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Toxoplasma gondii, a protozoan parasite, is responsible for toxoplasmosis. The available therapy for patients with toxoplasmosis involves a combination of pyrimethamine and sulfadiazine, which have several adverse effects, including bone marrow suppression, megaloblastic anemia, leukopenia, and granulocytopenia. The development of therapeutic alternatives is essential for the management of toxoplasmosis, emphasizing the recent advances in nanomedicine. This study aimed to evaluate the in vitro effects of biogenic silver nanoparticles (AgNp-Bio) on tachyzoite forms and Leydig cells infected with T. gondii. We observed that the AgNp-Bio reduced the viability of the tachyzoites and did not exhibit cytotoxicity against Leydig cells at low concentrations. Additionally, treatment with AgNp-Bio reduced the rate of infection and proliferation of the parasite, and lowered the testosterone levels in the infected cells. It increased the levels of IL-6 and TNF-α and reduced the levels of IL- 10. Among the morphological and ultrastructural changes, AgNp-Bio induced a reduction in the number of intracellular tachyzoites and caused changes in the tachyzoites with accumulation of autophagic vacuoles and a decrease in the number of tachyzoites inside the parasitophorous vacuoles. Collectively, our data demonstrate that the AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and inflammatory mechanisms and could be a potential alternative treatment for toxoplasmosis.

Toxoplasma gondii is a protozoan from phylum Apicomplexa, which causes the toxoplasmosis infection; this one exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. Our goal was to verify whether the Toxoplasma gondii (RH strain) tachyzoites form pretreated with pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations could affect the infection processes, suggesting direct action on protozoa intracellular proliferation through the inhibition of isoprenoids in the parasite’s apicoplast. To have the adhesion, infection, and parasite proliferation during experimental infection investigated, HeLa cells (105) were subjected to a 24-hour infection by T. gondii tachyzoites forms of RH strain (5 × 105) pretreated for 30 min with pravastatin and/or simvastatin combined or not with pyrimethamine and sulfadiazine. Combined with conventional drugs at low concentrations pravastatin and simvastatin inhibit the adhesion, invasion, and intracellular proliferation of T. gondii in HeLa cells which are similar to the positive control. Pravastatin and simvastatin in association with pyrimethamine and sulfadiazine at low concentrations can be regarded as a promising, effective alternative to toxoplasmosis treatment with reduced side effects.

Aims To describe an anti-Strongyloides IgA, IgG and IgG immune complex antibody response profile in patients with pulmonary tuberculosis. Methods and results Saliva and serum samples were collected from 100 individuals: group I, 50 apparently healthy individuals; and group II, 50 pulmonary tuberculosis patients. The IgA, IgG and IgG immune complex detection were carried out via an ELISA immunoenzymatic test. Optical density medians in saliva samples of IgA antibody (median of 7.21) and IgG-IC (median of 4.95) were significantly higher in tuberculosis group compared to control individuals (median IgA of 3.93 and IgG-IC of 2.38). Conclusion This study presents antibody data to the field of pulmonary tuberculosis and strongyloidiasis coinfection, including saliva samples, and especially IgG immune complex detection.

Toxoplasma gondii é o protozoário causador da toxoplasmose, infecção que apresenta caráter grave em imunocomprometidos. Os fármacos convencionais para o tratamento desta infecção apresentam alta toxicidade. Peçonhas de escorpião são investigadas uma vez que apresentam efeito direto sobre microrganismos. Objetivo deste trabalho foi avaliar o potencial toxoplasmicida e imunomodulador da peçonha do escorpião Tityus bahiensis (pTb) sobre taquizoítas da cepa RH de T. gondii em células HeLa. Para averiguar índices de infecção e proliferação celular de formas taquizoítas de T. gondii (5x105) em células HeLa (1x105). Foi avaliada, também, a produção de citocinas nas células HeLa infectadas e tratados com pTb (15 µg/mL). Os resultados demonstram que, após 24 horas de tratamento, nenhuma das concentrações apresentou foi citoxicidade. A pTb não alterou os níveis de infecção das células. Na proliferação intracelular a pTb induziu redução de 17,92% na concentração 4 g/mL, 10,18%, utilizando 7,5 g/mL e 16,82% com 15 g/mL. A pTb modulou apenas a produção de IL-8, reduzindo sua produção em 87,32%. Concluímos que a pTb exerce ação imunomoduladora e anti-proliferativa sobre células HeLa infectadas com Toxoplasma gondii (cepa RH).

