In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools.

Background: Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods: The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results: The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion: Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.

Unbalanced relationships: insights into the interaction between gut microbiota, geohelminths, and schistosomiasis.

Hosts and their microbiota and parasites have co-evolved in an adaptative relationship since ancient times. The interaction between parasites and intestinal bacteria in terms of the hosts' health is currently a subject of great research interest. Therapeutic interventions can include manipulations of the structure of the intestinal microbiota, which have immunological interactions important for modulating the host's immune system and for reducing inflammation. Most helminths are intestinal parasites; the intestinal environment provides complex interactions with other microorganisms in which internal and external factors can influence the composition of the intestinal microbiota. Moreover, helminths and intestinal microorganisms can modulate the host's immune system either beneficially or harmfully. The immune response can be reduced due to co-infection, and bacteria from the intestinal microbiota can translocate to other organs. In this way, the treatment can be compromised, which, together with drug resistance by the parasites makes healing even more difficult. Thus, this work aimed to understand interactions between the microbiota and parasitic diseases caused by the most important geohelminths and schistosomiasis and the consequences of these associations.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

Doxycycline hyclate: A schistosomicidal agent in vitro with immunomodulatory potential on granulomatous inflammation in vivo.

We investigated the effect in vitro and in vivo of doxycycline hyclate (Dx), a broad-spectrum antibiotic inhibitor of matrix metaloproteinases (MMPs), on adult Schistosoma mansoni worms and granulomatous liver inflammation in infected mice. Adult S. mansoni worms in culture treated with different concentrations of Dx (50-180 µg/mL) were studied for eight days to assess its morphology, eggs production, and mortality. Uninfected mice and those infected with S. mansoni, untreated and treated with praziquantel (Pz; 200 mg/kg) or Dx (50 mg/kg), were evaluated for 60 days. Our results indicated that Dx induced dose-dependent integumentary lesions (bubbles, tubercle collapse, spicule disappearance, peeling, and erosion), reduced mating rate and eggs-laying in adult S. mansoni worms. The effective lethal dose required to kill 50% of worms was 112.0 µg/mL Dx (DL50). In mice, S. mansoni infection induced hepatomegaly, intense IL-4, IL-10, TNF-α and TGF-ß production, granulomatous inflammation and hepatic glycogen depletion. The number and size of the granulomas was similar in untreated and Dx-treated animals. Untreated animals showed a predominance of productive granulomas, and intense MMP-2 and MMP-9 activities. Dx-treated mice exhibited a significant increase in IL-4 levels, tissue inflammation, proportion of involutive granulomas, and hepatic collagenogenesis, as well as attenuated MMP-2 and MMP-9 activities. Our findings indicated that Dx is toxic to adult S. mansoni worms in vitro. However, in vitro beneficial effects were not reproduced in vivo, since Dx treatment increased liver granulomatous inflammation and collagenogenesis in S. mansoni-infected mice by a process potentially associated with Dx-mediated hepatic MMP-2 and MMP-9 inhibition.

Seroepidemiological aspects of human infection by Strongyloides stercoralis in Alfenas, southern Minas Gerais, Brazil.

INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS: The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

Seroepidemiological aspects of human infection by Strongyloides stercoralis in Alfenas, southern Minas Gerais, Brazil

Abstract INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

Could diet composition modulate pathological outcomes in schistosomiasis mansoni? A systematic review of in vivo preclinical evidence.

Schistosomiasis and malnutrition are often overlapped in poor communities, resulting in disproportionately high mortality rates. Currently, fragmented data make it difficult to define the relationship between diet and schistosomiasis. Thus, we systematically review the preclinical evidence on the impact of diet in Schistosoma mansoni infection. From a structured search, we recovered 27 original articles. All studies used mice and most of them investigated hypoproteic (70.37%), hyperlipidic (22.22%) or vitamin-deficient (7.41%) diets. Diets based on carbohydrate, zinc or milk supplementation were investigated at a reduced frequency (3.70% each). Hypoproteic diets attenuated parasitic load and granulomatous inflammation, but also reduced host resistance to S. mansoni infection, determining higher mortality rates. By stimulating steatohepatitis, parasitic load and granulomatous inflammation, hyperlipidic diets increase organ damage and mortality in infected animals. Although a high-sugar diet and vitamin restriction potentiate and zinc supplementation attenuates S. mansoni infection, the current evidence for these diets remains inconclusive. Analysis of methodological quality indicated that the current evidence is at high risk of bias due to incomplete characterization of the experimental design, diet composition and treatment protocols. From the bias analysis, we report methodological limitations that should be considered to avoid systematic reproduction of inconsistent and poorly reproducible experimental designs.

