1,2,3-Triazole derivatives: synthesis, docking, cytotoxicity analysis and in vivo antimalarial activity.

Malaria remains one of the most important parasitic diseases in the world. The multidrug-resistant Plasmodium strains make the treatment currently available for malaria less effective. Therefore, the development of new drugs is necessary to overcome therapy resistance. Triazole derivatives exhibit several biological activities and provide a moiety that is promising from the biological perspective. Due to the structural similarity to NADH, it is believed that triazoles can bind to the active site of the Plasmodium lactate dehydrogenase (pLDH) enzyme. The present work evaluates the antimalarial activity of 1,2,3-triazole derivatives by in silico, in vitro, and in vivo studies. Preliminary in silico ADMET studies of the compounds demonstrated good pharmacokinetic properties. In silico docking analysis against LDH of Plasmodium berghei (PbLDH) showed that all compounds presented interactions with the catalytic residue in the active site and affinity similar to that presented by chloroquine; the most common antimalarial drug. Cytotoxicity and hemolysis by these derivatives were evaluated in vitro. The compounds 1, 2, 5, 8, and 9 proved to be non-cytotoxic in the performed tests. In vivo antimalarial activity was evaluated using mice infected with Plasmodium berghei NK65. The five compounds tested exhibited antimalarial activity until nine days post-infection. The compound 5 showed promising activities, with about 70% parasitemia suppression. Considering the in vitro and in vivo studies, we believe the compound 5 to be the most promising molecule for further studies in antimalarial chemotherapy.

In vitro and in vivo antiplasmodial activity of novel quinoline derivative compounds by molecular hybridization.

Chloroquine (CQ) has been the main treatment for malaria in regions where there are no resistant strains. Molecular hybridization techniques have been used as a tool in the search for new drugs and was implemented in the present study in an attempt to produce compound candidates to treat malarial infections by CQ-resistant strains. Two groups of molecules were produced from the 4-aminoquinoline ring in conjugation to hydrazones (HQ) and imines (IQ). Physicochemical and pharmacokinetic properties were found to be favorable when analyzed in silico and cytotoxicity and antiplasmodial activity were assayed in vitro and in vivo showing low cytotoxicity and selectiveness to the parasites. Candidates IQ5 and IQ6 showed important values of parasite growth inhibition in vivo on the 5th day after infection (IQ5 15 mg/kg = 72.64% and IQ6 15 mg/kg = 71.15% and 25 mg/kg = 93.7%). IQ6 also showed interaction with ferriprotoporphyrin IX similarly to CQ. The process of applying condensation reactions to yield imines is promising and capable of producing molecules with antiplasmodial activity.

Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based permeability.

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to­lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 µM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV-vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.

What do we know about the role of regulatory B cells (Breg) during the course of infection of two major parasitic diseases, malaria and leishmaniasis?

Parasitic diseases, such as malaria and leishmaniasis, are relevant public health problems worldwide. For both diseases, the alarming number of clinical cases and deaths reported annually has justified the incentives directed to better understanding of host's factors associated with susceptibility to infection or protection. In this context, over recent years, some studies have given special attention to B lymphocytes with a regulator phenotype, known as Breg cells. Essentially important in the maintenance of immunological tolerance, especially in autoimmune disease models such as rheumatoid arthritis and experimentally induced autoimmune encephalomyelitis, the function of these lymphocytes has so far been poorly explored during the course of diseases caused by parasites. As the activation of Breg cells has been proposed as a possible therapeutic or vaccine strategy against several diseases, here we reviewed studies focused on understanding the relation of parasite and Breg cells in malaria and leishmaniasis, and the possible implications of these strategies in the course of both infections.

CCL2, CXCL8, CXCL9 and CXCL10 serum levels increase with age but are not altered by treatment with hydroxychloroquine in patients with osteoarthritis of the knees.