Detection of Strongyloides stercoralis larvae is particularly challenging because only a small number of larvae are released into the feces, regardless of infection stage. Our objective was to apply conventional polymerase chain reaction (PCR) to the detection of S. stercoralis DNA in feces samples to evaluate its performance in samples of patients with strongyloidiasis and compare results with those of immunodiagnosis. Stool, serum, and saliva samples were collected from each individual (n = 48) at the clinic hospital of the State University of Londrina, Brazil, for parasitological, immunological, and molecular tests. Stool samples were processed via parasitological methods. Serum samples were used for immunoglobulin G (IgG) detection and saliva samples for IgA detection by ELISA. For amplification by conventional PCR, two different primers were used: species specific (101 bp) and genus specific (392 bp). The results showed that 34 (97.1%) of the 35 copro-positive individuals for S. stercoralis were positive for serum IgG and 19 (54.3%) were positive for salivary IgA. Regarding molecular analysis, both primers (species and genus specific) demonstrated positivity in 100% of the samples, which was confirmed by sequencing the positive samples. Complementary examinations of the parasitological method demonstrated excellent results in the context of the diagnosis of strongyloidiasis, especially in asymptomatic patients with irregular larval release in the feces.

Toxoplasma gondii, an etiologic agent of toxoplasmosis, is an obligate intracellular parasite, which exhibits an apicoplast organelle which assists in the metabolism of isoprenoids and other pivotal mediators for the parasite survival. Statins are drugs that inhibit cholesterol synthesis, blocking the conversion of the substrate HMG-CoA to mevalonate, thus preventing the initial processes of the biosynthesis of these precursors, both in humans and parasite. In the light of this information, we determined the effect of pravastatin and simvastatin associated with the current drugs (pyrimethamine and sulfadiazine) as a possible alternative treatment for this infection. Cytotoxicity was evaluated in HeLa cells by MTT assay, which was observed the drug combinations did not affect cell viability. HeLa cells (105) were infected with T. gondii tachyzoites of RH strain (5 × 105) and treated with pravastatin and/or simvastatin combined with pyrimethamine and/or sulfadiazine for 24 h. Our data showed a significant reduction in cell adhesion, infection and mainly parasite proliferation in all treatments. Based on these results, the combination of statins with drugs used in current therapy showed to be a promising therapeutic alternative for toxoplasmosis treatment.

This review considers the advantages and disadvantages of parasitological techniques, methods of detecting antibodies and antigens, as well as molecular biology techniques in the diagnosis of human strongyloidiasis. In addition, it elucidates the potential of different techniques for rapid and effective detection of clinical cases, thus enabling early treatment and preventing fatal consequences of this helminthiasis.

Toxoplasma gondii is a protozoan parasite that can cause severe and debilitating diseases, especially in immunocompromised individuals. The available treatment is based on drugs that have low efficacy, high toxicity, several adverse effects, and need long periods of treatment. Thus, the search for therapeutic alternatives is urgently needed. Biogenic silver nanoparticles (AgNp-Bio) have been associated with several biological effects, as antiproliferative, pro-apoptotic, antioxidant, antiviral, antifungal, and antiprotozoal activity. Thus, the objective was evaluating AgNp-Bio effect on HeLa cells infected with T. gondii (RH strain). First, nontoxic AgNp-Bio concentrations for HeLa cells (1.5 - 6 µM) were determined, which were tested on cells infected with T. gondii. A significant reduction in infection, proliferation, and intracellular parasitic load was observed, also an increase in ROS and IL-6. Additionally, the evaluation of the action mechanisms of the parasite showed that AgNp-Bio acts directly on tachyzoites, inducing depolarization of the mitochondrial membrane, ROS increase, and lipid bodies accumulation, as well as triggering an autophagic process, causing damage to the parasite membrane, and phosphatidylserine exposure. Based on this, it was inferred that AgNp-Bio affects T. gondii by inducing immunomodulation and microbicidal molecules produced by infected cells, and acts on parasites, by inducing autophagy and apoptosis.

