Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target

BACKGROUND Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown. OBJECTIVE To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound. METHODS We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog. RESULTS KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog. CONCLUSIONS KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Efectividad del Oleozón® en el tratamiento de niños y adolescentes con giardiasis / Effectiveness of Oleozón® in the treatment of children and adolescents with giardiasis

Se realizó un ensayo clínico en fase IV para evaluar la efectividad del Oleozón® aplicado por vía oral en niños y adolescentes con giardiasis, atendidos en la interconsulta de Pediatría del Policlínico Universitario Julián Grimau García de Santiago de Cuba, desde enero hasta diciembre del 2015. El universo estuvo constituido por 116 pacientes (con igual número de casos y controles) en las edades de 1 a 18 años, infectados por Giardia lamblia, quienes presentaban dolor abdominal, vómito y anorexia como síntomas fundamentales. En la serie se obtuvo que el mayor número de los pacientes del grupo experimental, cuyo esquema terapéutico incluía el Oleozón®, resolvió el dolor abdominal y mejoró su estado general al disminuir el resto de los síntomas. Pudo concluirse que el Oleozón® resultó efectivo en el tratamiento contra la giardiasis, pues las muestras de heces fecales dieron negativo en todos los casos; además, no se produjeron reacciones adversas y su costo es bajo, lo que conlleva un impacto económico, social y medioambiental

A clinical assay in phase IV was carried out to evaluate the effectiveness of oral Oleozón® used in children and adolescents with giardiasis, assisted in the Pediatrics specialty consultation from Julián Grimau García University Polyclinic in Santiago de Cuba, from January to December, 2015. The universe was constituted by 116 patients (with the same number of cases and controls) and aged 1 to 18 years, infected by Giardia lamblia who presented abdominal pain, vomit and anorexy as fundamental symptoms. In the series it was obtained that the highest number in the patients of the experimental group whose therapeutic schedule included Oleozón® , solved the abdominal pain and it improved their general state when decreasing the rest of the symptoms. It could be concluded that the Oleozón® was effective in the treatment against giardiasis, because the samples of faeces were negative in all cases; besides, it produced no adverse reactions and its cost is low, what bears an economic, social and environmental impact

[Antiprotozoal potential of Anethum graveolens and Punica granatum plant extracts against Giardia Lamblia]

Giardiasis is worldwide spread disease caused by the protozoan Giardia species. Its treatment includes drugs which may produce side effects and, in addition, onset of chemical resistance of this pathogenic protozoan. With the purpose of searching for new natural anti-protozoal chemotherapy, Anethum graveolens and Punica granatum two plants of the family Apiaceae, and Lythraceae respectively,are used in traditional medicine against intestinal disorders were selected to evaluate their petroleum[pet.] ether, ethyl acetate, methanol and aqueous leaves extracts activity against G. lamblia trophozoite. petroleum[pet.] ether extract from -A. graveolens was strongly active against G. lamblia [IC[50] - 30.807 ml[-1]] and no activity of P. granatum. Ethyl acetate extracts from A. graveolens and P. granatum showed no activity against G. lamblia. Methanol extracts from A. graveolens showed a good activity against G. lamblia [IC[50] = 11.248 mg ml[-1]] and a strong activity of P. granatum [IC[50] = 0.719 mgl[-1]]. The aqueous extracts from A. graveolens showed no activity against G. lamblia and strong activity of P. granatum [IC50 = 0.12 mg] Such results indicate A. graveolens methanol extracts and the aqueous extracts from P. granatum as possible candidates for further investigations to isolate and characterize their active principles as possible natural anti-protozoal agents

Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability. .

[Anti-giardia activity of carum copticum on giardia lamblia cysts in vitro]

Giardiasis, a wide-spread, small intestine parasitic infection, is one of the main causes of human diarrhea, especially in young children. The prevalence of Giardia lamblia was reported to be between 1 and 25 percent; however, it is considerably higher under poor sanitary conditions. Metronidazole is used for the treatment of Giardiasis but it may have undesirable side effects. Carum copticum, a medicinal herb, has been used for its antibacterial, antifungal, and antiprotozoal effects for centuries. The present study was conducted to evaluate the in vitro effects of alcoholic extract, and essential oil from Carum copticum on Giardia lamblia. In this experimental study, Giardia lamblia cysts were incubated with the two different extracts of Carum copticum under standard laboratory conditions. After 60 minutes Minimum Inhibitory Concentrations [MIC] of Carum copticum alcoholic extracts and essential oil were 100 mg and 8 mg/ml, respectively. After 120 minutes, MIC of Carum copticum alcoholic extracts and essential oil were 75 mg and 6 mg/ml, respectively; and after 180 minute MIC of Carum copticum alcoholic extracts and essential oil were 75 and 4 mg/ml, respectively. Findings of this study indicate that Carum copticum is effective against Giardia lamblia in vitro

Giardiacidal activity of lemon juice, vinifer and vinegar on Giardia intestinalis cysts.