AIM: Osteoarthritis (OA) is a major cause of morbidity and incapacity in the elderly. This study evaluates serum levels of the chemokines CCL2, CXCL8, CXCL9, and CXCL10 in 16 patients with primary OA of the knees, and investigates how treatment with hydroxychloroquine (HCQ) for 4 months affects these chemokine levels. METHOD: Thirteen elderly patients received a placebo. Healthy control groups consisted of 10 elderly individuals (age > 60 years) with no clinical or radiological evidence of OA (CT-O), and 10 young adult individuals, (CT-Y group, age < 40 years). RESULTS: The CT-Y group presented lower levels of all chemokines studied, in comparison to the other groups. HCQ treatment did not alter the serum levels of CCL2 (P = 0.80), CXCL8 (P = 0.76), CXCL9 (P = 0.95) and CXCL10 (P = 0.74) in OA patients. CONCLUSION: Hydroxychloroquine treatment did not alter the serum levels of CCL2, CXCL8, CXCL9 or CXCL10 in patients with OA of the knees, although increased serum levels correlated with aging for all subjects, including controls.

New quinoline derivatives demonstrate a promising antimalarial activity against Plasmodium falciparum in vitro and Plasmodium berghei in vivo.

Malaria continues to be an important public health problem in the world. Nowadays, the widespread parasite resistance to many drugs used in antimalarial therapy has made the effective treatment of cases and control of the disease a constant challenge. Therefore, the discovery of new molecules with good antimalarial activity and tolerance to human use can be really important in the further treatment of the disease. In this study we have investigated the antiplasmodial activity of 10 synthetic compounds derived from quinoline, five of them combined to sulfonamide and five to the hydrazine or hydrazide group. The compounds were evaluated according to their cytotoxicity against HepG2 and HeLa cell lines, their antimalarial activity against CQ-sensitive and CQ-resistant Plasmodium falciparum strains and, finally, their schizonticide blood action in mice infected with Plasmodium berghei NK65. The compounds exhibited no cytotoxic action in HepG2 and HeLa cell lines when tested up to a concentration of 100 µg/mL. In addition, the hydrazine or hydrazide derivative compounds were less cytotoxic against cell lines and more active against CQ-sensitive and CQ-resistant P. falciparum strains, showing high SI (>1000 when SI was calculated using the CC50 from the 3D7 strain as reference). When tested in vivo, the hydrazine derivative 1f compound showed activity against the development of blood parasites similar to that observed with CQ, the reference drug. Interestingly, the 1f compound demonstrated the best LipE value (4.84) among all those tested in vivo. Considering the in vitro and in vivo activities of the compounds studied here and the LipE values, we believe the 1f compound to be the most promising molecule for further studies in antimalarial chemotherapy.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil.

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Essential oil from leaves of Lantana camara: a potential source of medicine against leishmaniasis

Leishmaniasis is an infection of viscera or tegument caused by protozoa Leishmania sp. The extensive period required for the treatment, which involves the use of toxic medicines, leads patients to drop treatment increasing the development of resistant forms of Leishmania sp. Lantana camara L., Verbenaceae, is a tropical plant native from America. Folk uses have been described for treatment of tumors, tetanus, rheumatism and malaria. This study evaluates the leishmanicidal activity of the essential oil of leaves from L. camara on promastigote forms of Leishmania chagasi and L. amazonensis and its toxic effects on Artemia salina (brine shrimp test), macrophage cultures and BALB/c mice. The chemical composition was evaluated using the gas chromatography coupled with mass spectrometer (GC-MS). Thirty substances, mostly mono and sesquiterpenes were identified. The most representative constituents were: germacrene D (24.90%), farnesene derivatives (22%) and (E)-cariophylene (14.31%). Bioassays revealed a significant leishmanicidal activity of essential oil against L. amazonensis (IC50 0.25 µg/ mL) and a potential toxic effect on Brine shrimp (LC50 10 µg/mL) and macrophage assays (CC50 4 µg/mL), while there was no toxic manifestation on mice. The data show the relevant potential of L. camara as a source of medicine for leishmaniasis treatment.