O objetivo deste estudo consistiu em analisar o perfil clínico de pacientes positivos para tuberculose através de um estudo epidemiológico, descritivo e retrospectivo, com base em dados secundários contidos em prontuários dos pacientes atendidos no Hospital Universitário de Londrina compreendendo de janeiro de 2010 a dezembro de 2014. Dos prontuários disponíveis para a análise no momento de estudo 86 casos eram positivos para tuberculose, sendo a maioria do sexo masculino (65/76%) com faixa etária compreendida entre 2 e 91 anos. Os principais sinais clínicos apresentados foram tosse (50/58%), febre (45/52%) e perda de peso (34/40%). Em relação à forma clínica, 58/67% dos pacientes apresentaram a forma pulmonar, e 28/33% a forma não pulmonar. Casos positivos para tuberculose em associação com HIV/AIDS corresponderam a 32/37%. Também foram relatados hábitos prejudiciais dos pacientes nos quais 30/34% eram tabagistas, 20/23% usuários de drogas e 14/16% etilistas. Diante da escassez de dados publicados referentes à tuberculose na nossa região e sua relevância para a saúde pública, nosso estudo contribui com os aspectos epidemiológicos principalmente em relação ao elevado número de casos de coinfecção com Vírus da Imunodeficiência Humana e pacientes com evolução a óbito, auxiliando assim, o desenvolvimento e implementação de campanhas ou projetos que visem o diagnóstico e tratamento precoce.

Human strongyloidiasis is caused by helminthStrongyloides stercoralis. It has a worldwide distribution, often neglected and cause of severe morbidity. The parasitological diagnosis is hindered by the low and irregular amount of larvae in feces. The goal of the present study was to detect IgG and IgG immune complex using conventional serum samples and saliva as alternative samples. We collected samples from 60 individuals, namely: group I composed of 30 healthy individuals; and group II composed of 30 individuals eliminatingS. stercoralislarvae in feces. We calculated the area under the curve, general index of diagnostic accuracy, Kappa index and determined the correlations between different diagnostic tests. The detection of IgG levels was performed by an immunoenzymatic assay with alkaline extract ofS. venezuelensislarvae as antigen. Positivity of anti-S. stercoralisIgG in serum samples from group I was 3·3%, and from group II 93·3%. The detection of immune complex indicated that group I exhibited 3·3% and group II 56·7%. In the saliva samples, IgG detection was 26·7% for group I and 43·3% for group II. Immune complex was detected in 20% of group I, and 30% of group II. IgG immune complex in conventional serum samples and saliva as alternative samples can be considered biomarkers for the diagnosis of active strongyloidiasis.

Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this study was to assess the activity of rosuvastatin in HeLa cells infected with the RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significant to promote further studies and the search for alternative treatment for toxoplasmosis.

The highlights of biogenic silver nanoparticles (AgNp-Bio) include low toxicity - depending on size and concentration - and efficient antiparasitic activity. Therefore, the objective of this study was to assess the action of the AgNp-Bio on HeLa cells in an infection with strain of RH Toxoplasma gondii. Firstly, we performed a cellular viability test and characterized the AgNp-Bio to proceed with the infection of HeLa cells with T. gondii to be treated using AgNp-Bio or conventional drugs. Subsequently, we determined the level of standard cytokines Th1/Th2 as well as the content of nitric oxide (NO) and reactive oxygen species (ROS). Results indicated a mean size of 69 nm in diameter for the AgNp-Bio and obtained a dose-dependent toxicity. In addition, the concentrations of 3 and 6 μM promoted a significant decrease in adherence, infection, and intracellular proliferation. We also found lower IL-8 and production of inflammatory mediators. Thus, the nanoparticles reduced the adherence, infection, and proliferation of ROS and NO, in addition to immunomodulating the IL-8. Therefore, our data proved relevant to introduce a promising therapeutic alternative to toxoplasmosis.

