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.

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.

In vitro effect of Acanthospermum australe (Asteraceae) extracts on Acanthamoeba polyphaga trophozoites / Efeito in vitro de extratos de Acanthospermum australe (Asteraceae) sobre trofozoitos de Acanthamoeba polyphaga

Acanthamoeba is a free-living protozoan widely distributed in the environment, occurring in vegetative trophozoite and resistance cyst stages during its life cycle. It constitutes an etiological agent of Acanthamoeba keratitis, a disease that may cause severe ocular inflammation and blindness. New drugs can be developed from molecules found in plants and thus help in its difficult treatment. Acanthospermum australe (Asteraceae), a plant used in folk medicine, had its effect tested on Acanthamoeba polyphaga. Aqueous and ethanolic extracts of A. austral were obtained from aerial parts for infusion and static maceration, respectively. Concentrations of 10, 5, 2.5, 1.25 and 0.625 mg/ml of the extract were tested against Acanthamoeba polyphaga trophozoites. The cytotoxic effect of the extracts was tested in mammalian cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The 10 mg/ml concentration of ethanolic extract was lethal to 100% of the A. polyphaga trophozoites in 24 h and both extracts presented cytotoxic effect against mammalian cells. These findings suggest that the A. austral ethanolic extract may have compounds with relevance to the development of new amoebicidal drugs.

Acanthamoeba é um protozoário de vida livre amplamente distribuído no ambiente, ocorrendo sob a forma trofozoítica (metabolicamente ativa) e cística (de resistência), durante seu ciclo de vida. O protozoário constitui um agente etiológico da Ceratite Amebiana, uma doença que pode causar inflamação ocular severa e cegueira. Novos fármacos podem ser desenvolvidos a partir de moléculas encontradas em plantas e assim ajudar em seu difícil tratamento. Aqui, Acanthospermum australe (Asteraceae), uma planta utilizada na medicina popular, teve seu efeito sobre trofozoítos de Acanthamoeba polyphaga testado. O extrato aquoso e etanólico de A. australe foram obtidos das partes aéreas por infusão e maceração estática, respectivamente. As concentrações 10, 5, 2,5, 1,25 e 0,625 mg/ml dos extratos foram testadas contra trofozoítos do protozoário. O efeito citotóxico dos extratos foi testado em células de mamífero utilizando o ensaio de brometo de 3-[4,5-dimetiltiazol-2-il]-2,5-difeniltetrazólio (MTT). A concentração de 10 mg/ml do extrato etanólico foi letal a 100% dos trofozoítos de A. polyphaga em 24 h e ambos os extratos apresentaram efeito citotóxico contra as células de mamífero. Estes resultados sugerem que o extrato etanólico de A. australe pode ter componentes com relevância para o desenvolvimento de novos fármacos amebicidas.

Farmacoterapia actual de la amibiasis, avances en nuevas drogas y diseño de una vacuna / Current pharmacotherapy of amebiasis, advances in new drugs, and design of a vaccine

Aunque existe una gran cantidad de fármacos amebicidas que actúan en la luz intestinal, las drogas de acción tisular usadas para tratar la amibiasis invasiva son aún relativamente limitadas. El advenimiento del metronidazol (MTZ), que es el fármaco de elección para la amibiasis invasiva, y otros nitroimidazoles en el tratamiento de la amibiasis, ha simplificado enormemente la quimioterapia de la infección. No obstante, la erradicación de ésta después de la administración del MTZ requiere terapia adicional con un amebicida de acción luminal como la paramomicina. Después de décadas desde la introducción de estas drogas en la terapia de la infección, se han hecho pocas innovaciones. Mientras tanto, esta parasitosis continúa siendo una causa importante de morbilidad y mortalidad en el mundo contemporáneo. Debido a los efectos tóxicos y los recientes fracasos en el tratamiento de algunos protozoos intestinales con el MTZ, es necesaria la búsqueda de nuevos compuestos amebicidas. Un avance reciente es la nitazoxanida que tiene una actividad de amplio espectro contra diversos agentes infecciosos y se ha demostrado recientemente su acción contra E. histolytica. Este fármaco podría ser clave como amebicida por su efectividad contra el parásito en la luz intestinal y en los tejidos. Sin embargo, el diseño de una vacuna protectora contra la infección sigue siendo deseable. Los estudios experimentales recientes en animales modelo son alentadores. El objetivo de esta revisión es examinar y discutir los aspectos más importantes de la farmacoterapia actual de la amibiasis, así como de los prospectos para el desarrollo de nuevas drogas y una vacuna protectora contra la infección.