Photodynamic therapy for Schistosoma mansoni: Promising outcomes.

The purpose of this study was to assess, for the very first time, the effects of photodynamic therapy (PDT) on Schistosoma mansoni in vitro by measuring reactive oxygen species (ROS) generation throughout the treatment, as well as the behavior of the parasites (mating, motility and contraction/shortening), and damage to their tegument and excretory systems. The parasites were divided into 4 groups: control, photosensitizer, laser and PDT. Light irradiation was delivered with an InGaAlP low-level laser device operating at 660nm, with 40mW and 100J/cm2. For PDT, different toluidine blue dye (TBO) concentrations and times of exposure were utilized. Interestingly, TBO-mediated PDT was able to kill S. mansoni (P<0.001) due to the significant amount of ROS released that inflicted damages in the tegument and excretory system, as well as contraction and cessation of motility. In addition, males of S. mansoni were shown to be more sensitive to PDT if compared to their corresponding females when the optimal TBO concentration of 31.2µL was considered (P=0.0126). PDT presents two major advantages: not inducing microbial resistance and also lacking adverse effects. Therefore, PDT may become a promising therapeutic alternative for schistosomiasis in the near future, especially for cases of allergy and resistance to praziquantel.

Prevalence and risk factors for intestinal parasitic infections in children attending daycare centers in Alfenas, southern Minas Gerais, Brazil

Intestinal parasitic infections are common among pre-school children in developing countries and they are often associated with gastrointestinal morbidity such as chronic diarrhea and malnutrition. Their circulation is mainly associated with lack of personal hygiene and environmental sanitation, as well as limited housing and food conditions. As the diagnosis of intestinal parasites is not a simple procedure, especially in population studies, due to difficulties encountered in strategies to obtain fecal samples, reliable prevalence data are scarce. Indeed, the epidemiological data on the prevalence of these parasites in different locations are important for the development of appropriate control measures. This study aimed to investigate the prevalence and risk factors associated with intestinal parasitic infection in children attending three public municipal daycare centers in Alfenas, MG. Three fecal samples from each child were collected on alternate days and processed by the spontaneous sedimentation technique and also through the commercially available centrifugal concentration technique, known as the TF-Test® (TFT). Information on the biological, social and physical environment, in which the children lived, were obtained through the application of a socio-epidemiological questionnaire to the parents or guardians and daycare staff. Giardia duodenalis was the parasite species most frequently detected among the children, with a positive rate of 27.8% (77/277). Entamoeba coli was detected in one of the daycare centers studied, with positivity rate of 43.7%, (7/16); and helminth infection in only two children. The present study showed that children of municipal daycare centers in Alfenas could be at risk of infection by intestinal parasites.

S. mansoni-T. cruzi co-infection modulates arginase-1/iNOS expression, liver and heart disease in mice.

Although Schistosoma species and Trypanosoma cruzi share common endemic areas, co-infections by these parasites remains overlooked. By using a murine model of S. mansoni and T. cruzi co-infection, we investigated if and to what extent these infections might interact to change the pathological outcomes typically observed when the host is infected by a single parasite species. Swiss mice were randomized into four groups: uninfected (NI) and those infected by S. mansoni (SM), T. cruzi (TC) or co-infected (SM + TC). After 120 days of S. mansoni infection, T. cruzi was concurrently inoculated and the infection occurred for 30 days. Taken together, we identified that the overlap of Th2 (schistosomiasis) and Th1 (Chagas disease) immunological patterns changes the host resistance against both pathogens. Beyond impairing the control of granulomatous inflammation, T. cruzi parasitemia and parasitism in co-infected animals, the Th2 inflammatory response against S. mansoni elicits the activation of the arginase-1 pathway to the detriment of inducible oxide nitric synthase (iNOS) expression and nitric oxide (NO) production, contributing to the liver damage, with minor effects on heart pathology.