Casos de pacientes com coinfecção de tuberculose e estrongiloidíase são pouco estudados e relatados. No entanto, ambos quadros patológicos possuem cenário relevante na epidemiologia da saúde pública apresentando populações vulneráveis coincidentes. Diante do aumento da incidência de tuberculose em regiões tropicais e subtropicais, regiões nas quais existe uma alta prevalência de parasitas gastrointestinais, sobretudo da estrongiloidíase, nosso objetivo foi realizar o diagnóstico coprológico, imunológico e molecular da estrongiloidíase humana. Para isso, aprimoramos o diagnóstico sorológico e salivar através da detecção de imunocomplexos IgG anti ? S. stercoralis. Além disso, através de um teste rápido e sensível, Dot-ELISA, realizamos o diagnóstico salivar para detecção de IgA específico. Utilizando a biologia molecular, foi possível comparar o diagnóstico imunológico, soro e saliva, com o diagnóstico molecular nas fezes, através de dois primers, gênero e espécie específico. A partir desses dados, realizamos um estudo epidemiológico, descritivo e retrospectivo e incluímos os pacientes com tuberculose, no diagnóstico imunológico e molecular. Calculamos através do software GraphPad, área sob a curva, índice geral de acurácia diagnóstica, índice Kappa e as correlações entre os diferentes testes diagnósticos. Para o teste de quimiluminescência os resultados foram interpretados com a ajuda do programa Image Quant. Nossos resultados demonstraram que, a detecção de imunocomplexos IgG no soro foi de 3% (n=30) para o grupo I (indivíduos saudáveis) e 93% (n=30) para o grupo II (copropositivos para S. stercoralis), enquanto que, para a saliva, a detecção foi de 20% grupo I e 30% grupo II. O Dot-ELISA para detecção de IgA na saliva em pacientes positivos para S. stercoralis (n=10), apresentou resultados semelhantes ao teste ELISA, com sensibilidade de 70% e 60% e especificidade de 85% e 90%, respectivamente. A comparação entre o diagnóstico imunológico e molecular demonstrou que 97,1% dos 35 indivíduos copropositivos para S. stercoralis foram positivos para IgG sérica e 19 54,3% foram positivos para IgA salivar. Em relação à análise molecular, ambos os primers demonstraram positividade em 100% das amostras. Dos prontuários disponíveis para a análise dos pacientes, 86 casos eram positivos para tuberculose, sendo a maioria do sexo masculino (76%) com faixa etária entre 2 e 91 anos. Os principais sinais clínicos apresentados foram tosse, febre e perda de peso. Em relação à forma clínica, 67% dos pacientes apresentaram a forma pulmonar, e 33% a forma não pulmonar. Casos positivos para tuberculose em associação com HIV/AIDS corresponderam a 37%. Em relação ao imunodiagnóstico em pacientes portadores de tuberculose, a detecção de IgA foi de 34% para o grupo I (n=50) e 66% para o grupo II (n=50), na saliva. Enquanto que IgG na saliva para o grupo I foi de 46% e IC-IgG 38%. Já no grupo II foi de 52% e 62%, respectivamente. No soro observamos 36% de positividade para IgA no grupo I e 60% grupo II. Em relação a detecção de IgG e IC-IgG no soro, grupo I foi de 46% e 34%, respectivamente. No Grupo II 58% para IgG e 68% para IC-IgG. Quanto ao diagnóstico molecular, a positividade foi de 30,7% (16/52), tanto pela qPCR quanto pela cPCR. Os valores de Cts variaram de 32.6-40.9 (média 38.2). Com isso, o uso do diagnóstico molecular juntamente com o imunodiagnóstico, podem ser considerados como uma ferramenta complementar na triagem da estrongiloidíase, especialmente, considerando a gravidade da coinfecção neste grupo de pacientes. Casos de pacientes em coinfecção com tuberculose e estrongiloidíase são pouco estudados e relatados, no entanto, a realização de exames que complementem o diagnóstico rápido e preciso dessas