Sensibilidad de trofozoítos de Entamoeba histolytica a ivermectina / Sensibility of Entamoeba histolytica trophozoites to ivermectin

La amibiasis producida por Entamoeba histolytica es un problema de salud pública. Las formas clínicas más frecuentes son la disentería y el absceso hepático amibiano. En el mundo se notifican anualmente 50 millones de casos y más de 100 000 muertes por esta enfermedad. El ciclo de vida de E. histolytica tiene dos fases: trofozoíto y quiste. Los trofozoítos son los responsables de producir enfermedad. El tratamiento actual para la amibiasis incluye medicamentos con efectos colaterales serios. La ivermectina es un macrólido con actividad contra endoparásitos y ectoparásitos causantes de strongiloidosis, filariasis, oncocercosis, sarna y pediculosis. Su uso está extendido a casi todo el mundo y se lo reconoce como un medicamento seguro. El objetivo de este trabajo fue determinar la sensiblidad in vitro de trofozoítos de E. histolytica al tratamiento con ivermectina. Para determinar su sensibilidad a la droga, se utilizaron trofozoítos de E. histolytica cultivados en medio PEHPS. Durante su fase de crecimiento logarítmico se expusieron a diferentes concentraciones de ivermectina. Como controles se usaron otras drogas antiparasitarias. Se prepararon diluciones seriadas de cada droga, luego se agregaron a tubos con parásitos (2 x 10(4) células/ml). Se incubó por 72 h y luego se determinó el porcentaje de inhibición de crecimiento calculado por análisis Probit. La ivermectina tiene actividad contra trofozoítos de E. histolytica. La dosis de ivermectina que produjo el 50% de inhibición de crecimiento fue de 6.40 mg/ml. Esta dosis fue mayor a la encontrada con otras drogas antiparasitarias. Falta demostrar su actividad in vivo en modelos animales.

Amebiasis caused by Entamoeba histolytica is a problem of public world health. The most frequent clinical presentation are the dysentery and the amebic liver abscess. Fifty millions of cases and more than 100.000 deaths for this disease are reported annually worldwide. The life cycle of E. histolytica has two phases: trophozoite and cyst. Trophozoites are the causal agent of disease. The effective treatment for the amebiasis includes drugs with serious collateral effects. Ivermectin is a macrolid with activity against endoparasites and ectoparasites causing strongiloidosis, filariasis, oncocercosis, scabiasis and pediculosis. The use of ivermectin has been extended almost worldwide; it is recognized as a safe drug. The main objective of this study was to determine in vitro sensibility of trophozoites of E. histolytica was to the treatment with ivermectin. To determine the sensibility of the parasites to the drug, E. histolytica was cultivated in PEHPS medium. During its logarithmic growth phase the trophozoites were exposed to different concentrations of ivermectin. As controls other antiparasitic drugs were used. For each drug, serial dilutions were prepared, and mixed in culture tubes with parasites (2 x 10(4) cells/ml). They were incubated for 72 h and then the percentage of growth inhibition was calculated by Probit analysis. Ivermectin showed activity against trophozoites of E. histolytica. The 50% of growth inhibition of ivermectin was 6.40 mg/ml. This dose was higher than for other anti parasitic drugs. Its activity in vivo in animal models remains to be demonstrated.

Effect of Iron on Adherence and Cytotoxicity of Entamoeba histolytica to CHO Cell Monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.

Effect of Iron on Adherence and Cytotoxicity of Entamoeba histolytica to CHO Cell Monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.

Inhibitory Effects of Iranian Thymus vulgaris Extracts on in Vitro Growth of Entamoeba histolytica

One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25 degrees C for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica.

Effects of antacid therapy in asymptomatic patients with presence of amoeba in stool report.

Amoebic infection caused by the protozoan parasite Entamoeba histolytica is a prevalent infection in the developing countries. Milder form of this infection is associated with loose stool, flatulence and borborygmi, may or may not be associated with pain in abdomen and treated symptomatically by some physicians by antacid. To find out the effects of antacid (sorbacid) therapy in patients with amoeba in stools by examining the changes in the stool report, a study was conducted among 25 patients enrolled in the study with complaints of "gas" in the abdomen with stool reports positive for amoeba. Antacid (sorbacid) in a dose of one teaspoonful (5 ml) was given three times a day for 3 days and stool examination was repeated. The report showed a significant reduction in the amoeba and mucus in stool (p<0.05) and a trend towards reduction in the presence of occult blood. Other parameters in stool reports did not change. Moreover, all the patients gave the history of passing formed stools and no complaints of "gas" in abdomen thus providing the symptomatic benefit. Antacids may have some beneficial effects in amoebiasis. More studies are required to confirm the above finding and to find out the place of antacid as an adjuvant therapy along with the standard anti-amoebic drugs.