Prevalência de parasitoses intestinais em comunidade quilombola no município de Bias Fortes, estado de Minas Gerais, Brasil, 2008 / Prevalence of parasitic intestinal diseases in a quilombola community, in the Municipality of Bias Fortes, State of Minas Gerais, Brazil, 2008

Objetivo: investigar a prevalência e os fatores associados às parasitoses intestinais na população de Colônia do Paiol, comunidade quilombola na Zona da Mata de Minas Gerais, Brasil. Metodologia: procedeu-se um estudo de corte transversal por censo, sendo que dos 425 moradores, 391 (92 por cento) foram avaliados mediante resposta a questionário estruturado e exame coproparasitológico. Resultados: a positividade para pelo menos uma espécie parasitária foi de 63,8 por cento, sendo as espécies patogênicas mais frequentes Ascaris lumbricoides (22,4 por cento) e Trichuris trichiura (17,9 por cento); o poliparasitismo ocorreu em 36,5 por cento dos investigados; predominaram casos no sexo feminino e na faixa etária de escolares de seis a 14 anos de idade. Conclusão: observou-se associação entre a presença de parasitos intestinais e determinadas condições ambientais, confirmando a necessidade de melhoria das condições de saneamento básico e de acesso ao serviço de saúde, de utilização de medicações de fácil administração no tratamento das enteroparasitoses e de educação em saúde para os membros da comunidade.

Objective: the study aims to investigate prevalence and factors associated to intestinal parasitic diseases in the population of Colônia do Paiol, a quilombola community in the municipality of Bias Fortes, located in the Zona da Mata region of the State of Minas Gerais, Brazil. Methodology: cross-sectional census study was conducted, and 391 (92 per cent) of the 425 inhabitants were interviewed through a questionnaire and evaluated by means of a coproparasitologic test. Results: the test positivity rate for at least one parasite specie was of 63.8 per cent, and the pathogenic species more often found were Ascaris lumbricoides (22.4 per cent) and Trichuris trichiura (17.9 per cent); multiparasitism was diagnosed in 36.5 per cent of those who took part in the study; prevalence was higher for females compared to males, also for school children aged from 6 to 14 years old. Conclusion: a significant association was observed between the presence of intestine parasites and a number of environment conditions such as sewage collection and disposal, access to basic health services, use of easily administered medicines for treatment of enteroparasitosis, and education policies on basic health knowledgeamong population members.

4-aminoquinoline analogues and its platinum (II) complexes as antimalarial agents.

The high incidence of malaria and drug-resistant strains of Plasmodium have turned this disease into a problem of major health importance. One of the approaches used to control it is to search for new antimalarial agents, such as quinoline derivates. This class of compounds composes a broad group of antimalarial agents, which are largely employed, and inhibits the formation of ß-haematin (malaria pigment), which is lethal to the parasite. More specifically, 4-aminoquinoline derivates represent potential sources of antimalarials, as the example of chloroquine, the most used antimalarial worldwide. In order to assess antimalarial activity, 12 4-aminoquinoline derived drugs were obtained and some of these derivatives were used to obtain platinum complexes platinum (II). These compounds were tested in vivo in a murine model and revealed remarkable inhibition of parasite multiplication values, whose majority ranged from 50 to 80%. In addition they were not cytotoxic. Thus, they may be object of further research for new antimalarial agents.

Prevalence and risk factors for giardiasis and soil-transmitted helminthiasis in three municipalities of Southeastern Minas Gerais State, Brazil: risk factors for giardiasis and soil-transmitted helminthiasis.

Giardiasis and soil-transmitted helminthiasis (STH) are parasitic diseases that are among the major health concerns observed in economically disadvantaged populations of developing countries, and have clear social and environmental bases. In Brazil, there is a lack of epidemiologic data concerning these infections in the study area, whose inhabitants have plenty of access to health care services, including good dwelling and adequate sanitary conditions. In this survey we investigated the risk factors for giardiasis and STH in three municipalities with good sanitation, situated in Minas Gerais state, Brazil. A cross-sectional survey was conducted in the municipalities of Piau, Coronel Pacheco and Goianá, in both urban and rural areas. The fieldwork consisted of a questionnaire and the examination of 2,367 stool samples using the Hoffmann, Pons and Janer method. Of all individuals from the population sample, 6.1% were found infected with the parasitic diseases included in this work. Hookworm infection was the most prevalent disease, followed by giardiasis, trichuriasis and ascariasis. Infection was more prevalent in males (8.1%, p < 0.001; odds ratio [OR] = 1.975) and in individuals living in rural areas (8.6%, p = 0.003; OR = 1.693). Multivariate analysis showed that variables such as inadequate sewage discharge (p < 0.001), drinking of unsafe water (p < 0.001), lack of sanitary infrastructure (p = 0.015), and host sex (p < 0.001) were the risk factors more strongly associated with infection status (95% confidence interval [CI]). In this study we demonstrate that giardiasis and STH still persist, infecting people who have good housing conditions and free access to public health care and education.