infecções são essenciais para o bom prognóstico dos pacientes. Cases of patients with co-infection of tuberculosis and strongyloidiasis are poorly studied and reported; however, both pathologies have a relevant scenario in the epidemiology of public health, presenting vulnerable populations. In view of the increased incidence of tuberculosis in tropical and subtropical regions, where there is a high prevalence of gastrointestinal parasites, especially strongyloidiasis, our objective was to carry out the coprological, immunological and molecular diagnosis of human strongyloidiasis. For this, we improved the serological and salivary diagnosis through the detection of immunocomplex IgG anti - S. stercoralis. In addition, through a rapid and sensitive Dot-ELISA test, we performed the salivary diagnosis for specific IgA detection. Using molecular biology, it was possible to compare the immunological diagnosis, serum and saliva, and the molecular diagnosis in the feces, through two primers, genus and specific species. From these data, we conducted an epidemiological, descriptive and retrospective study and included patients with tuberculosis, both in immunological and molecular diagnosis. We calculated using the GraphPad software, area under the curve, general index of diagnostic accuracy, Kappa index and correlations between the different diagnostic tests. For the chemiluminescence test the results were interpreted using the Image Quant program. Our results demonstrated that the detection of serum IgG immunocomplexes was 3% for group I (healthy individuals) (n=30) and 93% for group II (copropositives for S. stercoralis) (n=30), whereas, for saliva, the detection was of 20% group I and 30% group II. The Dot-ELISA for the detection of IgA in saliva in S. stercoralis positive patients (n=10), presented results similar to the ELISA test, with sensitivity of 70% and 60% and specificity of 85% and 90%, respectively. The comparison between immunological and molecular diagnosis showed that 97.1% of the 35 copropositive individuals for S. stercoralis were positive for serum IgG and 19 54.3% were positive for salivary IgA. In relation to the molecular analysis, both primers demonstrated positivity in 100% of the samples. From the charts available for the patients analysis of the patients, 86 cases were positive for tuberculosis, the majority being male (76%) with ages ranging from 2 to 91 years. The main clinical signs presented were cough, fever and weight loss. Regarding the clinical form, 67% of the patients presented the pulmonary form, and 33% the non-pulmonary form. Positive cases for tuberculosis in association with HIV/AIDS accounted for 37%. Regarding immunodiagnosis in patients with tuberculosis, the IgA detection was 34% for group I (n=50) and 66% for group II (n=50), in saliva. While saliva IgG for group I was 46% and 38% for IC-IgG. In group II, it was 52% and 62%, respectively. In serum we observed 36% positivity for IgA in group I and 60% group II. Regarding the detection of IgG and IC-IgG in saliva, group I was 46% for IgG and 34% for IC-IgG. In Group II 58% for IgG and 68% for IC-IgG. Regarding the molecular diagnosis, the positivity was 30.7% (16/52), both by qPCR and by cPCR. Cts values ranged from 32.6-40.9 (mean 38.2). Therefore, the use of molecular diagnostics and iimmunological diagnosis can be considered as a complementary tool in the screening of strongyloidiasis, especially considering the severity of coinfection in this group of patients. Cases of patients in co-infection with tuberculosis and strongyloidiasis are poorly studied and reported; however, examinations that complement the rapid and accurate diagnosis of these infections are essential for the good prognosis of patients.