Although many drugs destroy Entamoeba histolytica within the colonic lumen, the number of tissue amebicides used to treat invasive amebiasis is still relatively limited. Metronidazole (MTZ), which is the drug of choice for invasive amebiasis, and other nitroimidazoles have greatly simplified the chemotherapy of this disease. However, eradication of E. histolytica infection after completion of MTZ therapy requires additional treatment with luminal amebicides, such as paramomycin. After decades of the introduction of MTZ and other nitroimidazoles in the therapy of amebiasis, there have been few innovations in treating amebic infections. Meanwhile, amebiasis remains among the leading causes of morbidity and mortality in the contemporary world. The toxic effects of MTZ and recent failures in the treatment of several intestinal protozoan parasites, has led to a search for other amebicidal drugs. A recent advance is the demonstration of the effect of nitazoxanide, which has broad spectrum of antiparasitic activity, against E. histolytica. This compound could be the key in the therapy of amebiasis by its action against both luminal and invasive parasite forms. However, the design of an effective vaccine against the infection is still being desirable. Work is underway to develop a vaccine and recent experimental studies are promising. The aim of this review is to examine and discuss the most important aspects of current antiamebic pharmacotherapy and the prospects for development of new drugs and a vaccine.

Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

Padronização de condições experimentais no cultivo e quantificação de Entamoeba histolytica que otimizem ensaios de compostos potencialmente amebicidas / Standardization of experimental conditions in the cultivation and quantification of Entamoeba histolytica that optimize test compounds potentially amebicides

O protozoário, Entamoeba histolytica, constitui a etiologia de milhares de óbitos anuais e, em muitos casos, a falta de saneamento, o grau de instrução e a falta de higiene da população podem favorecer a transmissão e a manutenção desses patógenos em uma comunidade. Por causar tantas mortes e problemas na saúde pública trabalhos que facilitem o estudo deste parasito fazem-se importantes. Uma vez que a padronização de cultivo de E. histolytica em placas de poços vai Existem indicações que mostram que este parasito pode se tornar resistente ao medicamento utilizado no tratamento desta protozoose, por isso, a busca por novas substâncias que possam atuar como tratamento alternativo é de suma importância. Portanto, o objetivo do presente trabalho foi otimizar e padronizar o cultivo e a contagem deste parasito in vitro, além de identificar substâncias com potencial amebicida, que possam ser utilizadas no futuro como fármacos no tratamento da amebíase, sugerindo também uma via possível de ação das substâncias que apresentaram os melhores efeitos. Para tanto, os trofozoítos foram cultivados em placas de 24 poços sobre diferentes condições, quatro métodos de contagem de células foram comparados e 74 (setenta e quatro) substâncias foram testadas. Destas 13 (treze) apresentaram uma inibição na proliferação axênica dos trofozoítos de cerca de 70%. Destas, três compostos foram estudados em mais detalhes, os mesoiônicos derivados da piperina (as MII, MVI e MIX). Estas substâncias pertencem ao grupo dos compostos mesoiônicos, substâncias formadas por um anel heteroatômico composto por nitrogênio, carbono e enxofre, capazes de atravessar membranas e interagir com biomoléculas. Além disso, alguns mesoiônicos são doadores de radicais NO e tais grupamentos são capazes de induzir uma morte celular semelhante à apoptose em E. histolytica, como sugerido pela expressão de fosfatidil-serina revelada por anexina-V. Confirmando os resultados descritos na literatura, estas substâncias foram capazes de induzir uma morte programada, porém observações da ultra-estrutura, tais como figuras de mielina, das células tratadas apontaram para autofagia que também foi evidenciada por testes com MDC gerando apoptose tipo II, que pode ser iniciada pela presença de ROS, que neste caso foram por DCFDA.

A randomized, controlled, open-label trial evaluating the efficacy and safety of chloroquine in the treatment of giardiasis in children / Estudio de etiqueta abierta, randomizado y controlado, para evaluar la eficacia y seguridad de la cloroquina en el tratamiento de la giardiasis en niños