The giardiacidal efficacy of simple disinfecting materials, ie lemon juice, vinifer, and vinegar, for uncooked foods with Giardia cysts was investigated to help travelers in Giardia-endemic areas. The cysts were obtained from stools of individuals with Giardia intestinalis infection by modified sucrose gradient procedure. A pooled batch of 3 x 10(4)/ml Giardia cysts was made from all specimens. The cysts were kept at 4 degrees C until use. Before each experiment, the number of cysts was determined by hemocytometer. Two sets of Eppendorf tubes were used for the experiments, one set at 4 degrees C and one at 24 degrees C. One thousand microliters each of lemon juice, vinifer, or vinegar was poured into each tube, and 1,000 microl of Giardia cysts were added. Variables were disinfectant materials, temperature, and time of exposure. Cyst viability 140 was determined by eosin inclusion procedure. Viability of at least 250 cysts in each tube at 0, 0.5, 1, 2 and 3 hours after the beginning of the experiments was determined. The mean giardiacidal activity at 4 degrees C after 3 hours for lemon juice, vinifer, and vinegar was 18.9, 12.8, and 28.4%, and at 24 degrees C, 28.3, 16.2, and 40.6%, respectively. In conclusion, the giardiacidal activity of vinegar was more than the other materials, and as exposure time and temperature increased, giardiacidal activity also increased; the highest giardiacidal activity of vinegar was at 3-hours exposure at 24 degrees C.

Comparison of metronidazole and furazolidone against giardia lamblia in children

This study was conducted on children infected by Giardia lamblia to evaluate the effectiveness and side effects of Metronidazole and Furazolidone drugs in Kashan. One hundred twenty two individuals infected with Giardia lamblia were assigned in 2 groups. They were 5-12 years old. Patients were surveyed four weeks following the therapy with Metronidazole and Furazolidone. Results were compared using chi-square method. Of 122 infected patients, 68.8% were urban residents, 43.4% were females and 56.6% were males. Abdominal pain was the most common clinical symptoms [84.2%]. Four weeks after the therapy, the efficacy of metronidazole and furazolidone were 87 and 81.6%, respectively. Malaise [12.9%] and dark urine [25%] was the most frequents side effects of metronidazole and furazolidone, respectively. With respect to the efficacy of antiparasite drugs in the treatment of giardiasis, further studies in different parts of the country are highly recommended

Eficacia y seguridad de mebendazol contra nitazoxanida en el tratamiento de Giardia lamblia en niños / Efficacy and safety of mebendazole versus nitazoxanide in the treatment of Giardia lamblia in children

Objetivo: comparar la eficacia y seguridad de mebendazol contra nitazoxanida en el tratamiento de Giardia lamblia en niñosLa giardiasis es una protozoosis intestinal de distribución mundial que afecta con mayor frecuencia a la población infantil. En México se ha encontrado una frecuencia de tres a 60 por ciento. Se han utilizado diferentes fármacos para su tratamiento, pero la experiencia con mebendazol y nitazoxanida es escasa..Método: estudio experimental tipo ensayo clínico. Se incluyeron niños de cuatro a doce años de edad, que tuvieran un estudio de heces positivo para quistes de Giardia lamblia. Los niños se dividieron en dos grupos: A, se les administró mebendazol 100 mg cada 12 h, por tres días. B, recibieron nitazoxanida 100 mg cada 12 h, por tres días. Se les realizaron estudios de heces de control a los tres, cinco y siete días posterior al tratamiento. Al final del tratamiento se les preguntó a los padres si los niños habían presentado reacciones secundarias durante la administración del medicamento. Para el análisis estadístico se utilizó la t de Student's y chi cuadradaResultados: se estudiaron 82 niños, 41 (50 por ciento) para cada grupo. En el grupo A, los estudios de heces de una control fueron negativos en 33 para dar una eficacia de 80.4 por ciento; en el B, fueron negativos en 32 para dar una eficacia de 78.0 por ciento, sin ser estadísticamente significativo con una p = 0.8. Las reacciones secundarias fueron encontradas en 9 (22 por ciento) niños del grupo A, y 16 (39 por ciento) en el grupo B, no hubo diferencia estadísticamente significativa con una p = 0.09, sin embargo, el dolor abdominal fue encontrado con mayor frecuencia en los niños que se les administró nitazoxanida..Conclusiones: podemos concluir que tanto el mebendazol como la nitazoxanida tienen buena eficacia contra la infección por Giardia lamblia, sin embargo, las reacciones secundarias por nitazoxanida son más frecuentes que con mebendazol

Giardia lamblia: in-vitro sensitivity to some chemotherapeutic agents.

The susceptibility of Giardia lamblia to eight chemotherapeutic agents was studied in vitro. The criteria of viability of the parasite was the ability of the parasite to attach to the coverslip surface in situ, and to multiply in drug free media on subcultures. The giardicidal and 50% inhibitory concentrations of each drug were measured. The activity of metronidazole in vitro was found to be similar to that of mepacrine, whilst that of chloroquine was inferior. The sensitivity of G. lamblia to mepacrine and chloroquine is markedly greater than that reported for Entamoeba histolytica.