This study evaluates the inclusion of the IgG avidity index in ELISA to detect anti-Strongyloides stercoralis IgG. The ELISA index revealed 70% of specificity. With the inclusion of screening AI, specificity increased to 80%. IgG avidity complemented traditional IgG ELISA by eliminating some of the suspected or false positive cases.

This study aimed to evaluate the use of saliva samples in the Dot-ELISA test for immunodiagnosis of human strongyloidiasis. The Dot-ELISA presented similar results to the ELISA test, with 70% and 60% sensitivity and 85% and 90% specificity, respectively, for IgA in the saliva. The Dot-ELISA with alternative saliva samples may be a suitable tool for diagnosing human strongyloidiasis, especially in populations with high levels of exposure to helminth.

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×105) were infected with T. gondii tachyzoites (5×105) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×105) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses.

Estrongiloidíase é uma parasitose de prevalência mundial e está entre as seis primeiras dentre as infecções causadas por geohelmintos. Os indivíduos infectados são assintomáticos na maioria dos casos, enquanto que os outros podem apresentar desconfortos gastrointestinais. Porém, em pacientes imunocomprometidos as manifestações promovem quadros graves potencialmente fatais. Nestes casos, o diagnóstico precoce e tratamento adequado são essenciais. O diagnóstico parasitológico da estrongiloidíase apresenta dificuldades uma vez que a liberação de larvas nas fezes é pequena e irregular, além de necessitar de análise por método e equipe especializada. Diante disso, nosso objetivo foi realizar a análise retrospectiva da prevalência de Strongyloides stercoralis e outros parasitos a partir da análise de 11.641 prontuários coproparasitológicos de indivíduos provenientes da cidade de Londrina-PR do período de fevereiro 2009 a dezembro de 2012. Além disso, realizamos um estudo prospectivo para pesquisa de S. stercoralis e outros parasitos em amostras de fezes de indivíduos provenientes da cidade de Londrina-PR no período de abril de 2013 a maio de 2014, utilizando quatro diferentes métodos parasitológicos (Hoffman, Pons e Janer, Faust, Kato-Katz e Rugai). Complementarmente realizamos o imunodiagnóstico através do método de ELISA para pesquisa de anticorpos IgG e IgA em amostras pareadas de soro e saliva, divididas em três grupos (Grupo I: positivos para S. stercoralis; Grupo II: indivíduos aparentemente saudáveis, com parasitológico negativo e Grupo III: parasitológico positivo para outras parasitoses). Para averiguar a afinidade funcional da IgG específica para Strongyloides em amostras de soro, foi realizado também o ensaio de avidez como complemento ao ELISA clássico. O teste Qui-quadrado foi utilizado para realizar a análise estatística dos estudos retrospectivo e prospectivo. Para o diagnóstico imunológico utilizou-se variações estatísticas usando análise de variância, seguida pelo teste de Kruskal-Wallis onde valores p < 0.05 foram considerados significativos. Índice avidez (IA) foi calculado para cada diluição considerando: IA triagem (diluição 1:80) e IA médio. Para diferenciar os grupos nos IAs de triagem e médio foi estabelecido o limiar de 75% (p < 0,001). Do total de 11.641 laudos analisados, 19,1% (n=2.221) indivíduos apresentaram-se positivos para diversos parasitos, dentre os protozoários patogênicos, Giardia lamblia foi mais prevalente com 19,1% (n=425) de positividade, enquanto que, ancilostomídeos foram os mais destacados entre os helmintos, com 7,8% (n=174) casos, com predominância de positividade de 27,1% (n=601) entre crianças de 0 a 10 anos. Para o estudo prospectivo foram analisados 2.315 amostras de fezes, sendo detectados parasitos em 19,3% (n=447) indivíduos, com maior ocorrência de S. stercoralis quando utilizado o método de Rugai. A região sul apresentou maior número de ocorrência de parasitos, com 61,3% (p < 0,003). No imunodiagnóstico observou-se que no G1, foram detectados anticorpos IgG no soro de 96,7% (n=29) amostras e em 6,7% (n=2) amostras de saliva (p < 0,001). Não foi detectado IgG no G2. No G3, observou-se reação cruzada em 26,7% (n=8) amostras para a presença de IgG no soro, e 6,7% (n=2) para IgG na saliva. Em relação à detecção de IgA, no grupo S. stercoralis positivo, foi detectado IgA em 76,7% (n=23) amostras de soro e em 56,7% (n=17) amostras de saliva. No grupo controle negativo apenas 3,3% (n=1) se mostrou positivo para IgA no soro. O grupo outras parasitoses apresentou positividade