Thiopurine derivatives containing triazole and steroid: synthesis, antimalarial and antileishmanial activities.

A series of novel 6-thiopurine derivates containing 1,2,3-triazole were synthesized and their in vivo antimalarial activity and in vitro antileishmanial activity were examined. The compounds 10, 11, 12 and 14 presented higher values of inhibition of parasite multiplication than chloroquine. For antileishmanial activity, the compound 14 showed activity against the three species of Leishmania tested. None of compounds showed cytotoxicity against mammalian cells.

Increased IgG1, IFN-gamma, TNF-alpha and IL-6 responses to Mycobacterium tuberculosis antigens in patients with tuberculosis are lower after chemotherapy.

Detection of specific antibodies may represent an additional tool in diagnosis of tuberculosis (TB). Herein, levels of serum IgG antibodies against early secreted antigenic target (ESAT-6), culture filtrate antigen-10 (CFP-10) and 16 kDa Mycobacterium tuberculosis antigens were measured in 33 active pulmonary TB patients (0M-TB), in 47 patients after 1-3 months of treatment (3M-TB) and in 22 patients who had completed 6 months of chemotherapy (6M-TB). The control group consisted of 38 BCG-vaccinated healthy controls (HC). In addition, IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-6, IL-2, IL-4 and IL-10 production in PBMC cultures from 20 patients were measured following stimulation with the M. tuberculosis-specific fusion protein ESAT-6/CFP-10. Elevated levels of IgG against ESAT-6, CFP-10 and 16 kDa antigens were detected in 0M-TB and 3M-TB patients in comparison to the HC and 6M-TB groups. Receiver operating characteristic analysis indicated sensitivity of 85, 94 and 61% and specificity of 89, 87 and 89% for serum IgG against ESAT-6, CFP-10 and 16 kDa, respectively. A predominant IgG1 response to ESAT-6 and CFP-10 was observed in 0M-TB patients, together with ESAT-6/CFP-10-specific IFN-gamma, TNF-alpha and IL-6 that were produced at lower levels in the 6M-TB group. These data indicate that a T(h)1 phenotype against early phase Mtb antigens appears to be dominant in the peripheral blood of patients with active pulmonary TB that is reduced after chemotherapy. Taken together, ESAT-6/CFP-10 cytokine tests together with detecting IgG antibodies specific to ESAT-6 and CFP-10 may be the useful TB disease biomarkers in monitoring treatment success.

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array.

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Anti-mycobacterial treatment reduces high plasma levels of CXC-chemokines detected in active tuberculosis by cytometric bead array

Chemokines recruit and activate leukocytes, assisting granuloma formation. Herein, we evaluated plasma chemokines in patients with active tuberculosis (ATB) and after completing treatment (TTB) and compared them to BCG-vaccinated healthy controls (HC). Levels of chemokines were measured by cytometric bead array. Levels of CXCL8, CXCL9 and CXCL10 were higher in ATB patients compared to HC, but they decreased in TTB. Levels of CCL2 and CCL5 in ATB patients were similar to those observed in HC. Thus, the high levels of CXC-chemokines detected during ATB, which can modulate the trafficking of immune cells from the periphery to the site of infection, were reversed by anti-mycobacterial treatment.

Validação do número de lâminas para realização do método de sedimentação espontânea das fezes / Validation of the number of slides for accomplishment of the faecal spontaneous sedimentation method

