Importância epidemiológica dos roedores como reservatórios de Leptospira spp. na cidade de Bissau (Guiné-Bissau) e áreas limítrofes

A leptospirose é uma zoonose de importância mundial, causada por espiroquetídeos do género Leptospira, com implicações na Medicina Veterinária e Humana (One Health) e impacte significativo na Saúde Pública e na Economia Pecuária. Embora de reconhecida importância em zonas tropicais, pouco se sabe sobre a leptospirose em África. Na Guiné-Bissau existem apenas dois estudos de seroprevalência em humanos, datados das décadas de 50 e 80 do século XX, que indicam a existência da doença. Os roedores, em particular os murinos (géneros Rattus e Mus, entre outros), constituem os principais reservatórios da bactéria, a partir dos quais as leptospiras são transmitida aos humanos e/ou a outros animais. A inespecificidade das manifestações clínicas da leptospirose nos humanos leva a que esta seja facilmente confundida com malária, septicemia, hepatite viral, dengue, ou outras doenças que cursam com síndrome febril, pelo que o doente deixa assim de receber o tratamento adequado e em tempo útil. Para o conhecimento da epidemiologia da leptospirose, em qualquer região e/ou país, são indispensáveis a identificação dos serovares mais prevalentes e a caracterização dos respetivos reservatórios/hospedeiros. Estes conhecimentos são críticos para a implementação de medidas de controlo eficazes da doença. O presente estudo teve como objetivo contribuir para um melhor conhecimento da importância epidemiológica e ecológica dos roedores e o seu papel como reservatório de Leptospira spp. na cidade de Bissau e áreas limítrofes, pela determinação da taxa da prevalência bacteriológica de Leptospira spp. na população de roedores capturados. Entre Maio e Agosto de 2013 foram capturados 104 roedores em sete (7) bairros da cidade de Bissau. Procedeu-se à necropsia dos roedores e os rins foram colhidos e conservados para posterior análise no Instituto de Higiene de Medicina Tropical em Lisboa. Foi determinada a taxa de infeção bacteriológica pela deteção e identificação de DNA leptospírico com recurso a técnicas moleculares (PCR e sequenciação). Foram utilizados dois protocolos, ambos com primers específicos para leptospiras patogénicas, um PCR convencional com primers baseados no gene hap1 e o outro, um nested-PCR com primers obtidos do gene lipL32. Em 10% das amostras tentou-se ainda o método de cultura para obtenção de isolados de Leptospira. Os roedores foram identificados como pertencentes às espécies Rattus norvegicus (63,5%), Mus musculus (34.6%) e Rattus rattus (1.9%). Trinta dos 104 roedores capturados (29%) mostraram presença de DNA leptospírico, sendo que (20/30; 67%) pertenciam à espécie R. norvegicus, (9/30; 30%) à espécie M. musculus e (1/30; 3%), pertencia à espécie R. rattus. Foram sequenciadas 12 das 30 amostras positivas, cuja análise por BLAST, permitiu a identificação dos serovares Icterohaemorrhagiae e Pomona, pertencentes respetivamente às genoespécies L. interrogans e L. kirschneri, ambas reconhecidamente de elevada patogenicidade para os humanos. Não houve até à data crescimento bacteriano nas sementeiras efetuadas. Em conclusão, este trabalho permitiu, pela primeira vez, identificar leptospiras patogénicas em roedores na cidade de Bissau, pelo que teve um carácter pioneiro na determinação do papel destes mamíferos silváticos como reservatórios de leptospiras na Guiné-Bissau. Os resultados obtidos justificam a realização de estudos adicionais e mais exaustivos de investigação epidemiológica sobre esta zoonose em Bissau e no território nacional. Os ganhos para a Saúde Humana e Veterinária no país poderiam ser significativos, nomeadamente em termos do benefício no acompanhamento clínico dos doentes com leptospirose e na implementação de medidas adequadas para o controlo desta zoonose.“Health is priceless”, however knowing the real costs of a disease allows a more efficient allocation in the management of the health care resources. In Portugal the current Government expenditure on health has increased over the years, and in 2012 reached 9,5% of the gross domestic product, which means that the Government has spent about 1 474,03 € per capita (Instituto Nacional de Estatística, 2013). Cardiovascular diseases are the first cause of mortality in the Portuguese population, with a significant economic impact: health care, hospitalizations, medications, lost work days, among many others. In 2011 the cardiovascular group of medicines represented 29,25% of the burden on the National Health Service, when considering the ambulatory market, of which 69,85% corresponded to antihypertensive drugs (Direção Geral da Saúde, 2013c; INFARMED, 2011; Nichols et al., 2012). Hypertension is one of the diseases that has most contributed to this scenario: several studies has demonstrated the association between hypertension and various diseases (Apifarma & Doentes, 2011; Nicolau et al., 2009). It is also the risk factor with the highest prevalence in the Portuguese population. The most recent studies estimate 11,3 hospital admissions per 100 000 people, under 70 years old, but it only refers to hypertension, not considering the consequences of this disease when left untreated or adequately monitored, nor estimate the total costs of the hospitalizations associated with this pathology. The aim of this thesis was to estimate the costs of hospitalizations associated with hypertension in mainland Portuguese population, in 2006-2012, and study some of the variables that may be associated with the evolution of these costs, with special focus on mean vintage, which is a variable that measures the impact of technological innovation of medicines. An observational, analytical, longitudinal study was conducted, with a quantitative approach. The time period under consideration corresponds to 2006-2012, being considered the population of continental Portugal. The hospitalization episodes were characterized in terms of age, gender, total cost, average cost per episode, health region, total days of hospitalization, type of episode and average length of stay. It was used multivariate linear regression to assess the effect of the explanatory variables tested: mean vintage, gender, education, age and medicated patients. The results showed that increasing age has a positive relationship with the costs of hospitalization; also the male gender is associated with higher costs. Individuals with less education are associated with lower costs of hospitalization, adjusted for other variables. The mean vintage is also inversely related with total hospitalization costs: the higher the mean vintage, which means, that more recent drugs are being used in the treatment of hypertension, lower are the estimated admissions costs. This study provides relevant information for estimating the total costs of hospitalizations associated with hypertension, on the other hand allows to better understanding of the nature of these costs, particularly for newer drugs, which cost impact is more relevant, so this relationship should be depth in order to optimize its use in preventing hospitalizations associated with hypertension and reduces costs

