Antigiardiasic activity of Cu(II) coordination compounds: Redox imbalance and membrane damage after a short exposure time.

Giardiasis is a widespread illness that affects inhabitants of underdeveloped countries, being children and seniors the highest risk population. The several adverse effects produced by current therapies besides its increasing ineffectiveness due to the appearance of resistant strains evidence the urgent need for new therapeutic approaches. We present the antigiardiasic effect of eight Cu(II) coordination compounds, which belong to the family Casiopeínas. Two of them, 4,7-diphenyl-1,10-phenanthroline(acetylacetonato)copper(II) nitrate (CasIII-Ha,36 µM) and 4,7-diphenyl-1,10-phenanthroline(glycinato)copper(II) nitrate (CasI-gly,36 µM) have shown the best antiproliferative effect in Giardia intestinalis trophozoite cultures, both with the higher lipophilic character of the series. The antiproliferative effect of these coordination compounds is attributable to its capacity to interact with the cellular membrane and to increase reactive oxygen species (ROS) concentration within the parasite since the first hours of exposure, (2-6 h). We found that these compounds mainly induced the cell death of trophozoites by apoptosis, contrary to metronidazole, which induces apoptosis and necrosis in the same ratio. The cytotoxic effects on lymphocytes and macrophages isolated from human peripheral blood allowed us to establish a selectivity index and in turn, identify and propose the best candidates to continue with the assays in animal models. The selected molecules do not include the most active compounds against trophozoites, instead of that, we propose the compounds 4',4'-dimethyl-2,2'-bipyridine(acetylacetonato)copper(II) nitrate (CasIII-ia,IC50 = 156 µM) and 4,7-dimethyl-1,10-phenanthroline(acetylacetonato) copper(II) nitrate (CasIII-Ea,IC50 = 125 µM), which possess an antiproliferative efficacy comparable with Metronidazole but also are those that produce the lowest effect on the viability of human lymphocytes and macrophages.

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

CONTEXT: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. OBJECTIVE: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. MATERIALS AND METHODS: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 µg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. RESULTS: Trinervinol and piquerol showed antigiardial activity with an IC50 = 2.03 and 2.42 µg/mL, and IC90 = 13.03 and 8.74 µg/mL, respectively. The concentrations of trinervinol (CC50 = 590 µg/mL) and piquerol (CC50 = 501 µg/mL) were not cytotoxic to human fibroblasts. CONCLUSIONS: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

Giardia duodenalis genotypes among schoolchildren and their families and pets in urban and rural areas of Sinaloa, Mexico.

INTRODUCTION: Giardiasis is a human health concern worldwide, especially among schoolchildren. Giardia duodenalis genotypes A and B are infective to humans, but their zoonotic potential remains controversial. In Mexico, the most prevalent genotype is A, but B was also detected in southeastern Mexico. In Sinaloa state, northwestern Mexico, giardiasis is highly prevalent, but Giardia genotypes have been poorly studied. METHODOLOGY: This study aimed to investigate the distribution and clinical-epidemiological correlation of G. duodenalis genotypes in schoolchildren and their families and pets in urban and rural areas of Sinaloa state, Mexico. RESULTS: Among 395 schoolchildren (274 urban, 121 rural), 76 (49 urban, 27 rural) were infected with G. duodenalis. In total, 22 families (15 urban, 7 rural) of infected schoolchildren, consisting of 60 family members (41 urban, 19 rural) and 21 pet dogs (15 urban, 6 rural) were examined; 10 family members (5 urban, 5 rural) and 5 pet dogs (3 urban, 2 rural) of 10 families (6 urban, 4 rural) were infected. After PCR-RFLP analyses of vsp417 and gdh genes, genotype prevalence among infected urban schoolchildren was 79.5% AI, 12.8% AII, and 7.7% mixed AI+B. However, only AI genotype was found in family members and pets. In the rural area, only the AI genotype was detected. Genotypes were not correlated with clinical manifestations. CONCLUSIONS: This paper shows the presence of B genotype in northwestern Mexico for the first time. Detection of AI genotype in dogs suggested the possible role of dogs as the reservoir for human giardiasis in Sinaloa, Mexico.

