The Amoebicidal Activity of Diferrocenyl Derivatives: A Significant Dependence on the Electronic Environment.

Amoebiasis is the second leading cause of death worldwide associated with parasitic disease and is becoming a critical health problem in low-income countries, urging new treatment alternatives. One of the most promising strategies is enhancing the redox imbalance within these susceptible parasites related to their limited antioxidant defense system. Metal-based drugs represent a perfect option due to their extraordinary capacity to stabilize different oxidation states and adopt diverse geometries, allowing their interaction with several molecular targets. This work describes the amoebicidal activity of five 2-(Z-2,3-diferrocenylvinyl)-4X-4,5-dihydrooxazole derivatives (X = H (3a), Me (3b), iPr (3c), Ph (3d), and benzyl (3e)) on Entamoeba histolytica trophozoites and the physicochemical, experimental, and theoretical properties that can be used to describe the antiproliferative activity. The growth inhibition capacity of these organometallic compounds is strongly related to a fine balance between the compounds' redox potential and hydrophilic character. The antiproliferative activity of diferrocenyl derivatives studied herein could be described either with the redox potential, the energy of electronic transitions, logP, or the calculated HOMO-LUMO values. Compound 3d presents the highest antiproliferative activity of the series with an IC50 of 23 µM. However, the results of this work provide a pipeline to improve the amoebicidal activity of these compounds through the directed modification of their electronic environment.

The association between rLiHyp1 protein plus adjuvant and amphotericin B is an effective immunotherapy against visceral leishmaniasis in mice.

Treatment of visceral leishmaniasis (VL) is compromised by drug toxicity, high cost and/or the emergence of resistant strains. Though canine vaccines are available, there are no licensed prophylactic human vaccines. One strategy to improve clinical outcome for infected patients is immunotherapy, which associates a chemotherapy that acts directly to reduce parasitism and the administration of an immunogen-adjuvant that activates the host protective Th1-type immune response. In this study, we evaluated an immunotherapy protocol in a murine model by combining recombinant (r)LiHyp1 (a hypothetical amastigote-specific Leishmania protein protective against Leishmania infantum infection), with monophosphoryl-lipid A (MPLA) as adjuvant and amphotericin B (AmpB) as reference antileishmanial drug. We used this protocol to treat L. infantum infected-BALB/c mice, and parasitological, immunological and toxicological evaluations were performed at 1 and 30 days after treatment. Results showed that mice treated with rLiHyp1/MPLA/AmpB presented the lowest parasite burden in all organs evaluated, when both a limiting dilution technique and qPCR were used. In addition, these animals produced higher levels of IFN-γ and IL-12 cytokines and IgG2a isotype antibody, which were associated with lower production of IL-4 and IL-10 and IgG1 isotype. Furthermore, low levels of renal and hepatic damage markers were found in animals treated with rLiHyp1/MPLA/AmpB possibly reflecting the lower parasite load, as compared to the other groups. We conclude that the rLiHyp1/MPLA/AmpB combination could be considered in future studies as an immunotherapy protocol to treat against VL.

Super aggregated amphotericin B with a thermoreversible in situ gelling ophthalmic system for amoebic keratitis treatment.

Acanthamoeba spp. are the causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK). Amphotericin B - deoxycholate (AB) is used in the treatment of infectious keratitis, however, its topical administration has side effects as blepharitis, iritis, and painful instillation. In this context, the preheating of AB can decrease its toxicity by the formation of super aggregates (hAB). hAB associated with a thermoreversible in situ gelling ophthalmic system is a promising option due to the latter biocompatibility, low toxicity, and high residence time on the ocular surface. Our objective was to develop a topical ocular formulation of hAB for the treatment of AK. After heating at 70°C for 20 min, hAB was incorporated into a thermoreversible gelling system. The amebicidal activity of AB and hAB was evaluated against trophozoites and cysts of A. castellanii (ATCC 50492) and a regional clinical isolate (IC01). The results showed that the preheating of AB did not change the pharmacological action of the drug, with the amebicidal effect of AB and hAB under trophozoites and cysts of Acanthamoeba spp. The thermoreversible system remained stable, allowing the increase of drug retention time. For assessment of cytotoxicity, HUVEC (ATCC® CRL-1730) cells were challenged with AB and hAB for 48h. Cell viability was assessed, and hAB did not show cytotoxicity for HUVEC cells. As far as we know this was the first study that showed the preheated AB associated with a thermoreversible in situ gelling ophthalmic system as a promising system for topical ocular topical administration of hAB for AK therapy.

Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Heteroleptic NiII complexes: Synthesis, structural characterization, computational studies and amoebicidal activity evaluation.

In this work, we present the synthesis, characterization, electrochemical studies, DFT calculations, and in vitro amoebicidal effect of seven new heteroleptic NiII coordination compounds. The crystal structures of [H2(pdto)](NO3)2 and [Ni(pdto)(NO3)]PF6 are presented, pdto = 2,2'-[1,2-ethanediylbis-(sulfanediyl-2,1-ethanediyl)]dipyridine. The rest of the compounds have general formulae: [Ni(pdto)(NN)](PF6) where N-N = 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (44dmbpy), 5,5'-dimethyl-2,2'-bipyridine (55dmbpy), 1,10-phenanthroline (phen), 4,7-dimethyl-1,10-phenanthroline (47dmphen) and 5,6-dimethyl-1,10-phenanthroline (56dmphen). The size of NN ligand and its substituents modulate the compound electronic features and influence their antiproliferative efficiency against Entamoeba histolytica. 56dmphen derivative, shows the biggest molar volume and presents a powerful amoebicidal activity (IC50 = 1.2 µM), being seven times more effective than the first-line drug for human amoebiasis metronidazole. Also, increases the reactive oxygen species concentration within the trophozoites. This could be the trigger of the E. histolytica growth inhibition. The antiparasitic effect is described using NiII electron density, molar volume, estimated by DFT, as well as the experimental redox potential and diffusion coefficients. In general, amoebicidal efficiency is directly proportional to the increment of the molar volume and decreases when the redox potential becomes more positive.

In Vitro Amoebicidal Activity of Imidazolium Salts Against Trophozoites.

INTRODUCTION: Several strains of the free-living genus Acanthamoeba can cause granulomatous amoebic encephalitis (GAE), a rare chronic and slowly progressive infection of the central nervous system (CNS), and Acanthamoeba keratitis (AK), a sight-threatening eye infectious disease. AK incidence has increased with the popularization of the contact lens wear and its treatment is currently limited and frequently unsuccessful. As imidazolium salts (IS), cationic imidazole derivatives, have promising antimicrobial potential. MATERIALS AND METHODS: The present study evaluated the amoebicidal activity of four IS; 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS), chloride (C16MImCl) and bis (triluoromethylsulfonyl) imide (C16MImNTf2 ), and 1-methyl-3-n-octadecylimidazolium chloride (C18MImCl), against the Acanthamoeba castellanii (ATCC30010) environmental strain and a clinical isolate (genotype T4). RESULTS: Three IS showed being lethal to 100% of the Acanthamoeba trophozoites at the minimum inhibitory concentrations of 125 and 62.5 µg/mL (C16MImMeS), 31.25 and 62.5 µg/mL (C16MImCl), and 125 and 125 µg/mL (C18MImCl) for ATCC30010 and isolate T4, respectively. C16MImNTf2 did not demonstrate amoebicidal activity. All active IS caused the hemolysis of erythrocytes. The cytotoxic effect of the IS was tested in RAW macrophages and human brain microvascular endothelial cells, which demonstrated cytotoxicity in all concentrations tested against both cell lines. As a consequence, these IS with amoebicidal activity presented low selectivity index values (SI) (SI < 1.0), demonstrating lack of parasite selectivity. CONCLUSION: Thus, C16MImMeS, C16MImCl, and C18MImCl seem to hold greater promise as components for contact lens cleaning and disinfection solutions, instead of direct human application.

Tinidazole efficacy for amebic colitis therapeutic prophylaxis in patients with de novo acute leukemia receiving intensive chemotherapy.

INTRODUCTION: In Mexico, seroprevalence of Entamoeba histolytica is 8.4%. The intestinal amebiasis in patients with acute leukemia of novo, after the start of chemotherapy (CT) in the Hematology Service of the CMN 20 de Noviembre is 12%, even if patients show a negative baseline coprological test. OBJECTIVE: To find out if the administration of tinidazole, in patients with acute leukemia and negative coprological test, at the beginning of the CT, decreases the incidence of amoebic colitis during the induction to remission. METHOD: Prospective and not comparative study. Patients with de novo diagnosis of acute leukemia who initiate induction and initial coprological CT. Tinidazole was indicated, 2 g/day for 5 days in the first week of CT started. They were monitored until the induction was concluded and hematopoietic recovery started. RESULTS: 38 patients, 15 women and 23 men with a mean age of 44 years (16-72), with acute lymphoblastic leukemia 19, myeloblastic 16 and promyelocytic 3. Cases without and with intestinal amebiasis were 35 and 3, respectively. Patients with amebiasis only received tinidazole for 3 days and it was given 2 days after the CT started. CONCLUSION: Tinidazole, in patients with acute de novo leukemia who initiate induction CT, is effective in the prevention of intestinal amebiasis, during the induction stage, if administered at 2 g/day, for five days, starting on day 1 of the CT.