Aspectos epidemiológicos da esquistossomose em área do sudoeste de Minas Gerais, Brasil / Epidemiological aspects of schistosomiasis in area at the southwest of Minas Gerais, Brazil

This study was developed aiming at contributing to the schistosomiasis surveillance, within the scope of the Regional Health Superintendence of Alfenas, MG, in the South/Southwest mesoregion of the state, considered not endemic for schistosomiasis, unlike North and Northeast areas of the state. During the year of 2015, schoolchildren and migrants from two municipalities of this region, Arceburgo and Guaranésia, underwent parasitological and serological surveys. In the parasitological survey, no case of schistosomiasis was detected in Arceburgo. In Guaranésia, S. mansoni eggs were detected among the migrants, with a positivity rate of 13.6% (9/66), and in only one schoolchild. Seven members of his family, who were classified as residents of Guaranésia, but were determined as coming from Timbaúba, PE, when investigated by the epidemiological surveillance, they were also positive for S. mansoni. In the serological survey, the positivity for schistosomiasis was 18.5% among migrants from Guaranésia. Concerning the other intestinal parasites, the positivity rates ranged from 12.5% to 32.3%. The results suggest differences in the risk of exposure to S. mansoni and the importance of epidemiological surveillance, even in non-endemic areas, with a focus on migrants when they come from endemic regions for schistosomiasis.

Este estudo foi desenvolvido com o objetivo de contribuir com a vigilância da esquistossomose, no âmbito da Superintendência Regional de Saúde de Alfenas, MG, na mesorregião Sul/Sudoeste do estado, considerada não endêmica para a esquistossomose, ao contrário de outras áreas ao norte e nordeste do estado. Durante o ano de 2015, os escolares e migrantes de dois municípios dessa região, Arceburgo e Guaranésia, foram submetidos aos inquéritos parasitológico e sorológico. No inquérito parasitológico, nenhum caso de esquistossomose foi detectado em Arceburgo. Em Guaranésia, ovos de S. mansoni foram detectados entre os migrantes, com taxa e positividade de 13,6% (9/66), e em um único estudante. Sete membros da família, classificada como moradora de Guaranésia, mas determinada como oriunda de Timbaúba, PE quando investigada pela vigilância epidemiológica, foram também positivos para S. mansoni. No inquérito sorológico, a positividade para esquistossomose foi de 18,5% entre os migrantes de Guaranésia. Em relação às demais parasitoses, as taxas de positividade variaram de 12,5% a 32,3%. Os resultados sugerem diferenças em relação ao risco de exposição a S. mansonie a importância da vigilância epidemiológica, mesmo em áreas não endêmicas, com foco nos migrantes, quando estes são oriundos de regiões endêmicas para esquistossomose.

Aspectos epidemiológicos da esquistossomose em área dosudoeste de Minas Gerais, Brasil / Epidemiological aspects of schistosomiasis in area at the southwest of Minas Gerais, Brazil

Este estudo foi desenvolvido com o objetivo de contribuir com a vigilância da esquistossomose,no âmbito da Superintendência Regional de Saúde de Alfenas, MG, na mesorregião Sul/Sudoestedo estado, considerada não endêmica para a esquistossomose, ao contrário de outras áreas ao norte e nordeste do estado. Durante o ano de 2015, os escolares e migrantes de dois municípios dessa região, Arceburgo e Guaranésia, foram submetidos aos inquéritos parasitológico esorológico. No inquérito parasitológico, nenhum caso de esquistossomose foi detectado emArceburgo. Em Guaranésia, ovos de S. mansoni foram detectados entre os migrantes, com taxa de positividade de 13,6% (9/66), e em um único estudante. Sete membros da família, classificadacomo moradora de Guaranésia, mas determinada como oriunda de Timbaúba, PE quando investigada pela vigilância epidemiológica, foram também positivos para S. mansoni. No inquéritosorológico, a positividade para esquistossomose foi de 18,5% entre os migrantes de Guaranésia.Em relação às demais parasitoses, as taxas de positividade variaram de 12,5% a 32,3%. Os resultados sugerem diferenças em relação ao risco de exposição a S. mansoni e a importância da vigilância epidemiológica, mesmo em áreas não endêmicas, com foco nos migrantes, quando estes são oriundos de regiões endêmicas para esquistossomose.