Multidrug resistance in amoebiasis patients.

BACKGROUND & OBJECTIVES: Amoebiasis, caused by Entamoeba sp. a protozoan parasite, is a major public health problem in tropical and subtropical countries. The symptomatic patients are treated by specific chemotherapy. However, there are reports of treatment failure in some cases suggesting the possibility of drug resistance. The present study was therefore planned to assess the presence and expression of mRNA of multidrug resistance (MDR) gene in clinical isolates of Entamoeba histolytica and E. dispar. METHODS: Forty five clinical isolates of Entamoeba sp. [E. histolytica (15) and E. dispar (30)] were maintained in polyxenic followed by monoxenic medium. DNA and total RNA were extracted from clinical isolates of Entamoeba sp. and from sensitive strain of E. histolytica (HM1: IMSS) and subjected to polymerase chain reaction (PCR) and multiplex reverse transcription (RT)-PCR techniques. RESULTS: The 344 bp segment of E. histolytica DNA was seen by PCR using primers specific to EhPgp1 in all clinical isolates and sensitive strain of E. histolytica. Over expression of EhPgp1 was observed only in resistant mutant of E. histolytica; however, transcription of EhPgp1 was not seen in any clinical isolates and sensitive strain of E. histolytica. INTERPRETATION & CONCLUSION: The findings of the present study indicate that, so far, drug resistance in clinical isolates of E. histolytica does not seem to be a major problem in this country. However, susceptibility of clinical isolates of E. histolytica against various antiamoebic drugs needs to be investigated for better management.

Drug resistance in amoebiasis.

Amoebiasis caused by Entamoeba histolytica, is a major public health problem in developing countries. Morphologically similar E. dispar is non pathogenic. Because of the redefinition of E. histolytica and E. dispar, and the limited number of antiamoebic drugs available, a new approach to treat such individuals is necessary. The cost of treating asymptomatic individuals is highly exorbitant and not justifiable. The indiscriminate use of antiamoebic drugs can result in increased minimum inhibitory concentration (MIC) values against Entamoeba species, and treatment failure may emerge as an important public health problem. Development of new antiamoebic drugs is still in infancy and vaccine development appears to be distant dream. In future, the development of drug resistance may seriously affect the control of disease. This review discusses the factors involved in drug resistance mechanisms developed by the parasite.

Massive amebic liver abscess--an unusual presentation.

A 40-year-old woman presented with a huge lump in the right half of the abdomen and irregular menses. Ultrasonography showed a cystic lump with septations, extending from the upper abdomen to the pelvis; the right ovary was not seen. On exploration, there was a large cyst arising from the right lobe of the liver; the aspirate was bilious. Since the cyst wall was adherent to retroperitoneal structures, complete excision was not possible. A roux-en-Y loop of jejunum was anastomosed to the cyst wall. Biopsy of the wall showed inflammatory granulation tissue with trophozoites of Entamoeba histolytica. She was treated with metronidazole, and recovered uneventfully.

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.

Comparative study of the efficacy and tolerability of Secnidazole suspension (single dose) and Tinidazole suspension (two days dosage) in the treatment of amebiasis in children

Amebiasis caused by Entamoeba histolytica may be considered the most aggressive parasitic disease affecting human intestine, causing acute amoebic colitis and extra-intestinal diseases of high morbidity and mortality. 5-nitroimidazoles are the drugs of choice. In this multicenter, open and randon clinical trial, the efficacy and tolerability of secnidazole suspension in a single oral dose of 1ml/kg was compared with 0.5ml/kg doses of tinidazole suspension given for 2 consecutive days to 303 Entamoeba histolytica-positive children aged 2 to 13. Patients with extra-intestinal complications were excluded from the study. Clinical and parasitological follow-up using the Faus and Kato-Katz method were carried out 7, 14, and 21 days after treatment. Clinical improvement/cure was observed in 93 percent of the patients in the secnidazole group and 91 percent in the tinidaloze group. Parasitological sucess was reported for 77 percent and 63 percent of the secnidazole and tinidazole patients, respectively, showing a significant statistical difference between the two groups (p=0.007). Both drugs were well tolerated, and the adverse effects reported were mild, consisting mainly of digestive disturbances. This comparative study showed that a single oral dose of 1ml/kg of secnidazole produced a significantly higher parasitological cure rate than 2 doses of tinidazole. Secnidazole is a safe and effective drug for the treatment of uncomplicated intestinal amebiasis.

