(‒)-Epicatechin reveals amoebicidal activity against Acanthamoeba castellanii by activating the programmed cell death pathway.

BACKGROUND: Acanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (‒)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled. METHODS: The amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated. RESULTS: EC revealed amoebicidal activity against A. castellanii trophozoites with an IC50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation. CONCLUSION: EC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections.

The amoebicidal effect of Torreya nucifera extract on Acanthamoeba lugdunensis.

As the number of contact lens users increases, contact lens induced corneal infection is becoming more common. Acanthamoeba keratitis (AK) is a type of those which is caused by Acanthamoeba species, and may cause severe ocular inflammation and visual loss. We evaluated whether Torreya nucifera (T. nucifera) extract has an anti-amoebic effect and studied its mechanism of action on Acanthamoeba lugdunensis (A. lugdunensis). Cell viability was tested using the alamarBlue™ method, and the cell death mechanism was confirmed using the Tali® Apoptosis Kit. The SYTOX® Green assay was performed to check the plasma membrane permeability. The JC-1 dye was used to measure the mitochondrial membrane potential. A CellTiter-Glo® Luminescent Assay was used to measure the adenosine-triphosphate (ATP) level. Morphological changes in the mitochondria were examined by transmission electron microscopy (TEM). Cystic changes and a decrease in cell viability after treatment with T. nucifera were observed. Both apoptotic and necrotic cells were found in the Tali® Apoptosis assay. There was no significant difference in plasma membrane permeability between the control and T. nucifera treated groups. The collapse of the mitochondrial membrane potential and reduced ATP level in A. lugdunensis was confirmed in the groups treated with T. nucifera. Structural damage to the mitochondria was observed on TEM in the groups treated with T. nucifera. T. nucifera showed an anti-amoebic effect on A. lugdunensis, by inducing the loss of mitochondrial membrane potential. Thus, it could be a future therapeutic agent for AK.

Acanthamoeba Keratitis: an update on amebicidal and cysticidal drug screening methodologies and potential treatment with azole drugs.

Introduction: Acanthamoeba encompasses several species of free-living ameba encountered commonly throughout the environment. Unfortunately, these species of ameba can cause opportunistic infections that result in Acanthamoeba keratitis, granulomatous amebic encephalitis, and occasionally systemic infection.Areas covered: This review discusses relevant literature found through PubMed and Google scholar published as of January 2021. The review summarizes current common Acanthamoeba keratitis treatments, drug discovery methodologies available for screening potential anti-Acanthamoeba compounds, and the anti-Acanthamoeba activity of various azole antifungal agents.Expert opinion: While several biguanide and diamidine antimicrobial agents are available to clinicians to effectively treat Acanthamoeba keratitis, no singular treatment can effectively treat every Acanthamoeba keratitis case.Efforts to identify new anti-Acanthamoeba agents include trophozoite cell viability assays, which are amenable to high-throughput screening. Cysticidal assays remain largely manual and would benefit from further automation development. Additionally, the existing literature on the effectiveness of various azole antifungal agents for treating Acanthamoeba keratitis is incomplete or contradictory, suggesting the need for a systematic review of all azoles against different pathogenic Acanthamoeba strains.

Clinical study on the efficacy of Amoebex (coded herbal drug) compared with Metronidazole for the treatment of Amoebic dysentery.

Amoebiasisis an infectious disease, which originated with the single-celled parasitic protozoan Entamoeba histolytica. The parasitic amoeba infects the liver and intestine and may cause mild diarrhea and serious dysentery with bloody and mucoid stool. A study was conducted to evaluate the efficacy of Amoebex (400mg), a herbal formulation for the treatment of amoebiasis infections as compared to that of Metronidazole (400mg). The therapeutic evaluations of these medicines were carried out on 184 clinically diagnosed cases of the amoebiasis infection. Sample sizes of Ameobex for this study included a total of 93 patients and for Metronidazole a total of 91 were registered and treated. Comparison of the data recorded for the participants relating to sign and symptoms variables showed significant differences of efficacy between test and control groups (p<0.0357) and no side effects were at all recorded in test group. According to observation, there was a difference in the overall clinical success of both treatment groups, however, the efficacy of the test treated medication (Amoebex) was superior to that of Metronidazole as (p<0.03), and on the basis of the statistical analysis done by the chi square test, the null hypothesis was rejected. `It is clearly evident that Amoebex possesses therapeutic value for the treatment of amoebiasis associated symptoms but also the eradication rate of amoebiasis is superior by Amoebex as compared to that of Metronidazole (Control drug).

Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis.

Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.

[Diabetic foot infections. Prevalence and antibiotic sensitivity of the causative microorganisms]. / Infecciones del pie diabético. Prevalencia de los distintos microorganismos y sensibilidad a los antimicrobianos.

BACKGROUND: Foot infections are a common reason for hospitalization and a cause of complications in patients with diabetes. The aim of this study was to determine the prevalence of microorganisms found on culture in complicated diabetic foot infections in hospitalized patients, and the sensitivity of the causative microorganisms to antimicrobial agents. METHODS: Between December 2001 and December 2005 in our department, 84 samples in 62 diabetic patients with moderate/severe infection were collected for microbiological study. RESULTS: At least one microorganism was isolated in 88% of samples. The most frequently isolated germ group was gram-positive bacteria (55% of the samples), with Staphylococcus aureus (33%) in the first position, followed by Pseudomonas aeruginosa (12%), Enterococcus spp. (9%), and Escherichia coli (8%). Culture for anaerobic microorganisms was only performed in half the samples; 25% were positive, and Peptostreptococcus spp. predominated. Among the multiresistant microorganisms, methicillin-resistant staphylococci aureus (MRSA) were the most common, accounting for 38% of the isolated strains of S. aureus, ie, 12% of all samples. As to the gram-negative microorganisms, nearly 30% of E. coli strains were resistant to amoxicillin/clavulanic acid and ciprofloxacin. CONCLUSION: Most of the cultures in our study were monomicrobial, with S. aureus being the most prevalent microorganism, followed by enterobacteria and P. aeruginosa. The main resistant microorganism in diabetic foot infections requiring hospitalization was methicillin-resistant golden staphylococcus, which was found in 12% of the series.

Therapeutic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri.

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

Cutaneous leishmaniasis.

Cutaneous leishmaniasis is a widespread tropical infection caused by numerous different species of Leishmania protozoa that are transmitted by sandflies. Its clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood. Diagnosis should aim to identify the exact species involved, but this requires laboratory investigations that are not widely available. No single ideal treatment has been identified, and those available are limited by variable success rates and toxicity. Clinical guidelines are needed to make better use of the investigations and treatments that do exist. Prevention is currently limited to bite prevention measures.

Antiamoebic activity of iridoids from Morinda morindoides leaves.

An aqueous decoction (dried extract), an 80 % methanolic extract from Morinda morindoides (Rubiaceae) leaves, and five iridoids isolated from the 80 % methanolic extract were evaluated in vitro for their activity against Entamoeba histolytica and their cytotoxicity. The aqueous decoction and the 80 % methanolic extract exhibited a promising antiamoebic activity with IC (50) values of 3.1 +/- 1.7 and 1.7 +/- 0.6 microg/mL, respectively. All tested iridoids displayed antiamoebic activity, the most active being epoxygaertneroside (IC (50): 1.3 +/- 0.4 microg/mL) and methoxygaertneroside (IC (50): 2.3 +/- 0.7 microg/mL) followed by gaertneroside, acetylgaertneroside and gaertneric acid with IC (50) values of 4.3 +/- 1.8, 5.4 +/- 1.4 and 7.1 +/- 1.4 microg/mL, respectively. Synergistic effects between the iridoids tested, or with other constituents, may explain the high activity of the extracts. All extracts and iridoids were devoid of any cytotoxic effect against MT-4 cells at the highest test concentration of 250 microg/mL. These findings support at least in part the traditional use of Morinda morindoides leaves for the treatment of amoebiasis in the Democratic Republic of Congo.