BACKGROUND: Giardia duodenalis is among the commonest protozoan parasites in the intestinal tract of humans and may cause significant morbidity worldwide. Although there are several antigiardial agents, treatment failures have been commonly reported. OBJECTIVE: To compare the efficacy and safety of chloroquine (CQ) versus metronidazole (MTZ) in the treatment of children with confirmed G duodenalis mono-infection. METHODS: A randomized, controlled, open-label trial was carried out at the Cuban Institute of Gastroenterology. One hundred and twenty-two children were randomly assigned to receive either CQ (10 mg/Kg bodyweight twice a day for five days) or MTZ [15 mg/Kg bodyweight divided in three daily does for five days]. All children were asked to provide three faecal samples on days 3, 5 and 7 after treatment completion. Children were considered to be cured, if no Giardia trophozoites or cysts were found in any of the three post-treatment faecal specimens evaluated by direct wet mounts and/or after Ritchie concentration techniques. RESULTS: The frequency of cure was a little higher for CQ than for MTZ but the difference was not statistically significant. Headache was more common in patients treated with CQ as was bitter taste. Yellowish colouration of the urine was more frequent in the MTZ treated group. CONCLUSION: Chloroquine, for five days, is as efficacious as the recommended treatment with MTZ in children infected with G duodenalis.

ANTECEDENTES: La giardia lamblia (giardia duodenalis) se halla entre los parásitos protozoos más comunes del tracto intestinal de los seres humanos, y puede causar una morbilidad significativa a nivel mundial. Aunque existen varios agentes antigiardiales, se han reportado fracasos en el tratamiento OBJETIVO: Comparar la eficacia y seguridad de la cloroquina (CQ) con el metronidazol (MTZ) en el tratamiento de los niños con mono-infección de G duodenalis. MÉTODOS: En el Instituto Cubano de Gastroenterología, se llevó a cabo un estudio de etiqueta abierta, randomizado y controlado. Ciento veintidós niños fueron aleatoriamente designados para recibir bien CQ (10 mg/Kg peso corporal dos veces por día durante cinco días) o MTZ (15 mg/Kg peso corporal dividido en tres dosis diarias por un período de cinco días). A todos los niños se les tomaron tres pruebas fecales los días 3, 5 y 7 después de terminado el tratamiento. Los niños se daban por curados, si no había presencia de tropozoítos o quistes de giardia en ninguno de los tres especimenes fecales post-tratamiento, evaluados directamente con portaobjetos húmedos y/o después de técnicas de concentración de Ritchie. RESULTADO: La frecuencia de la cura fue un poco más alta para CQ que para MTZ, pero la diferencia no fue estadísticamente significativa. El dolor de cabeza fue más común en pacientes tratados con CQ que el sabor amargo. La coloración amarillenta de la orina fue más frecuente en el grupo tratado con MTZ. CONCLUSIÓN: La cloroquina, administrada durante cinco días, es tan eficaz como el tratamiento recomendado con MTZ en niños infectados con giardias lamblias.

Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: Experience of over a decade.

Purpose: To review the epidemiological characteristics, microbiological profile, and treatment outcome of patients with suspected microbial keratitis. Materials and Methods: Retrospective analysis of a non-comparative series from the database was done. All the patients presenting with corneal stromal infiltrate underwent standard microbiologic evaluation of their corneal scrapings, and smear and culture-guided antimicrobial therapy. Results: Out of 5897 suspected cases of microbial keratitis 3563 (60.4%) were culture-proven (bacterial – 1849, 51.9%; fungal – 1360, 38.2%; Acanthamoeba – 86, 2.4%; mixed – 268, 7.5%). Patients with agriculture-based activities were at 1.33 times (CI 1.16–1.51) greater risk of developing microbial keratitis and patients with ocular trauma were 5.33 times (CI 6.41–6.44) more likely to develop microbial keratitis. Potassium hydroxide with calcofluor white was most sensitive for detecting fungi (90.6%) and Acanthamoeba (84.0%) in corneal scrapings, however, Gram stain had a low sensitivity of 56.6% in detection of bacteria. Majority of the bacterial infections were caused by Staphylococcus epidermidis (42.3%) and Fusarium species (36.6%) was the leading cause of fungal infections. A significantly larger number of patients (691/1360, 50.8%) with fungal keratitis required surgical intervention compared to bacterial (799/1849, 43.2%) and Acanthamoeba (15/86, 17.4%) keratitis. Corneal healed scar was achieved in 75.5%, 64.8%, and 90.0% of patients with bacterial, fungal, and Acanthamoeba keratitis respectively. Conclusions: While diagnostic and treatment modalities are well in place the final outcome is suboptimal in fungal keratitis. With more effective treatment available for bacterial and Acanthamoeba keratitis, the treatment of fungal keratitis is truly a challenge.

Rare presentation of a common disease of tropics.

A 19 years male presented with fever, oliguria and purpuric lesions involving both hands. The patient was diagnosed as a case of purpura fulminans with disseminated intravascular coagulation due to complicated falciparum malaria. The case is presented to sensitize the physicians to keep malaria as a differential in cases of fever with purpura fulminans.