In vitro activity of the F-6 fraction of oregano against Giardia intestinalis.

Giardiosis is a neglected parasitic disease that produces diarrhoea and different degrees of malabsorption in humans and animals. Its treatment is based on derivatives of 5-nitroimidazoles, benzimidazoles, nitrofuranes, acridine and nitrotiazoles. These drugs produce undesirable secondary effects, ranging from a metallic taste in the mouth to genetic damage and the selection of resistant strains; therefore, it is necessary to develop new therapeutic alternatives. We demonstrated that a 2-h treatment with 2·87 µg ml(-1) of fraction 6 of Lippia graveolens (F-6) was sufficient to kill half of an experimental Giardia intestinalis (Syn. G. duodenalis, G. lamblia) population, based on the reduction of MTT-tetrazolium salt levels. F-6 breaks the nuclear envelope and injures the ventral suckling disc. The major compounds of F-6 were characterized as naringenin, thymol, pinocembrin and traces of compounds not yet identified. The results suggest that Lippia is a potential source to obtain compounds with anti-Giardia activity. This knowledge is an important starting point to develop new anti-giardial drugs. Future studies will be required to establish the efficacy of F-6 in vivo using an animal model.

A simplified method for hatching and isolating Toxocara canis larvae to facilitate excretory-secretory antigen collection in vitro.

Human toxocariasis causes several dangerous syndromes that can involve the viscera, vision and central nervous system. Diagnosing toxocariasis requires the identification of antibodies against Toxocara canis or Toxocara cati excretions and secretions (ES). To obtain ES it is necessary to collect a large number of larvae. However, since the earliest work describing the culture of Toxocara larvae, few advances in the method have been made. It has been suggested that carbon dioxide triggers molecular mechanisms that enable nematode hatching. A similar hypothesis has been made regarding Giardia excystation. To test the hypothesis we used the Giardia excystation HBSS method to hatch embryonated T. canis eggs. We found that the HBSS method was more effective than the original De Savigny method. Our results suggest that both parasites require stimulation in an acidic environment, and the abrupt change to a basic milieu in duodenum. This physiological adaptation is successful to exploit the intestinal habitat.

Goblet cells: are they an unspecific barrier against Giardia intestinalis or a gate?

Giardiosis is one of the major intestinal parasitic diseases of human beings as well as wild and domesticated animals. Several protective mechanisms against infection have been described. However, specific information about relationship between giardiosis and the increased proliferation of goblet cells (GC) in patients infected with Giardia intestinalis (Syn. G. duodenalis, G. lamblia) is scarce. In this work, we compare and quantify the number of GC, and have inferred their metabolic state in the small intestine of dogs parasitized with Giardia intestinalis compared to dogs without parasites. Small intestine segments were processed using routine methods for histology and electron microscopy; areas and cells were screened with an Axiovision Ver. 4.0 system. Data were analyzed by ANOVA and comparison of averages. Parasitized dogs showed higher GC numbers than nonparasitized ones. Averages were: 20+/-0.81 GC/25 microm(2) with independent mucin granules and 11+/-1.53 GC/25 microm(2) that were expelling mucus, compared to 11+/-0.94 GC/25 microm(2) and 1+/-0.27 GC/25 microm(2), respectively, in nonparasitized dogs (Tukey, p<0.001). The increases in GC number seem to be an unspecific defensive mechanism against Giardia trophozoites. However, we found some evidence supporting that GC hyperplasia could be a prejudicial to epithelial barrier that gives rise to gates allowing for Giardia-tissue invasion.

A simple and inexpensive in vitro method for retrieving fertilized Toxocara canis eggs.