Activity of isoflavans of Dalea aurea (Fabaceae) against the opportunistic ameba naegleria fowleri.

One new and one known isoflavan, 3 S(+)-7-methoxymanuifolin K (1) and manuifolin K (2), respectively, were isolated from methanolic extracts of Dalea aurea (Fabaceae). Isoflavans 1 and 2 exhibited significant in vitro activity against the ameba Naegleria fowleri, an organism responsible for an infrequent but rapidly fatal form of primary amebic meningoencephalitis (PAM). At concentrations of 30 microM, both 1 and 2 caused growth inhibition of N. fowleri at a level comparable to amphotericin B (at 0.1 microM), the currently preferred treatment for this disease. Over a seven-day growth period, 1 and 2 (30 microM) exhibited superior growth inhibition of N. fowleri than amphotericin B after day 4. Isoflavan 2 was evaluated in a mouse model of PAM at a dose of 25 mg/kg/day for five days. While amphotericin B (2.5 mg/kg/day) offered 12.5 % protection of the mice, compound 2 did not protect the mice from PAM infection compared to controls.

Effects of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall on caecal amoebiasis in mice.

The anti-amoebic effects of crude methanol extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall against Entamoeba histolytica infecting the caecum of mice were studied. Caecal amoebiasis in mice was induced by injection of Entamoeba histolytica trophozoites directly into the caecum. The mice were then treated orally with the extract, a standard drug (metronidazole), or vehicle p.o. for five consecutive days, beginning 24 h after the infection and were examined on the sixth day. At a dose of 1000 mg/kg per day, the extracts of Piper longum fruit, Piper sarmentosum root and Quercus infectoria nut gall had a curative rate of 100, 40 and 26%, respectively. At a concentration of 500 and 250 mg/kg/day, extract from Piper longum fruit was still effective in 93 and 46% of the cases, respectively, while extract from Piper sarmentosum root at a dose of less than 1000 mg/kg per day did not cure any mice from amoebiasis. Extract of Quercus infectoria nut gall at a concentration of 500 and of 250 mg/kg per day cured 26 and 13% of mice, respectively. Metronidazole at a concentration of 125 and of 62.5 mg/kg per day had a curative rate of 100 and 60%, respectively. The severity of caecal wall ulceration was reduced in mice which received the extract and metronidazole as compared to the control animals.

Common intestinal parasites.

Intestinal parasites cause significant morbidity and mortality. Diseases caused by Enterobius vermicularis, Giardia lamblia, Ancylostoma duodenale, Necator americanus, and Entamoeba histolytica occur in the United States. E. vermicularis, or pinworm, causes irritation and sleep disturbances. Diagnosis can be made using the "cellophane tape test." Treatment includes mebendazole and household sanitation. Giardia causes nausea, vomiting, malabsorption, diarrhea, and weight loss. Stool ova and parasite studies are diagnostic. Treatment includes metronidazole. Sewage treatment, proper handwashing, and consumption of bottled water can be preventive. A. duodenale and N. americanus are hookworms that cause blood loss, anemia, pica, and wasting. Finding eggs in the feces is diagnostic. Treatments include albendazole, mebendazole, pyrantel pamoate, iron supplementation, and blood transfusion. Preventive measures include wearing shoes and treating sewage. E. histolytica can cause intestinal ulcerations, bloody diarrhea, weight loss, fever, gastrointestinal obstruction, and peritonitis. Amebas can cause abscesses in the liver that may rupture into the pleural space, peritoneum, or pericardium. Stool and serologic assays, biopsy, barium studies, and liver imaging have diagnostic merit. Therapy includes luminal and tissue amebicides to attack both life-cycle stages. Metronidazole, chloroquine, and aspiration are treatments for liver abscess. Careful sanitation and use of peeled foods and bottled water are preventive.