em 6,7% (n=2) amostras de soro (P 75% (exclusão de infecção ativa) foram, predominantemente, detectadas em G3. Os parâmetros diagnóstico de sensibilidade e especificidade revelam 100% de sensibilidade para o G1 em IE, e um aumento na especificidade quando associado IE a IA, tanto para IA triagem (80%) quanto para IA médio (85%). Esses dados demonstram a importância tanto do diagnóstico parasitológico da estrongiloidíase por se tratar de um indicador de prevalência do parasito, e do diagnóstico imunológico utilizando amostras de soro e saliva que demonstrou ser importante ferramenta diagnóstica na estrongiloidíase. Além disso, IgG avidez em amostras de soro pode ser utilizado para distinguir entre infecção ativa por S. stercoralis de casos suspeitos ou falsos positivos casos. Strongyloidiasis is a parasitic disease of worldwide prevalence and is among the six first infections caused by geohelminth. Infected individuals are asymptomatic in 50% of the cases, while others may have intestinal discomfort. However, in immunocompromised patients the extra intestinal manifestations promote severe clinical conditions potentially fatal. In such cases, early diagnosis and appropriate treatment are essential. The parasitological diagnosis of strongyloidiasis presents complications and difficulties since the release of larvae in the feces is small and irregular besides requiring analysis method and specialized staff. Therefore, our objective was to perform the retrospective analysis of the prevalence of Strongyloides stercoralis and other parasites from the analysis of 11,641 fecal records of individuals from the periphery of the city of Londrina-PR from February 2009 to December 2012. Furthermore, we conducted a prospective study for researching S. stercoralis and other parasites in feces samples of individuals from the periphery of the city of Londrina from April 2013 to May 2014, using four different parasitological methods (Hoffman, Pons and Janer, Faust, Kato-Katz and Rugai). Complementary, we conducted the immunodiagnostic through the ELISA method for research of IgG and IgA antibodies in paired samples of serum and saliva, divided into three groups (Group I: positive for S. stercoralis; Group II: apparently healthy individuals with negative parasitological and Group III: positive parasitological for other parasitic diseases). To determine the functional affinity of specific IgG for Strongyloides in serum samples, the avidity assay was performed as a complement to classical ELISA. The chi-square test was used for statistical analysis of retrospective and prospective studies. For the immunological diagnosis, we used statistical variations using analysis of variance followed by Kruskal-Wallis test in which the P values < 0.05 were considered significant. Avidity index (AI) was calculated to each serum considering: screening AI (serum 1:80) and mean AI at different dilutions. In order to differentiate groups at screening and mean AI a value of 75% was stablished (p < 0.001). From the total of 11,641 reports analyzed, 19.1% (n = 2,221) individuals were positive for multiple parasites, among pathogenic protozoan, Giardia lamblia was the most reported with 19.1% (n = 425) of positivity, while, ancylostomids were the most prominent among the helminths, with 7.8% (n = 174) patients, with a predominance of positivity of 27.1% (n = 601) among 0-10 years children. In the prospective study were analyzed 2,315 samples of feces, parasites were detected in 19.3% (n = 447) of the individuals, in which most of S. stercoralis detection was observed when using the Rugai method. The southern region of the city of Londrina had a higher number of occurrences, with 61.3% (P 75% (exclusion of active infection) were, predominantly, detected in G3. The diagnostic parameters of sensitivity and specificity showed 100% sensitivity for the G1 in IE, and an increased specificity when IE and IA were associated, both for screening IA (80%) and for mean IA (85%). In group 2 IA antibody levels ranged from 17% to 33% (mean 33%) at dilutions 1:80, 1:160, 1:320 and 1:640. In G3 ranged from 42% to 86% (mean 58%) in the same dilutions. These data demonstrate not only the importance of parasitological diagnosis of strongyloidiasis like an indicator of parasite prevalence, but also as an immunological diagnosis using samples of serum and saliva which showed like a important diagnostic tool in strongyloidiasis. Furthermore, avidity IgG in serum samples can be used to distinguish active infection by S. stercoralis cases to suspected or false positives.