This study was developed aiming at contributing to the schistosomiasis surveillance, within the scope of the Regional Health Superintendence of Alfenas, MG, in the South/Southwestmesoregion of the state, considered not endemic for schistosomiasis, unlike North and Northeast areas of the state. During the year of 2015, schoolchildren and migrants from two municipalitiesof this region, Arceburgo and Guaranésia, underwent parasitological and serological surveys.In the parasitological survey, no case of schistosomiasis was detected in Arceburgo. In Guaranésia, S. mansoni eggs were detected among the migrants, with a positivity rate of 13.6% (9/66),and in only one schoolchild. Seven members of his family, who were classified as residents of Guaranésia, but were determined as coming from Timbaúba, PE, when investigated by theepidemiological surveillance, they were also positive for S. mansoni. In the serological survey, the positivity for schistosomiasis was 18.5% among migrants from Guaranésia. Concerningthe other intestinal parasites, the positivity rates ranged from 12.5% to 32.3%. The results suggest differences in the risk of exposure to S. mansoni and the importance of epidemiologicalsurveillance, even in non-endemic areas, with a focus on migrants when they come from endemic regions for schistosomiasis.

Curcumin Enhances the Anti-Trypanosoma cruzi Activity of Benznidazole-Based Chemotherapy in Acute Experimental Chagas Disease.

Although curcumin can increase the effectiveness of drugs against malaria, combination therapies using the molecule have never been investigated in Chagas disease (ChD). Therefore, we evaluated the efficacy of curcumin as a complementary strategy to benznidazole (Bz)-based chemotherapy in mice acutely infected with Trypanosoma cruzi Eighty-four 12-week-old Swiss mice were equally randomized into seven groups: uninfected (NI), T. cruzi infected and untreated (INF), infected and treated with 100 mg/kg of body weight Bz (B100), 50 mg/kg Bz (B50), 100 mg/kg curcumin (C100), 100 mg/kg Bz plus 100 mg/kg curcumin (B100 plus C100), and 50 mg/kg Bz plus 100 mg/kg curcumin (B50 plus C100). After microscopic identification of blood trypomastigotes (4 days after inoculation), both drugs were administered by gavage once a day for 20 days. Curcumin showed limited antiparasitic, anti-inflammatory, and antioxidant effects when administered alone. When curcumin and Bz were combined, there was a drastic reduction in parasitemia, parasite load, mortality, anti-T. cruzi IgG reactivity, circulating levels of cytokines (gamma interferon [IFN-γ], interleukin 4 [IL-4], and MIP1-α), myocardial inflammation, and morphological and oxidative cardiac injury; these results exceeded the isolated effects of Bz. The combination of Bz and curcumin was also effective at mitigating liver toxicity triggered by Bz, increasing the parasitological cure rate, and preventing infection recrudescence in noncured animals, even when the animals were treated with 50% of the recommended therapeutic dose of Bz. By limiting the toxic effects of Bz and enhancing its antiparasitic efficiency, the combination of the drug with curcumin may be a relevant therapeutic strategy that is possibly better tolerated in ChD treatment than Bz-based monotherapy.

Potent Schistosomicidal Constituents from Garcinia brasiliensis.

Praziquantel is the drug of choice for the treatment of schistosomiasis. However, several strains of Schistosoma mansoni are resistant to praziquantel, making it necessary to discover new drugs that might be used for its treatment. With this in mind, the properties of a schistosomicidal ethanolic extract of Garcinia brasiliensis Mart. epicarp, the fractions obtained by partitioning this extract, including the hexane fractions, ethyl acetate fraction, and the aqueous fraction, and the isolated compounds 7-epiclusianone, a major component from these fractions, and fukugetin were tested in vitro on adult worms of S. mansoni. Mortality, damage to membranes, and excretory system activity were observed at 100.0, 50.0, 75.0, and 14.0 µg/mL for the ethanolic extract of G. brasiliensis Mart. epicarp, its hexane fractions, the ethyl acetate fraction, and 7-epiclusianone, respectively. For 7-epiclusianone, these data were confirmed by fluorescent probe Hoechst 33 258 and resorufin. Additionally, the biocidal effect of 7-epiclusianone was even higher than the hexane fractions. Moreover, an inhibitory effect of 7-epiclusianone on the egg laying of female adult S. mansoni worms was observed in cercariae and schistossomula. Thus, 7-epiclusianone is a promising schistosomicidal compound; however, more studies are needed to elucidate its mechanism of toxicity and to evaluate the in vivo activity of this compound.