Potential antiamoebic property of the roots of Piper longum Linn.

In an earlier study we reported the effect of ethanol extracts of Piper longum fruit against caecal amoebiasis in rats. A follow-up study to determine whether the roots possess similar antiamoebic activity was carried out. The ethanolic extract of roots was amoebicidal at 1,000 microg/mL in vitro and cured 88% of caecal amoebiasis cases, suggesting that both the root and fruit of P. longum possess antiamoebic activity to approximately the same extent.

[Therapeutic evaluation of artesunate in experimental Schistosoma mansoni infection]. / Avaliação terapêutica do artesunato na infecção experimental pelo Schistosoma mansoni.

Mice experimentally infected with Schistosoma mansoni were treated orally with artesunate (Lactab) in a single dose of 300 or 500 mg/kg or over a period of five consecutive days. The animals were sacrificed 7, 30, 60 or 90 days after treatment. Statistically significant differences were found in the distribution and mortality of the worms and in the alterations of the oogram in the treated group when compared to control in all of the tested schemes when the animals were sacrificed 30 days after treatment. Morphological analysis of female worms showed a reduction of ovarian volume and rarefaction of the vitelline follicles. These modifications were more marked after treatment with the higher dose, explaining the alteration of the oogram which reached 100%. However, when the animals were sacrificed 60 or 90 days after treatment, the differences and alterations were smaller, showing that the surviving worms recovered and restarted oviposition.

Effect of alpha,beta-arteether against primary amoebic meningoencephalitis in Swiss mice.

Artemisinin and its derivative alpha, beta-arteether have been evaluated for activity against experimental primary amoebic meningoencephalitis. In vivo experiments have shown that amphotericin B at dose of 2.5 mg/kg for 5 days produced 100% protection. Artemisinin and alpha, beta-arteether, even when tested at a high doses (60-120 mg/kg x 5 days and 90-180 mg/x 5 days) respectively, were not curative and showed only slight protection as indicated by extension of mean survival time.

Antiamoebic activity of 3,3'-fluro-4,4'-di-(pyrrolidine-2-ylidene amino)-diphenyl (liroldine), against experimentally infected intestinal and hepatic amoebiasis.

HL 707, Liroldine, a novel synthetic compound, was found effective against both extraintestinal and intestinal amoebiasis in animal models. Its activity against hepatic infection in golden hamsters is comparable with that of different derivatives of nitroimidazoles used for human treatment. Against intestinal amoebiasis in Wistar rats, the activity was superior to nitroimidazoles and chloroquine. Paramomycin was comparable and diloxanide furoate was marginally superior. The comparative in vitro and in vivo studies with standard marketed drugs and Liroldine indicate an excellent profile of the compound against experimental amoebiasis. LD50 of Liroldine determined in mice is 910 mg/kg x 1, po and 940 mg/kg x 1 ip).

In vivo study of artemisinin and its derivatives against primary amebic meningoencephalitis caused by Naegleria fowleri.

Artemisinin and its derivatives, beta-arteether and sodium artesunic acid, have been evaluated for activity against experimental primary amebic meningoencephalitis and the efficacy of these compounds has been compared with that of the standard drug amphotericin B. In vivo experiments in Swiss mice have shown that amphotericin B at a dose of 2.5 mg/kg for 5 days produced 100% protection in the mice infected intranasally with Naegleria fowleri. Artemisinin, beta-arteether, and sodium artesunic acid, even when tested at high doses (60-180 mg/kg x 5 days), were not curative and showed only slight protection as indicated by extension of mean survival time.

Amoebicidal compounds from medicinal plants.

A series of the natural constituents with amoebicidal activity isolated from several medicinal plants is shown. A list of the medicinal plants potentially active as amoebicide and/or against dysentery also is demonstrated. The present data grouping of the natural compounds and medicinal plants can be an important source of information for the selection of research plant material by the investigators interested in the discovery of new biologically active compounds as amoebicide.