Development of a Novel Ex-vivo 3D Model to Screen Amoebicidal Activity on Infected Tissue.

Amoebiasis is a parasitic disease that causes thousands of deaths every year, its adverse effects and resistance to conventional treatments have led to the search of new treatment options, as well as the development of novel screening methods. In this work, we implemented a 3D model of intestine and liver slices from hamsters that were infected ex vivo with virulent E. histolytica trophozoites. Results show preserved histology in both uninfected tissues as well as ulcerations, destruction of the epithelial cells, and inflammatory reaction in intestine slices and formation of micro abscesses, and the presence of amoebae in the sinusoidal spaces and in the interior of central veins in liver slices. The three chemically synthetized compounds T-001, T-011, and T-016, which act as amoebicides in vitro, were active in both infected tissues, as they decreased the number of trophozoites, and provoked death by disintegration of the amoeba, similar to metronidazole. However, compound T-011 induced signs of cytotoxicity to liver slices. Our results suggest that ex vivo cultures of precision-cut intestinal and liver slices represent a reliable 3D approach to evaluate novel amoebicidal compounds, and to simultaneously detect their toxicity, while reducing the number of experimental animals commonly required by other model systems.

Acanthamoeba keratitis in patients wearing scleral contact lenses.

PURPOSE: To report a series of cases of Acanthamoeba keratitis (AK) in scleral lens wearers with keratoconus to determine whether this type of contact lens presents a greater risk for development of infection. METHODS: This study reports three patients who wore scleral contact lenses to correct keratoconus and developed AK. The diagnoses of AK were established based on cultures of the cornea, scleral contact lenses, and contact lens paraphernalia. This study investigated the risk factors for infections. RESULTS: The possible risks for AK in scleral contact lens wearers are hypoxic changes in the corneal epithelium because of the large diameter and minimal tear exchange, use of large amounts of saline solution necessary for scleral lens fitting, storing the scleral lens overnight in saline solution rather than contact lens multipurpose solutions, not rubbing the contact lens during cleaning, and the space between the cornea and the back surface of the scleral lens that might serve as a fluid reservoir and environment for Acanthamoeba multiplication. Two patients responded well to medical treatment of AK; one is still being treated. CONCLUSIONS: The recommendations for use and care of scleral contact lenses should be emphasized, especially regarding use of sterile saline (preferably single use), attention to rubbing the lens during cleaning, cleaning of the plunger, and overnight storage in fresh contact lens multipurpose solutions without topping off the lens solution in the case.

Diloxanide furoate binary complexes with ß-, methyl-ß-, and hydroxypropyl-ß-cyclodextrins: inclusion mode, characterization in solution and in solid state and in vitro dissolution studies.

The purpose of this study was to investigate the effect on solubility and dissolution rate of binary complexes of ß-(ßCD), methyl-(MßCD) and hydroxypropyl-ß-cyclodextrin (HPßCD) with diloxanide furoate (DF). The complexation in solution was evaluated by phase solubility studies and 1H nuclear magnetic resonance (NMR). Enhanced water solubility of DF was obtained with the DF:MßCD system (61-fold). The mode of inclusion was supported by NMR experiments, which indicated that real inclusion complexes were formed between DF and MßCD or HPßCD. Solid state analysis was performed using infrared and thermal methods, which suggested the formation of true inclusion complexes of DF with two derivatized cyclodextrins, MßCD and HPßCD, and an exclusion complex with ßCD when the systems were prepared by freeze-dried technique. Dissolution studies conducted in simulated gastric fluid (2 h) and subsequent simulated intestinal fluid (next 4 h) showed increased dissolution rate of DF from the freeze-dried systems with ßCD, MßCD, and HPßCD (85; 77 and 75% of dissolved drug at 5 min, respectively) and 100% of the drug dissolved at 150 min for the three systems. The enhancement of the solubility and the dissolution of DF observed make these complexes promising candidates for the preparation of oral pharmaceutical formulations.