Experimental evidence for limited in vivo virulence of Mycobacterium africanum

Funding Information: The authors thank the excellent support from the i3S scientific platforms, namely Animal Facility and Translational Cytometry. Funding. This work was supported by Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 ? Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Funda??o para a Ci?ncia e a Tecnologia, Minist?rio da Ci?ncia, Tecnologia e Inova??o in the framework of the project ?Institute for Research and Innovation in Health Sciences? (POCI-01-0145-FEDER-007274), and by grants FCT ? Aga Khan Development Network (ref 333197025), POCI-01-0145-FEDER-028955 (to MS), PTDC/BIA-MIC/30692/2017, and UID/Multi/04413/2013 (to DM and MV). BC and KF were funded by FCT Ph.D. scholarships SFRH/BD/114403/2016 and SFRH/BD/114405/2016, respectively. The Gulbenkian Foundation is acknowledged for a field work research grant to BC, Bolsas de apoio ? investiga??o para estudantes de doutoramento dos PALOP, Ref. P-146397. DM and MS were supported by FCT through Estimulo Individual ao Emprego Cient?fico. Publisher Copyright: © Copyright © 2019 Cá, Fonseca, Sousa, Maceiras, Machado, Sanca, Rabna, Rodrigues, Viveiros and Saraiva.Tuberculosis remains a public health problem and a main cause of death to humans. Both Mycobacterium tuberculosis and Mycobacterium africanum cause tuberculosis. In contrast to M. tuberculosis, which is geographically spread, M. africanum is restricted to West Africa. Differences have also been found in the growth rate and type of disease caused by M. africanum, globally suggesting an attenuation of this bacteria. In this study, we used the mouse model of infection to follow the dynamics of M. africanum infection in terms of bacterial burdens and tissue pathology, as well as the immune response triggered. Our findings support a lower virulence of M. africanum as compared to M. tuberculosis, including in mice lacking IFN-γ, a major protective cytokine in tuberculosis. Furthermore, the lung immune response triggered by M. africanum infection in wild-type animals was characterized by a discrete influx of leukocytes and a modest transcriptional upregulation of inflammatory mediators. Our findings contribute to elucidate the pathogenesis of M. africanum, supporting the hypothesis that this is an attenuated member of the tuberculosis-causing bacteria. Understanding the biology of M. africanum and how it interacts with the host to establish infection will have implications for our knowledge of TB and for the development of novel and better tools to control this devastating disease.publishersversionpublishe

