Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

Clinical and epidemiological profiles of patients with American cutaneous leishmaniasis from the states of Pernambuco and Amazonas, Brazil.

INTRODUCTION: Brazil has a high number of cases of American cutaneous leishmaniasis (ACL) in the north and northeast regions. Therefore, continuous surveillance of environmental and socioeconomic factors in endemic areas is needed to develop strategic control measures. This study aimed to describe the clinical and epidemiological profiles of patients with ACL. METHODS: All patients were from the states of Amazonas and Pernambuco, and examinations were carried out between 2015 and 2018. All patients had a clinical and epidemiological history compatible with ACL after positive diagnostic tests. Information obtained from medical records included gender, employment activity, level of education, age, and number and sites of lesions. RESULTS: A total of 213 patients were included, of whom 30.98% were female and 69.02% were male. The main employment activity was agriculture (27.56%). The most common level of education was elementary (62.42%). The average age was approximately 39 years. The majority of the patients presented only with one lesion (54.87%), and legs/feet were the most commonly affected area (48.25%), followed by the arms/hands (44.75%). CONCLUSIONS: These data demonstrated that irrespective of the patients' places of origin, interventions need to be focused on men of economically productive age, in view of the high risk of exposure to the vector in this group. Education activities need to be directed to farmers about the importance of protection against ACL vectors during work. Such information must also be directed to employers as a way of implementing and maintaining appropriate working conditions and stepping up vector control.

Clinical and epidemiological profiles of patients with American cutaneous leishmaniasis from the states of Pernambuco and Amazonas, Brazil

Abstract INTRODUCTION: Brazil has a high number of cases of American cutaneous leishmaniasis (ACL) in the north and northeast regions. Therefore, continuous surveillance of environmental and socioeconomic factors in endemic areas is needed to develop strategic control measures. This study aimed to describe the clinical and epidemiological profiles of patients with ACL. METHODS: All patients were from the states of Amazonas and Pernambuco, and examinations were carried out between 2015 and 2018. All patients had a clinical and epidemiological history compatible with ACL after positive diagnostic tests. Information obtained from medical records included gender, employment activity, level of education, age, and number and sites of lesions. RESULTS: A total of 213 patients were included, of whom 30.98% were female and 69.02% were male. The main employment activity was agriculture (27.56%). The most common level of education was elementary (62.42%). The average age was approximately 39 years. The majority of the patients presented only with one lesion (54.87%), and legs/feet were the most commonly affected area (48.25%), followed by the arms/hands (44.75%). CONCLUSIONS: These data demonstrated that irrespective of the patients' places of origin, interventions need to be focused on men of economically productive age, in view of the high risk of exposure to the vector in this group. Education activities need to be directed to farmers about the importance of protection against ACL vectors during work. Such information must also be directed to employers as a way of implementing and maintaining appropriate working conditions and stepping up vector control.

Neonatal malnutrition programs the oxidant function of macrophages in response to Candida albicans.

Experimental maternal nutrition restriction models are used to investigate short or long-term consequences of nutritional deficiency on puppies' growth. By assuming that the immune function is directly related to host's nutritional status, the current study aims to investigate the effects of neonatal malnutrition on oxidative stress and on the cell death of the alveolar macrophage after in vitro infection by Candida albicans. Wistar rats were suckled by mothers fed on diets containing 17% protein (Nourished group) or 8% protein (Malnourished group) in the current assay. Both groups received the standard diet used in the vivarium until adulthood, after weaning. The results showed that the offspring from mothers fed on low-protein diet presented lower body weight from 5 days of life on. Their low weight remained until adulthood when it was compared to that of rats in the nourished group. Superoxide and nitric oxide production was lower in malnourished animals and it was accompanied by low inducible nitric oxide synthase gene expression levels in systems in which the alveolar macrophages were challenged by immunogenic stimulus. No significant differences were observed in comparisons performed between the nourished and malnourished groups in any of the analyzed cell viability (apoptosis/necrosis) parameters. The fungal inoculum-stimulated system induced higher oxidative stress and cell death by necrosis. The current study demonstrated that dietary restriction during lactation alters the oxidant function of alveolar macrophages in puppies; It happens from the gene transcription step to the release of mediators, thus compromising the host's defenses against Candida albicans. It raises the possibility that Candida albicans may cease to be a commensal fungus to become a pathogen in offspring that have suffered nutritional deficiency during critical developmental periods, due to impaired immune responses.

Identification of phlebotomine sand fly blood meals by real-time PCR.

BACKGROUND: Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. METHODS: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 µl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. RESULTS: The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. CONCLUSIONS: The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.

Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco / Leishmaniose cutânea no nordeste do Brasil: uma avaliação crítica dos estudos realizados no Estado de Pernambuco

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

A leishmaniose cutânea americana (LCA) é uma doença complexa com características clínicas e epidemiológicas que podem variar de região para região. De fato, pelo menos, sete diferentes espécies de Leishmania, incluindo Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi e Leishmania (Leishmania) amazonensis, têm sido incriminadas na etiologia da LCA no Brasil, e numerosas espécies de flebotomíneos do gênero Lutzomyia, foram considerados vetores suspeitos ou comprovados. Devido ao seu caráter focal, a compreensão da dinâmica da LCA a nível local é imprescindível para a implementação de medidas de controle eficazes. Este trabalho consiste de uma revisão narrativa sobre a epidemiologia da LCA em Pernambuco, nordeste do Brasil. Além disso, se enfatiza a necessidade de maior efetividade no diagnóstico, tratamento, controle e estratégias de prevenção para as populações afetadas. Este artigo fornecerá aos pesquisadores uma avaliação crítica da LCA em Pernambuco. Espera-se também contribuir com as autoridades de saúde pública no aprimoramento das estratégias atuais de controle da LCA nos níveis estaduais e nacional.

Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco.

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

Avanços biotecnológicos para o diagnóstico das doenças infecciosas e parasitárias / Biotechnology advances for diagnosis of infectious and parasitic diseases

Os recentes surtos epidêmicos de doenças emergentes e reemergentes têm demonstrado a importância da aplicação de medidas de controle e prevenção. Para que tais medidas sejam eficazes, o desenvolvimento de métodos de diagnóstico acurados é essencial. Os métodos decorrentes do aprimoramento da biologia molecular e celular têm propiciado a utilização de técnicas diagnósticas que produzem um resultado confiável em poucos minutos ou horas. Os ensaios imunocromatográficos, a PCR e suas variações, a tecnologia de microarranjos de DNA, a citometria de fluxo e a análise do proteoma constituem exemplos. Alguns desses métodos já estão disponíveis em laboratórios, especialmente os da rede privada. Contudo, a interpretação dos resultados requer níveis diferentes de conhecimento, o que torna necessária a capacitação de recursos humanos para sua maior difusão e aproveitamento. Espera-se, portanto, que o surgimento de novas tecnologias resulte no desenvolvimento de novas drogas e terapias e, sobretudo, de métodos diagnósticos que auxiliem a investigação epidemiológica e promovam melhorias na qualidade de vida da população ao se implantar rapidamente o tratamento.

Desenvolvimento e avaliação de um sistema baseado em PCR em tempo real para o diagnóstico da infecção por Leishmania (Leishmania) infantum / Development and evaluation of system based on real-time in PCR for the diagnosis of Leishmania (Leishmania) infantum infection in dogs

O diagnóstico precoce da leishmaniose visceral (LV) é importante para evitar danos severos que podem levar o paciente à morte. Neste contexto, os métodos moleculares vêm sendo desenvolvidos com destaque para a tecnologia da reação em cadeia da polimerase (PCR). Recentemente, a PCR apresentou um significativo avanço em sua tecnologia; é a PCR quantitativa em tempo real (qPCR). Objetivouse desenvolver e avaliar um sistema baseado em qPCR para o diagnóstico da infecção por Leishmania infantum em cães, bem como efetuar uma análise comparativa com a PCR convencional utilizada no Serviço de Referência em Leishmanioses de Pernambuco. Com base na seqüência NCBI Z35273.1 de L. infantum disponível no BLAST-NCBI foram desenhados primers específicos para o complexo L. donovani. A combinação dos primers gerou sistemas de detecção, sendo o sistema Linf 1 B o mais promissor. A curva-padrão foi gerada resultando em limite de detecção de 10 fg de DNA genômico de L. infantum (7x10-2 parasitas), e = 0.9417, R2= 0.931 e slope= -3.47. O sistema desenvolvido foi avaliado em sangue de cães positivos e negativos para leishmaniose visceral canina, apresentando sensibilidade de 100 por cento e especificidade de 83,33 por cento. A análise comparativa com o PCR convencional mostrou que, utilizando-se amostras de sangue, o qPCR é mais sensível (sensibilidade PCR= 23,8 por cento, qPCR= 100 por cento). Em relação à especificidade, apesar da PCR convencional ter apresentado valor de 100 por cento, as análises por meio dos intervalos de confiança e o teste 2? mostraram que os dois testes (PCR convencional e qPCR) são equivalentes. Desta forma, conclui-se que os resultados obtidos para o sistema de qPCR, em amostra de sangue de cães e, a análise comparativa com a PCR convencional (RV1/RV2), sugerem sua utilização nas rotinas de diagnóstico da infecção por L. infantum.