Polycyclic ferrocenyl(dihydro)thiazepine derivatives: Diastereo-selective synthesis, characterization, electrochemical behavior, theoretical and biological investigation.

The reaction of E-2-ferrocenylmethylidenetetralones and E,E-2,6-bis-(ferrocenylmethylidene)-cyclohexanone with 2-aminothiophenol proceed with high diastereoselectivity, forming the ~4.5:1 mixture of trans- and cis-isomers of polycyclic ferrocenylthiazepines, respectively. The reactions of E,E-2,5-bis-(ferrocenylmethylidene)cyclopentanone and E,E-3,5-bis-(ferrocenylmethylidene)-1-methyl-4-piperidone with 2-aminothiophenol take place stereo specifically to form the diastereomeric tricyclic thiazepines of cis- and trans-configuration, respectively. The structures of the obtained compounds were established by IR, 1H and 13C NMR spectroscopy and mass-spectrometry. The structures of the trans-tetralino[1,2a]-, trans-5,7-dimethyltetralino[1,2a]-2-ferrocenyl [1,5]benzo-2,3-dihydrothiazepines and cis-5-ferrocenyl-methylidenecyclopentano[1,2a]-2-ferrocenyl- [1,5]benzo-2,3-dihydrothiazepine were confirmed by X-ray diffraction analysis. An electrochemical study reveals that the diferrocenyl derivatives belong to a Class I compounds of the Robin-Day classification. This behavior is explained by the analysis of frontier orbitals as calculated by density functional theory, showing that only one ferrocenyl unit participates in the generation of HOMO and LUMO orbitals. Compounds 4a and 4c showed similar capacity to inhibit the proliferation of HM1: IMSS trophozoite cultures than the first choice drug for human amoebiasis treatment, metronidazole. Morphological changes induced in the trophozoites after drug exposure suggest a redox in balance as the probable mechanism of the parasite death.

Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 µg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 µg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency.

Amoebicidal and citotoxic activity of green propolis aqueous extract against Acanthamoeba castellanii trophozoites and cysts

O gênero Acanthamoeba pertencente ao grupo das amebas de vida livre e é amplamente distribuído no ambiente. Estes protistas são conhecidos por causarem doenças graves, como a Encefalite Amebiana Granulomatosa em pacientes imunocomprometidos e ceratite amebiana, especialmente em usuários de lentes de contato imunocompetentes. Própolis verde é uma substância resinosa e balsâmica, conhecida na medicina alternativa por exibir várias atividades biológicas. Neste estudo avaliou-se a atividade amebicida de um extrato aquoso de própolis verde contra trofozoítos e cistos de A. castellanii. Nas concentrações de 10 e 20 mg/mL, o extrato foi capaz de inativar 100% de trofozoítos no prazo de 24 horas e 48 horas, enquanto a uma concentração de 5 mg/mL 100% dos trofozoítos foram inativados em 72 horas. Os cistos foram inativados após 24 horas de exposição ao extrato à concentração de 40 mg/mL. O efeito do extrato foi avaliado sobre células HCE (epiteliais de córnea humana), empregando-se ensaio de viabilidade baseado na redução do sal de tetrazólio MTT. O extrato não apresentou efeito citotóxico significativo sobre as células HCE, nas concentrações de 0,312, 0,625, 1,25 e 2,5 mg/mL. O teste de adesão realizado mostrou que a fixação de Acanthamoeba a células HCE apresenta comportamento dose- dependente em relação ao extrato de própolis. Assim, este estudo demonstrou a eficácia da própolis verde contra trofozoítos e cistos de Acanthamoeba e provou ser uma substância promissora especialmente para a formulação de soluções para desinfecção de superfícies. No entanto, mais estudos são necessários para entender seu mecanismo de ação.

The Amoebicidal Effect of Ergosterol Peroxide Isolated from Pleurotus ostreatus.