Desarrollo de una formulación de tabletas revestidas de secnidazol 500 mg / Development of a formulation of coated secnidazole tablets 500 mg

Se describe el desarrollo de una formulación de tabletas revestidas de secnidazol 500 mg. Durante el desarrollo de esta se evaluaron 2 métodos: la granulación seca y la granulación húmeda. En el revestimiento de las tabletas se empleó un sistema hidroalcohólico con el preparado comercial Policoat YS 1-7003 (Opadry)®. Los lotes sometidos a ensayos de estabilidad fueron elaborados a escala piloto, por la vía de granulación húmeda, lo que demostró la factibilidad del proceso de fabricación a esta escala. Las tabletas revestidas presentaron adecuadas propiedades físico-mecánicas y tecnológicas y estas fueron envasadas en frascos de polietileno de alta densidad y en sobres termoconformados de polivinilcloruro/aluminio. La estabilidad química y microbiológica de las tabletas de secnidazol fue estudiada durante 24 meses; los resultados demostraron que estas cumplían con los requisitos establecidos durante este periodo.

The development of a formulation of coated Secnidazole tablets 500mg was described. During its development, 2 methods were evaluated: dry granulation and humid granulation. A hydroalcoholic system was used with the Policoat YS 1-7003 (Opadry)® commercial preparation for coating the tablets. The batches subjected to stability assays were made at pilot scale by humid granulation, which showed the feasibility of the manufacturing process at this scale. The coated tablets had adequate physicomechanical and technological properties and they were packed in polyethylene flasks of high density and in polyvinyl chloride/aluminium thermoconformed envelopes. The chemical and microbiological stability of the secnidazole tablets were studied during 24 months. The results demonstrated that these tablets met the requirements established during this period.

Progress in research on the anti-tumor effect of artesunate / 中国结合医学杂志

It has been found in recent years that Artesunate (Art), a water soluble derivative of arteannuin, mainly previously used for its anti-malarial activity, has some other effects, e.g. it could act as an anti-tumor agent by way of inducing cell apoptosis, antagonizing angiogenesis, reversing immunosuppression of tumor cells, etc. More and more attention is being paid to the anti-tumor effects of Art. Such progress is reviewed in this paper.

Amoebiasis cutis in HIV positive patient.

Protozoan infections of the skin, particularly cutaneous amoebiasis, are rare in HIV-positive patients. We report a case of amoebiasis cutis in an HIV-positive truck driver with a history of frequent unprotected sexual exposures. He presented with multiple painful ulcers and sinuses with purulent discharge, necrotic slough and scarring in the perianal and gluteal region for the last 2 years. He was positive for HIV-1 and -2. Cutaneous biopsy revealed numerous Entamoeba histolytica in the trophozoite form, in addition to an inflammatory infiltrate and necrotic debris. He responded well to oral metronidazole and chloroquine. Amoebiasis cutis should be considered in the differential diagnosis of perianal ulcers, particularly in HIV-positive patients.

Differentiating amoebic ulcero-haemorrhagic recto-colitis from idiopathic inflammatory bowel disease: still a diagnostic dilemma / Diferenciar la Rectocolitis Úlcero-hemorrágica Amebiana de la Enfermedad Inflamatoria Idiopática del Intestino: un Dilema Diagnóstico sin Resolver

The colon responds monomorphically to a variety of insults thus making it difficult to differentiate invasive amoebic colitis and inflammatory bowel disease (IBD). The authors present a case with chronic dysentery, haematochezia, anaemia and hypoproteinaemia. The endoscopic findings were suggestive of IBD. The stool examination was negative for trophozoites or cysts of parasites. The recto-colonic biopsy specimens showed mucosal inflammation with exudates containing amoebic trophozoites. The patient was successfully treated with metronidazole and iodoquinol. He recovered within two weeks and repeat colonoscopy four weeks after the treatment showed a normal rectum and colon. Clinicians should have a high level of suspicion for amoebic colitis in cases of colitis especially in regions where amoebiasis is still present. Efforts should be made to find the amoebic trophozoites in multiple stool and colonic biopsy specimens