Effect of bacterial association on virulence of Entamoeba histolytica to baby hamster kidney cell monolayers.

Axenic E. histolytica trophozoite strain NIH:200 and HMI:IMSS when co-associated with aerobic bacteria Escherichia coli strain K12 and serotype 056 showed marked increase in virulence as observed by destruction of baby hamster kidney (BHK) monolayers. However, when incubated with anaerobic bacterial strains Clostridium perfringens and Bacteroides fragilis virulence remained unaltered. Further, adherence of E. histolytica to BHK monolayer was found to be mediated by N-acetyl-D-galactosamine.

Tratamiento profiláctico con quinfamida para el control comunitario de las infecciones intestinales por entamoeba histolytica / Prophilactic treatment with quinfamide for the community control of the intestinal entamoebosis

Estudio realizado para evaluar la utilidad profiláctico de la quinfamida en el control de la entamoebosis intestinal a nivel comunitario. Se trabajaron comparativamente dos comunidades similares, con alta prevalencia de infección por Entamoeba histolytica, la cual fue identificada mediante coproparasitoscopía. Las tasas iniciales eran de 41,7 por ciento y de 51,2 por ciento, respectivamente. A una de ellas (A), se le administró tratamiento mensual con quinfamida, por un solo día y durante 12 meses; a la segunda (B) solamente se le desparasitó al inicio del estudio y al año. Al término del estudio se observó que en la comunidad A el número de casos de infección amebiana disminuyó conforme transcurrieron los meses de terapia, llegándose a una tasa final de 6,3 por ciento, en tanto que en la comunidad B, de control, la prevalencia permaneció en 46,0 por ciento. Se concluye que el tratamiento profiláctico con quinfamida administrada cada mes, es útil para disminuir el problema de infección por E. histolytica en comunidades

Effect of afsanteen [artemisia absinthium linn.] in acute intestinal amoebiasis

Amoebiasis is the common intestinal ailment of human beings met with in clinical practice. It is usually a chronic disease often associated with loose motions, abdominal pain and mucous discharge with stool. In the Unani System of Medicine, there are many mufrid or murakkab drugs which are said to be beneficial in this disease. Afsanteen [Artemisia absinthium Linn.] is one of them. In the present study the drug has given about 84.66% overall relief in different clinical stages with disappearance of E. histolytica in 70% of cases, without any notable adverse effect

Physiology and molecular biology of multidrug resistance in Entamoeba histolytica

In this paper, we present the most relevant facts on multidrug resistance (MDR) in the protozoan parasite Entamoeba histolytica. MDR in E. histolytica presents characteristics similar to transformed mammalian cells. E. histolytica drug resistant mutants show cross-resistance to several drugs, and as in mammalian cells the resistance is reverted by verapamil. Six P-glycoprotein-like genes (EhPgp) have been cloned and characterized. Apparently, four of thses genes are transcribed in drug-resistance mutants (EhPgh1, EhPgp2, EhPgp5 and EhPgp6), although only Egpgp1, EhPgp5 and EhPgp6 transcripts were clearly detected. The open reading frame (ORF) of the four completely full length genes is about 1300 amino acids long. EhPgp1, EhPgp2 and EhPgp5 have between 64 and 67 percent of positional identity among them, while EhPgp6 shows 38 to 46 percent positional identity to the other ameba genes. Insterestingly, the phylogenetic tree suggested that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins. Differential gene expression in drug-resistant mutants was detected when specific probes for each EhPgp gene were used. To understand the differential expression of EhPgp genes we initiated the characterization of the upstream flanking regions of EhPgp1 and EhPgp5 genes. Upstream sequences showed between 53 and 66 percent of positional identity to Dictyostelium discoideum promoters

Estudio comparativo entre quinfamida y etofamida en el tratamiento de la infección amibiana asintomática / Comparative study in quinfamide and etofamide in the treatment of asymptomatic amoebic infection