Dysentery is an inflammation of the intestine caused by the protozoan parasite Entamoeba histolytica and is a recurrent health problem affecting millions of people worldwide. Because of the magnitude of this disease, finding novel strategies for treatment that does not affect human cells is necessary. Ergosterol peroxide is a sterol particularly known as a major cytotoxic agent with a wide spectrum of biological activities produced by edible and medicinal mushrooms. The aim of this report is to evaluate the amoebicidal activity of ergosterol peroxide (5α, 8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol isolated from 5α, 8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol) (Jacq.) P. Kumm. f. sp. Florida. Our results show that ergosterol peroxide produced a strong cytotoxic effect against amoebic growth. The inhibitory concentration IC50 of ergosterol peroxide was evaluated. The interaction between E. histolytica and ergosterol peroxide in vitro resulted in strong amoebicidal activity (IC50 = 4.23 nM) that may be due to the oxidatory effect on the parasitic membrane. We also tested selective toxicity of ergosterol peroxide using a cell line CCL-241, a human epithelial cell line isolated from normal human fetal intestinal tissue. To the best of our knowledge, this is the first report on the cytotoxicity of ergosterol peroxide against E. histolytica, which uncovers a new biological property of the lipidic compound isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida.

Potential amoebicidal activity of hydrazone derivatives: synthesis, characterization, electrochemical behavior, theoretical study and evaluation of the biological activity.

Four new hydrazones were synthesized by the condensation of the selected hydrazine and the appropriate nitrobenzaldehyde. A complete characterization was done employing 1H- and 13C-NMR, electrochemical techniques and theoretical studies. After the characterization and electrochemical analysis of each compound, amoebicidal activity was tested in vitro against the HM1:IMSS strain of Entamoeba histolytica. The results showed the influence of the nitrobenzene group and the hydrazone linkage on the amoebicidal activity. meta-Nitro substituted compound 2 presents a promising amoebicidal activity with an IC50 = 0.84 µM, which represents a 7-fold increase in cell growth inhibition potency with respect to metronidazole (IC50 = 6.3 µM). Compounds 1, 3, and 4 show decreased amoebicidal activity, with IC50 values of 7, 75 and 23 µM, respectively, as a function of the nitro group position on the aromatic ring. The observed differences in the biological activity could be explained not only by the redox potential of the molecules, but also by their capacity to participate in the formation of intra- and intermolecular hydrogen bonds. Redox potentials as well as the amoebicidal activity can be described with parameters obtained from the DFT analysis.

Antiprotozoal activity against Entamoeba histolytica of plants used in northeast Mexican traditional medicine. Bioactive compounds from Lippia graveolens and Ruta chalepensis.

Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.

Hypocholesterolemia in patients with an amebic liver abscess.

BACKGROUND/AIMS: Many parasites induce changes in the lipid profiles of the host. Cholesterol increases the virulence of Entamoeba histolytica in animal models and in vitro culture. This study aimed to determine, in patients with an amebic liver abscess, the correlation between cholesterol and other features, such as the size and number of abscesses, standard hematological and serum chemistry profiles, liver tests, and duration of hospital stay. METHODS: A total of 108 patients with an amebic liver abscess and 140 clinically healthy volunteers were investigated. Cholesterol and triglycerides were measured in the sera. The data from medical observations and laboratory tests were obtained from the clinical records. RESULTS: A total of 93% of patients with an amebic liver abscess showed hypocholesterolemia not related to any of the studied parameters. Liver function tests correlated with the size of the abscess. The most severe cases of amebic liver disease or death were found in patients whose cholesterol levels continued to decrease despite receiving antiamebic treatment and hospital care. CONCLUSIONS: Our results show that the hypocholesterolemia observed in patients with an amebic liver abscess is not related to any of the clinical and laboratory features analyzed. This is the first study relating hypocholesterolemia to severity of hepatic amebiasis.

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru.

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.

Entamoeba histolytica P-glycoprotein (EhPgp) inhibition, induce trophozoite acidification and enhance programmed cell death.

Programmed cell death (PCD) is induced in Entamoeba histolytica by a variety of stimuli in vitro and in vivo. In mammals, intracellular acidification serves as a global switch for inactivating cellular processes and initiates molecular mechanisms implicated in the destruction of the genome. In contrast, intracellular alkalinization produced by P-glycoprotein overexpression in multidrug-resistant cells has been related to apoptosis resistance. Our previous studies showed that overexpression of E. histolytica P-glycoprotein (PGP) altered chloride-dependent currents and triggered trophozoite swelling, the reverse process of cell shrinkage produced during PCD. Here we showed that antisense inhibition of PGP expression produced a synchronous death of trophozoites and the enhancement of biochemical and morphological characteristics of PCD induced by G418. The nucleus was contracted, and the nuclear membrane was disrupted. Moreover, chromatin was extensively fragmented. Ca(2+) concentration was increased, while the intracellular pH (ipH) was acidified. In contrast, PGP overexpression prevented intracellular acidification and circumvented the apoptotic effect of G418.