El colon responde de manera monomórfica a una variedad de insultos, lo cual hace difícil distinguir entre la colitis amebiana invasiva y la enfermedad intestinal inflamatoria (EII). Los autores presentan un caso con disentería crónica, hematoquexia, anemia e hipoproteinemia. Los resultados endoscópicos apuntaban a una EII. El análisis de las heces fecales arrojó resultados negativos en cuanto a presencia de trofozoitos o quistes de parásitos. Esto condujo a un diagnóstico erróneo y el paciente fue tratado por una EII. Sin embargo, los especímenes de la biopsia rectocolónica mostraron una inflamación mucosal con exudados en los que se hallaban presentes trofozoitos amebianos. El paciente tuvo un tratamiento exitoso con metronidazol y iodoquinol. Se recuperó en dos semanas, y se le repitió la colonoscopia cuatro semanas después de que el tratamiento mostró un recto y colon normales. Los clínicos debían mostrar un alto nivel de sospecha ante la colitis amebiana, especialmente en aquellas regiones donde la amebiasis todavía está presente. Deben hacerse esfuerzos por encontrar trofozoitos amebianos en múltiples especímenes de heces fecales y biopsia colónica.

Amebiasis in the newborn.

Infestation with Entamoeba histolytica is especially common in areas with low socioeconomic status. Extra intestinal invasive involvement is more frequent in young children with significant mortality. This disease is rarely reported in the newborns. This 19-day-old newborn who was infected with orally given surgar solution is presented. He was successfully treated with omidazole.

Naegleria meningitis: a rare survival.

Acute amebic meningoencephalitis caused by free-living amebae naegleria fowleri is extremely rare and uniformly fatal with only seven survivals reported till date. An interesting case of naegleria meningitis diagnosed by wet mount cytology of cerebrospinal fluid (CSF) and treated with amphoterecin B, rifampicin and ornidazole with complete recovery is presented. In cases of suspected pyogenic meningitis, if CSF staining, antigen detection or culture is negative for bacteria, a wet mount cytology of CSF for naegleria is suggested. Early treatment with amphoterecin B and rifampicin may improve survival.

In vitro effect of artesunate against Acanthamoeba spp.

The in vitro effects of artesunate, the antimalarial agent, and metronidazole against Acanthamoeba spp were studied. Acanthamoeba Group II and Acanthamoeba polyphaga-like were isolated from natural water courses in Buri Ram Province, northeastern Thailand. The trophozoites were axenically cultured in PPYG medium and treated with artesunate in a concentration of 5-700 microg/ml. Artesunate showed its ability to inhibit the growth of acanthamoeba trophozoites: 54% at 50 mg/ml (after six days of exposure) and 93.2% at 100 microg/ml (after two days). The 500-700 microg/ml concentration caused inhibition on the first day of more than 93.2%; excystation did not occur in drug-treated medium. The present study shows that artesunate is amebastatic rather than amebicidal in an axenic culture of trophozoites at the highest concentration of 100 microg/ml. Metronidazole, in concentrations of 5-1,000 microg/ml, had no effects on either trophozoites or cysts.

In vitro effect of antifungal drugs on pathogenic Naegleria spp.

An ameba of the genus Naegleria causing fatal meningoencephalitis in human subjects was investigated for its sensitivity to antifungal drugs: amphotericin B, ketoconazole, fluconazole and itraconazole. The efficacy of these antifungal drugs for pathogenic Naegleria spp was investigated in three strains isolated from patients who had died of primary amebic meningoencephalitis infection at Siriraj Hospital (1986), Ramathibodi Hospital (1987) and Chachoengsao Hospital (1987). All of the isolates were maintained in axenic culture in the Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand. The sensitivities of the antifungal drugs (MIC50) were: amphotericin B (0.05-0.5 microg/ml), ketoconazole (0.125 microg/ml), fluconazole (0.5-2.0 mg/ml), and itraconazole (10 mg/ml) (p < 0.05). It is important to explain that ketoconazole is slightly more effective than amphotericin B because its action is directed of the permeability of the amebic membrane. The amebae were more resistant ot fluconazole and itraconazole due to the action of the cytochrome P450 multienzyme (in the case of fluconazole) and the direct effect on heme-iron, blocking cytochrome P450-dependent chitin synthesis (in the case of itraconzole). We conclude that amphotericin B and ketoconazole remain the main drugs with proven activity against pathogenic Naegleria spp.

Isolation of emetine resistant clones of Entamoeba histolytica by petri dish agar method.

Emetine resistant clones of Entamoeba histolytica strain HM1:IMSS were isolated by using petri dish agar method after mutation with ethyl-methanesulphonate. Two emetine resistant clones were obtained and both were resistant to emetine at a concentration of 24 micrograms/ml of emetine. The 50 per cent inhibitory concentration (IC50) for both emetine sensitive and resistant clones was 5 and 14 micrograms/ml respectively. The colony forming efficiency of E. histolytica strain HM1:IMSS varied from 44 to 54 per cent. This method is useful for isolating clones from different strains of the parasite for molecular and immunological studies.