The erratic migration of nematode larvae is potentially deleterious for humans, especially those produced by Toxocara roundworms. Diagnosis of human toxocariosis: visceral larva migrans depends on antibody detection against excretions-secretions (E/S) from Toxocara larvae by enzyme-linked immunosorbent assay. In the present work, we wish to propose a simple and cheap culture procedure for keeping T. canis female worms alive. We removed living worms from the small intestine of euthanized puppies at the Canine Control Centre "Culhuacan" in the Southern part of Mexico City. To ascertain the utility of the method, we compared the population size of fertilized eggs from both female worms kept in culture (FCM) and those obtained using uterus excision method (UEM). Operationally, a fertilized egg develops a larva after 1 month. Our results show that the culture method was superior at a ratio of 3:1 over UEM. FCM had advantages over UEM, such as (1) low probability of contamination with foreign antigens, (2) greater safety for infective egg manipulation, and (3) gave rise to a higher percentage of fertile eggs.

Giardia intestinalis and other zoonotic parasites: prevalence in adult dogs from the southern part of Mexico City.

The protozoan Giardia intestinalis is a mammalian-infecting parasite. It produces diarrhoea and malabsorption in its hosts. There is growing evidence that dogs could be reservoirs and play an important role in transmission. In Mexico, there are few data on the frequency of G. intestinalis. Therefore, we studied the small intestine of stray dogs, euthanazed at the "Culhuacan" Control Canine Centre, towards the end of 1997 and during the summer of 1998. We microscopically analysed intestinal contents and mucus samples taken every 3cm. During the cold season (winter), parasites were not found in 38/100 dogs, in contrast to 8/100 through the warm season. We found that 42/100 in winter and 51/100 in summer harboured G. intestinalis. To our knowledge, these G. intestinalis frequencies are the highest found in adult dogs worldwide. The results showed a rise in Ancylostoma spp. from 23/100 to 67/100 during the cold and warm seasons. Toxocara canis frequencies varied between 12/100 and 18/100, respectively. The data suggest that the probability of infection is higher during the hottest months compared to the coldest months of the year. Both puppies and adult dogs are highly infected. Dogs are reservoirs for zoonotic parasites; for this reason, it is imperative for humans to avoid fecal contamination in streets, public gardens and parks.

Determinación in vitro de la susceptibilidad a la nitazoxanida de cuatro aislados de Giardia duodenalis obtenido de diferentes huéspedes / In vitro determination of the sensitivity to nitazoxanide of four isolates of Giardia duodenalis from different hosts

Antecedentes: En México la giardiosis es la protozoosis intestinal más frecuente en los niños, para el tratamiento se utilizan los nitrofuranos, nitroimidazoles y benzimidazoles. Recientemente se introdujo un antiparasitario polivalente: la Nitazoxanida (Ntz); del cual hay pocos estudios sobre su actividad antigiardiásica. Objetivo: Determinar in vitro la sensibilidad de G. duodenalis a la Nitazoxanida y compararla con el Tinidazol (Tnz). Material y métodos: La susceptibilidad se estudió en cuatro aislados axénicos de G. duodenalis: INP231087MM y INP210897-AXA1 (humanos), INP170693HG8 y INP300693-CP5 (gato y perro respectivamente). Se expusieron 500,000 trofozoítos a diferentes concentraciones de Ntz o Tnz. La viabilidad se determinó por el método colorimétrico de la reducción de sales de tetrazolio (MTT) a MTT-formazán y los resultados se analizaron por ANOVA. Los cambios estructurales se determinaron por microscopia electrónica de transmisión. Los experimentos se realizaron por triplicado y se repitieron cuatro veces en un diseño experimental ciego.Resultados: Para alcanzar el 100 por ciento de mortalidad (sensibilidad o susceptibilidad) se necesitó una concentración de 1 a 7 mg/mL de Ntz y de 1 a 4 mg/mL de Tnz. Cuando se comparó la susceptibilidad de los aislados a la Ntz se encontró que hubo diferencias significativas entre ellos (P<0.001). Los fármacos produjeron aumento de volumen y menor electrodensidad del citoplasma y núcleos. Conclusiones: Este es el primer reporte de la sensibilidad, in vitro, de G. duodenalis a Ntz, en aislados de diferentes huéspedes. Se observó mayor susceptibilidad al Tnz que a la Ntz. Debido a la deformación de los trofozoítos, se sugiere que la Ntz induce modificaciones en la membrana y lisis celular.