Se realizó un estudio para evaluar la eficacia de la quinfamida y de la etofamida en el tratamiento de la infección amibiana asintomática. Se estudiaron 655 niños con coproparasitoscópicos por concentración en serie de tres días consecutivos. En 501 niños se encontraron quistes de Entamoeba histolytica (76.48 por ciento). Con estos niños de integraron dos grupos, en forma aleatoria; el primero recibió quinfamida y el segundo se le administró etofamida. Se realizaron coproparasitoscópicos al concluir el tratamiento. En el grupo de lka quinfamida se obtuvo una eficacia de 89.3 por ciento y en la etofamida de 89.9 por ciento (p>0.05). Se concluye que la quinfamida y la etofamida son buenas opciones para el manejo del portador sano de Entamoeba histolytica

Effect of histamine and glucose on encystation of Entamoeba histolytica in vitro.

Isolates of Entamoeba histolytica grown and maintained in modified Boeck and Drbohlav's medium were used to examine the effect of histamine and glucose on encystation process in vitro. This revealed that 60-74% of the active and motile trophozoites of E. histolytica encysted within 72 hours in the presence of histamine and glucose, in contrast to 10-20% encystment in controls (with out histamine and glucose); a highly significant difference (P < 0.001). These observations also suggest the possibility of active role of histamine and glucose in encystation of E.histolytica in vivo.

Modulation of the chemotherapeutic action of some amoebicidal drugs by tin-N-phenylimin chloride

Tin-N-phenylimin chloride was tested for its amoebicidal effect on Entamoeba histolytica in vitro and in vivo. Its effects were investigated in combination with metronidazole or bacitracin and compared to the sole effects of each of these drugs or bacitracin zinc. Tin-N-phenylimin chloride or bacitracin zinc was more effective amoebicide than metronidazole or bacitracin on E. histolytica culture. Tin-N-phenylimin chloride synergized the amoebicidal effects of metronidazole or bacitracin to 1.5 and 7.9 times, respectively. Minimal inhibitory and minimal lethal concentrations [MICs, MLCs] were increased in axenic as compared to xenic cultures reaching 1.44-3.5 and 1.05-4.68 times, respectively. The tested drug regimens exhibited a direct amoebicidal effect on E. histolytica in vitro. Clinical effects of these drug regimens were directly proportional to the duration of treatment, reaching a full cure rate five days after treatment. These data indicated that amoebic dysentery cases were advantageously treatable with bacitracin zinc, tin-N-phenylimin chloride or a combination of the latter with metronidazole or bacitracin

In vitro effect of 5-hydroxytryptamine on concanavalin-A agglutinability and multiplication of Entamoeba histolytica.

Six isolates of E. histolytica isolated and maintained by serial passage in modified Boeck and Drbohlav's medium were used in this study. When five of the isolates were grown in the above medium with 5-hydroxytryptamine (5-HT) added to the overlay, the agglutinability of the amoebae by concanavalin- A (con-A) was significantly increased compared to corresponding control cultures. Four isolates of E. histolytica grown with 5-HT had 1.5 to 2 times higher counts than the control cultures. Similarly the con-A agglutinability and counts of cultures of NIH-200 (an axenic strain of E.histolytica) were enhanced when grown in association with 5-HT.

Immunogenicity of detergent membrane proteins of Entamoeba histolytica.

The lymphokine release and antibody production were assessed in the peripheral blood of 52 and 48 cases of amoebic liver abscess respectively, by employing detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200). Lymphokine release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was performed by leukocyte migration inhibition test. A highly increased release of LMIF and 100 per cent positivity was observed with DDMP where as the same for whole amoebic extract, was only 73 per cent. The difference between the means of the above two values with regards to release of LMIF, was found to be highly significant (P less than 0.005). Antibodies production in response to both DDMP and whole amoebic lysate was performed by indirect haemagglutination assay on blood samples from amoebic liver abscess cases. A 53 folds increased titres of IHA and cent percent positivity was observed with DDMP compared to WAL. The difference between mean titres of the above two with regards to detection of antibodies, was found to be highly significant (P less than 0.001). This shows that the patients, had high degree of leukocyte sensitization and production of antibodies which will not be assessed simply with WAL. These findings suggest that the shed material might have important role as a potent antigen in elicitation cell mediated and humoral immune response in amoebic liver abscess cases.