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1β is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1β production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities

Heterogeneous Streptomycin Resistance Level Among Mycobacterium tuberculosis Strains From the Same Transmission Cluster

12 páginas, 5 figuras.Widespread and frequent resistance to the second-line tuberculosis (TB) medicine streptomycin, suggests ongoing transmission of low fitness cost streptomycin resistance mutations. To investigate this hypothesis, we studied a cohort of 681 individuals from a TB epidemic in Portugal. Whole-genome sequencing (WGS) analyses were combined with phenotypic growth studies in culture media and in mouse bone marrow derived macrophages. Streptomycin resistance was the most frequent resistance in the cohort accounting for 82.7% (n = 67) of the resistant Mycobacterium tuberculosis isolates. WGS of 149 clinical isolates identified 13 transmission clusters, including three clusters containing only streptomycin resistant isolates. The biggest cluster was formed by eight streptomycin resistant isolates with a maximum of five pairwise single nucleotide polymorphisms of difference. Interestingly, despite their genetic similarity, these isolates displayed different resistance levels to streptomycin, as measured both in culture media and in infected mouse bone marrow derived macrophages. The genetic bases underlying this phenotype are a combination of mutations in gid and other genes. This study suggests that specific streptomycin resistance mutations were transmitted in the cohort, with the resistant isolates evolving at the cluster level to allow low-to-high streptomycin resistance levels without a significative fitness cost. This is relevant not only to better understand transmission of streptomycin resistance in a clinical setting dominated by Lineage 4 M. tuberculosis infections, but mainly because it opens new prospects for the investigation of selection and spread of drug resistance in general.This work has been funded by of the projects NORTE-01-0145- FEDER-000039, NORTE-01-0145-FEDER-000013, and NORTE01-0145-FEDER-00002, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also funded through the Foundation for Science and Technology (FCT) – project FCT IF/00474/2014, UIDB/50026/2020 and UIDP/50026/2020. DR and CM were funded by FCT Ph.D. scholarships (grant numbers SFRH/BD/135422/2017 and SFRH/BD/132797/2017, respectively). DR was partially funded by the PGCD – Graduate Program Science for Development. MS is supported by FCT through the Estimulo ao Emprego Científico.Peer reviewe

Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils

Modulation of immunity and disease by glycans is increasingly recognized. However, how host glycosylation shapes and is shaped by tuberculosis remains poorly understood. We show that deficiency in the glucosaminyl (N-acetyl) transferase 1 (Gcnt1), a key enzyme for core-2 O -glycans biosynthesis, drives susceptibility to Mycobacterium tuberculosis infection. The increased susceptibility of Gcnt1 deficient mice was characterized by extensive lung immune pathology, mechanistically related to neutrophils. Uninfected Gcnt1 deficient mice presented bone marrow, blood and lung neutrophilia, which further increased with infection. Blood neutrophilia required Gcnt1 deficiency in the hematopoietic compartment, relating with enhanced granulopoiesis, but normal cellular egress from the bone marrow. Interestingly, for the blood neutrophilia to translate into susceptibility to M. tuberculosis infection, Gnct1 deficiency in the stroma was also necessary. Complete Gcnt1 deficiency associated with increased lung expression of the neutrophil chemoattractant CXCL2. Lastly, we demonstrate that the transcript levels of various glycosyltransferase-encoding genes were altered in whole blood of active tuberculosis patients and that sialyl Lewis x, a glycan widely present in human neutrophils, was detected in the lung of tuberculosis patients. Our findings reveal a previously unappreciated link between Gcnt1, neutrophilia and susceptibility to M. tuberculosis infection, uncovering new players balancing the immune response in tuberculosis