Este estudo avaliou o número satisfatório de lâminas a serem examinadas pela técnica de sedimentação espontânea das fezes ou de Hoffmann, Pons e Janer (HPJ), com o intuito de contribuir para a eficiência no diagnóstico de exames coproparasitológicos no diagnóstico de parasitoses, objetivando menor custo e maior benefício. A partir de um estudo da prevalência de enteroparasitoses, cujo cálculo amostral aleatório teve como base toda a população residente no município de Piau -Minas Gerais, foram investigadas 925 amostras. O número máximo de lâminas examinadas para cada amostra de fezes foi de cinco, considerado como padrão-ouro. Cada lâmina foi avaliada por cinco examinadores diferentes. Na análise de apenas uma lâmina, a frequência de positividade para enteroparasitos foi inferior a 60% e aumentou para 79,7% na análise de uma segunda lâmina. Na observação da terceira e quarta lâminas, a positividade das amostras foi 87,5% e 95,3%, respectivamente. Verificou-se que a sensibilidade e especificidade aumentaram com o número de lâminas examinadas e que na observação de três lâminas por amostra, a sensibilidade foi de 87,5% e a especificidade de 99,1%. Entre os enteroparasitos, os ancilostomídeos e Giardia lamblia foram os mais prevalentes, representando 13,7% de positividade para ambos. A análise de apenas uma lâmina por amostra elevou o risco de resultado falso negativo. Os resultados encontrados sugerem a realização de três lâminas por amostra pela técnica do HPJ na tentativa de aumentar a acurácia do método diagnóstico com custos sustentáveis para a gestão junto ao Sistema Único de Saúde.

This work analyzed the optimal number of slides necessary in the spontaneous fecal sedimentation method, also known as the Hoffmann, Pons e Janer (HPJ) technique, with the objective of increasing efficiency and reducing cost of the diagnosis of parasitic infections using the stool test. In a study of the prevalence of intestinal parasite infection in a random sampling of the resident population of Piau, Minas Gerais, 925 samples were analyzed. The maximum number of slides analyzed for each fecal sample was five, considered as the gold standard. Each slide was evaluated by five different analysts. Upon examination of only one slide, the positive frequency for enteroparasites was less than 60.0% and increased to 79.7% when analyzing a second slide. After analysis of a third and fourth slide, the number of enteroparasite positive samples increased to 87.5% and 95.3%, respectively. It was verified that the sensitivity and specificity increased with the number of slides examined and, after observation of three slides per stool sample, the sensitivity was 87.5% and the specificity was 99.1%. Among the enteroparasites detected, the most prevalent were hookworms and Giardia lamblia, with a frequency of 13.7% for both. The analysis of only one slide per sample increased the risk of false negative result. The findings suggest that the examination of three slides per stool sample using the HPJ fecal spontaneous sedimentation technique is optimal in order to increase the efficiency of the fecal diagnostic method without substantially increasing costs for the public health care system.

Immunological diagnosis of tuberculosis: problems and strategies for success.

Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB(R) and T SPOT.TB(R), which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.

Diagnóstico imunológico da tuberculose: problemas e estratégias para o sucesso / Immunological diagnosis of tuberculosis: problems and strategies for success

A tuberculose continua sendo um grave problema social e de saúde, afetando milhões de pessoas anualmente. A vacina Bacille Calmette-Guerin (BCG), usada no controle profilático, é incapaz de conter a progressão da doença, que usualmente se manifesta através da queda da imunidade celular do indivíduo. O diagnóstico da tuberculose em seus estágios iniciais, aliado à poliquimioterapia, pode contribuir para o controle da disseminação da infecção. Os atuais métodos de diagnóstico apresentam problemas, como: baixa sensibilidade da baciloscopia; longo tempo de realização das culturas microbiológicas; e baixa especificidade do teste cutâneo com o derivado protéico purificado do M. tuberculosis. Novos métodos de diagnóstico que utilizam antígenos específicos (por exemplo, os conhecidos em inglês como o early secreted antigenic target 6-kDa e o culture filtrate protein 10-kDa), estão sendo testados. Os genes que codificam esses antígenos estão localizados na região de diferença 1 do M. tuberculosis, M. africanum e M. bovis, mas estão ausentes no M. bovis (BCG) e na maioria das micobactérias do meio ambiente. Métodos de diagnóstico baseados na produção de interferon-gama por linfócitos T, em resposta a esses antígenos, como o QuantiFERON-TB® e o T SPOT.TB®, estão sendo testados, e superam o teste cutâneo com o derivado protéico purificado nas seguintes características: maior sensibilidade; menor reatividade cruzada devido à vacinação com o BCG ou infecção por micobactérias do meio ambiente; e tempo de execução. A introdução de métodos de diagnóstico mais específicos e sensíveis, assim como um maior entendimento dos mecanismos moleculares e celulares que regulam a interação parasito-hospedeiro, pode contribuir para um eficiente combate à tuberculose.

Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB® and T SPOT.TB®, which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients.

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.