Human strongyloidiasis is an infection caused by the helminth Strongyloides stercoralis that can be fatal, especially in immunosuppressed patients. The aim of this study is to evaluate parasite-specific IgG and IgA levels using S. venezuelensis third-stage (L3) infective larvae alkaline extract as a heterologous antigen by ELISA in paired serum and saliva samples with improved sensitivity and specificity. Individuals from northern Parana state, Brazil were divided into three groups: 30 patients copropositive for S. stercoralis (Group I); 30 clinically healthy individuals (Group II); and 30 patients copropositive for other parasites (Group III). The area under ROC curve (AUC), an overall index of diagnostic accuracy, and Kappa index were calculated. Data were analyzed using analysis of variance (ANOVA) followed by a Kruskal-Wallis test. Probability (p) values of

A toxoplasmose é uma doença causada pelo protozoário Toxoplasma gondii. Possui importância na saúde pública principalmente por promover, muitas vezes, quadros graves na infecção congênita. O tratamento atual limita-se ao uso de espiramicina ou associação de sulfadiazina, pirimetamina e ácido folínico. Os efeitos tóxicos causados pelos fármacos, levam à necessidade de novas estratégias terapêuticas. Assim, o óleo essencial de orégano (Origanum vulgare L.) (OEO) apresenta-se como uma alternativa promissora devido ao seu potencial antibacteriano, antifúngico e antiparasitário. O objetivo deste trabalho foi analisar a capacidade antiproliferativa e mecanismos de ação diretos do OEO em taquizoítos de T. gondii (cepa RH). Para investigar a toxicidade do OEO em células BeWo foi realizado o ensaio de MTT utilizando concentrações de 0,7 a 150 µg/mL. Para avaliar a viabilidade de taquizoítos após tratamento com OEO utilizou-se método de exclusão por azul de tripan. Em seguida, foram calculados a concentração citotóxica (CC50), inibitória (CI50) e índice de seletividade (IS). A atividade antiproliferativa do tratamento com OEO foi determinada pelos índices de infecção e proliferação intracelular do parasito tratados com OEO utilizando concentrações 6,2; 12; 25 e 50 µg/mL e confirmados pela microscopia de luz por meio da coloração de azul de toluidina. Os mecanismos envolvidos na morte direta do parasito também foram avaliados, sendo eles, níveis de espécies reativas de oxigênio (ERO), potencial de membrana mitocondrial e gotículas lipídicas. A apoptose e a permeabilização da membrana plasmática foram investigados por citometria de fluxo através da marcação com Anexina-V e iodeto de propídio, além de autofagia por monodansilcadaverina. Para avaliar topologia (SEM) e ultraestrutura (TEM) foram realizadas análises de microscopia eletrônica de varredura e transmissão. O tratamento com OEO apresentou baixa citotoxicidade nas células BeWo (CC50 de 114,8 µg/mL ± 0,01), redução da viabilidade de taquizoítos (CI50 de 12,5 ± 0,06 µg/mL), e IS de 9,18. Houve redução da proliferação intracelular de 55% e 84% nas concentrações de 25 e 50 ?g/mL, respectivamente. Em taquizoítos livres, o tratamento causou aumento dos níveis de EROs, despolarização da membrana mitocondrial e formação de gotículas lipídicas, bem como tumefação da mitocôndria observadas em TEM. Estas alterações estão atreladas à indução do processo de autofagia e permeabilização de membrana plasmática, com extravasamento do conteúdo citoplasmático e alterações nucleares, confirmadas através de TEM e SEM, culminando em um processo de morte celular. O tratamento com OEO demonstrou atividade anti-T. gondii direcionando a morte do parasito por alterações metabólicas sem causar toxicidade às células, podendo ser um composto promissor no tratamento da toxoplasmose. Toxoplasmosis is a disease caused by the protozoan Toxoplasma gondii. It has in public health mainly for promoting, sometimes, serious conditions in the public majority. Current treatment is limited to the use of spiramycin or the association of sulfadiazine, pyrimethamine and folinic acid. The effects of medicines lead to the need for new therapies. Thus, the essential oil of oregano (Origanum vulgare L.) (OEO) presents itself as a promising alternative due to its antibacterial, antifungal and antiparasitic potential. The objective of this work was to analyze the antiproliferative capacity and the mechanisms of the direct action of OEO in tachyzoites of T. gondii (RH strain). To investigate the toxicity of OEO in BeWo cells, the MTT assay was performed using concentrations from 0.7 to 150 µg/mL. To evaluate the viability of tachyzoites after treatment with OEO, the trypan blue exclusion method was used. Then, the cytotoxic (CC50), inhibitory (IC50) and selectivity index (SI) concentrations were calculated. The antiproliferative activity of the OEO treatment was determined by the rates of infection and intracellular proliferation of the parasite treated with OEO using concentrations 6.2; 12; 25 and 50 µg/mL and confirmed by light microscopy using toluidine blue staining. The mechanisms involved in the direct death of the parasite were also evaluated, namely, levels of reactive oxygen species (ROS), mitochondrial membrane potential and lipid droplets. Apoptosis and plasma membrane permeabilization were investigated by flow cytometry by labeling with Annexin-V and propidium iodide, in addition to autophagy by monodansylcadaverine. To evaluate topology (SEM) and ultrastructure (TEM) scanning and transmission electron microscopy analyzes were performed. OEO treatment showed low cytotoxicity on BeWo cells (CC50 of 114.8 µg/mL ± 0.01), reduced tachyzoite viability (IC50 of 12.5 ± 0.06 µg/mL), and IS of 9. 18. There was a reduction in intracellular proliferation of 55% and 84% at concentrations of 25 and 50 ?g/mL, respectively. In free tachyzoites, treatment caused increased levels of ROS, mitochondrial membrane depolarization and lipid droplet formation, as well as mitochondrial swelling seen in TEM. These alterations are linked to the induction of the process of autophagy and permeabilization of the plasma membrane, with extravasation of the cytoplasmic content and nuclear alterations, confirmed by TEM and SEM, culminating in a process of cell death. OEO treatment demonstrated anti-Toxoplasma directing the death of the parasite by metabolic changes without causing toxicity to the cells, which may be a promising compound in the treatment of toxoplasmosis.