of metal exposure.

Metal determination in human tissues is the most common application of biological monitoring for screening, diagnosis and assessment of metal exposures and their risks. Various biopsy-materials may be used. This paper deals with the quantitative determination of Cd, Pb, Cr, Mn, Fe, Ni, Cu, and Zn concentrations in nails of male subjects exposed to these metals along with their respective controls, while working in locomotive, carriage and road ways workshops, and lead battery factories. The levels of Cd, Pb, Cr, Mn, Fe, Ni, Cu and Zn in fingernails, assayed by atomic absorption spectrophotometry, were compared with their respective controls by student 't' test. All the obtained values were correlated to the personal and medical history of the subjects under study. Significantly high levels of Cd, Pb, Cr, Fe, Ni, Cu and Zn were present in smokers, compared to nonsmokers. The concentrations of Cd, Pb, Cr, Mn and Fe were not significantly high in vegetarian subjects. It was also observed that there is no contribution of liquor towards nail-metal concentration. Significant correlations were observed between skin disease and Cr, Mn, Fe, Cu; hypertension and Cd, Mn, Cu; mental stress and Cd, Pb, Mn, Ni, Cu, Zn; diabetes and Cr, Mn, Ni; chest pain and Pb; respiratory trouble and Cr, Mn, Fe, Ni, Zn; tuberculosis and Zn; acidity and Cd; and ophthalmic problems and Mn, Fe, Ni, and Zn.

Biochemical & histochemical changes relating to fibrosis following infection with Mycobacterium tuberculosis in the guinea pig.

Guinea pigs infected with M. tuberculosis were studied for parameters relating to fibrosis following infection. The infected animals were followed up to a period of 44 wk and the changes that occurred in the lung, liver and spleen were studied. Corresponding tissues from animals injected with bleomycin, an anti-mitotic drug which has the ability to produce pulmonary fibrosis, served as positive controls. Tissue collagen, elastin and hexosamines were estimated biochemically. The presence of granuloma and stainable collagen in paraffin sections of these tissues was also studied. Establishment of the infection was assessed bacteriologically by culturing the viable organisms from the spleen. It was observed that a self-limiting infection was established in the guinea pigs and none of the animals died of the infection. In the infected animals, collagen, elastin and hexosamines showed an initial decrease followed by an increase. While the elastin and the hexosamine levels returned to the basal levels in all the three organs, collagen levels increased in the lung and were comparable to those of the bleomycin control. Collagen stainable by Van Gieson's method was found to be increased in the lung from the 4th wk onwards. The present report indicates the potential of adopting this system for studying mechanisms of fibrogenesis in tuberculous infection.

Tincion original del bacilo de la tuberculosis a mas de 100 años de su descubrimiento / Original staining of tuberculosis bacilli after two years of its discovery

Se produjo la tecnica de tincion de Koch (1882) para el bacilo tuberculoso. Ya que gracias a este aporte cientifico se logro visualizar el agente etiologico de tan devastadora y denigrante enfermedad para la humanidad y consolidar el inicio de la bacteriologia diagnostica. Originalmente las muestras estudiadas y tratadas por Koch que pertenecian a tejidos y organos infectados (animales de laboratorio) eran sumergidas en vesubina durante dos minutos. La variante en este trabajo es la utilizacion de muestras de esputo con su posterior inclusion rapida en vesubina. Del procedimiento: Frotis, fijacion (calor, tiempo), solucion de azul de metileno alcalino 24 horas, lavado, vesulina rapida, lavado, secado, observar. De la observacion al microscopio: Un fondo marron y los bacilos de un bello color azul. Concluimos que esta tecnica de tincion es morosa, compleja y dificilmente reproducible, puesto que es sensible y no especifica por lo que recomendamos la tecnica de Ziehl Neelsen por su bajo costo, facil realizacion y su aplicabilidad a un diagnostico. (Baciloscopia)

Release of superoxide anion from activated mouse peritoneal macrophages during Mycobacterium tuberculosis infection.

Mouse peritoneal macrophage monolayers infected with M. tuberculosis were cultured in RPMI up to 7 days. Release of superoxide was assayed on different days in presence or absence of Phorbol myristate acetate (PMA), a known stimulator of NADPH oxidase which is involved superoxide production. Basal level of superoxide release was significantly higher in M. tuberculosis infected peritoneal mouse macrophages (P < 0.01) as compared to normal mouse macrophages. When normal and tuberculoid macrophage cultures were stimulated with PMA, increased superoxide anion release was observed in both the cultures but the increase of superoxide was significantly higher in normal macrophages as compared to tuberculoid stimulated macrophages. Superoxide release was maximum in 4 day old cultured macrophages and gradually it declined in older cultures by day 7, both in vitro and in vivo. A defective macrophage function in killing of M. tuberculosis bacilli was observed after 4 days of in vitro and in vivo cultures.

Interaccion farmacocinetica entre Ketoconazol Isoniacida y Rifampicina / Pharmacokinetic interaction of ketoconazole, Isoniacid and Rifampicin

A 8 enfermos tuberculosos, varones, entre 20 y 60 años de edad, se les dio Isoniacida, 5 mg/Kg, y Ketoconazol, 200 mg, primero uno por vez y luego, asociados y se midió la concentración plasmática de las drogas a las horas 0,2 y de la toma. Isoniacida se midió por espectrofotometría y de Ketoconazol, por método microbiológico con Candida albicans como cepa testigo. Cuando se dieron ambas drogas asociadas se comprobó un marcado descensio de la concentración de Ketoconazol pero no hubo modificaciónes en la concentración de isoniacida. Un estudio análogo se efectuócon 11 pacientes tuberculosos a quienes se les dio Rifampicina, 10 mg/Kg, y Ketoconazol mg. Las concentraciones de ambas drogas se midieron a las mismsas horas. La rifampicina se midió por cromatografía líquida de alta presión. Al recibir las drogas asociadas se comprobó un marcado descenso en las concentraciones plasmáticas tanto para la Rifampicina como para el ketoconazol. La investigación de una probable interacción quimica entre Isoniacida y Ketoconazol por un lado y entre Rifampicina y Ketoconazol, por otro lado, dio resultados negativos. Se concluye que existe una marcada interacción farmacocinética del Ketoconazol con Isoniacida y Rifampicina, hecho que debe ser tenido en cuenta si se deben administrar tratamiento antimicótico y antiinfecciosos simultáneos