Efecto in vitro de prazicuantel sobre la actividad de Giardia intestinalis / In vitro effects of praziquantel over the trophozoites of Giardia intestinalis

Se realizó un estudio in vitro para conocer el efecto farmacológico de prazicuantel sobre los trofozoítos de Giardia intestinalis cepa P-1. Después de cultivarlos axénicamente, con cinco diferentes concentraciones de prazicuantel (de 0.32 hasta 1.62 nM/mL) durante 24 h a 37ºC, la viabilidad y la CI50 evaluada por la captación de colorante fluorogénico con citometría de flujo mostró un desplazamiento logarítmico de 10º a 10 a la tercera lo que correspondió a una concentración de 1.28 nM/mL. La incorporación de H3 metil-timidina como parámetro de crecimiento celular a 50 por ciento alcanzó 1.20 nM/mL de prazicuantel y el efecto citotóxico calculado por la capacidad de lisar células CHO (células de ovario de hámster chino) marcados con Cr51 se observó para la CI50 en una concentración de 1.05 nM/mL. Con estos tres parámetros se puede inferir qué viabilidad a 50 por ciento se observó a concentraciones más altas de prazicuantel comparadas con la capacidad para dividirse, así como el efecto citolítico, en donde la concentraciones más altas de prazicuantel comparadas con la capacidad para dividirse, así como el efecto citolítico, en donde la concentración de prazicuantel fue la más baja a lo que se atribuye la cualidad de apagar radicales libres, sin embargo, esto no fue proporcional al incrementar la concentración suponiéndose una importante capacidad detoxificante en Giardia. Se concluye que prazicuantel desarrolló actividad farmacológica en protozoarios, lo que le hace susceptible de emplearse contra la giardiasis

Role of nitric oxide and superoxide in Giardia lamblia killing

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (~60 per cent dead trophozoites). This effect was inhibited (>90 per cent) by an NO synthase inhibitor (200 muM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors S-nitroso-acetyl-penicillamine(SNAP)and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 muM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentration up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35 per cent dead trophozoites at 1 mM). The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite.

Effect of ivermectin on combined intestinal protozoal infection [giardiasis and cryptosporidiosis]

Ivermectin, a broad spectrum antiparasitic agent, has become a promising drug for treating parasitic infection and infestation. This study was carried out to investigate its activity against 2 common protozoal infection, giardiasis and cryptosporidiosis, in a rat model. The results of this study showed that ivermectin therapy is effective against both parasites at a dose of 200 mug/kg

Respuesta al tratamiento con secnidazol en niños parasitados por Giardia Lambia / Response to treatment with secnidazole in children infected with giardia lamblia

Se informa de los resultados del tratamiento son secnidazol de 100 niños entre uno y 12 años. El medicamento se dio a razón de 30 mg/Kg/día, por tres días. El resultado del tratamiento se valoró 10 días después en 95 niños; en ocho la G. lamblia aún estaba presente en las heces por lo que se estimó una efectividad de 84 por ciento. La manifestación indeseable al medicamento que se presentó con mayor frecuencia (36.8 por ciento) fue el ®mal sabor¼

Effect of Nigella sativa extract on experimental giardiasis

Antiprotozoal effect of alcoholic extract of Nigella sativa seeds was studied in experimental giardiasis in comparison with metronidazole drug. A single oral dose of Nigella sativa seeds alcoholic extract [40 mg/kg body weight] was found to be as effective as a single oral dose of metronidazole [40 mg/kg body weight] as both gave a cure rate of 80%. In addition, Nigella sativa seeds extract showed some protective action against further infection with giardiasis in experimental animals, while metronidazole did not

Giardia lamblia: comparación de los métodos de diagnóstico y evaluación de la respuesta al tratamiento con metronidazol / Giardia lamblia: comparison of two diagnostic methods and evaluation of response to treatment with metronidazole

Entre Marzo y Noviembre de 1989, 70 niños que acudieron a la consulta externa del Departamento de Pediatría del Hospital Central de Valencia fueron diagnosticados para giardiasis mediante 2 métodos de diagnóstico: Examen directo de heces y aspirado duodenal, y tratados con tres regímenes terapéuticos de Metronidazol, de 15, 30 y 50mg/Kg/día por 10 días. En el estudio se observó un promedio de esta parasitos en la edad preescolar (60%), con una relación del sexo femenino al masculino de 1,05:1. El 72,8% de esta serie eran eutróficos, procediendo del área suburbana 55,7% de ellos. Los síntomas predominantes fueron dolor abdominal, diarrea, vómitos, distensión abdominal, estreñimiento y flatulencia. El síntoma más frecuente en los lactantes fue la diarrea 83,3%, en los preescolares y escolares fue el dolor abdominal con 78,5 y 100% respectivamente. El examen de heces fue positivo en el 97,1% de los niños, mientras que el aspirado duodenal lo fue en el 100% de ellos; en las muestras de heces predominaron quistes de Giardia (88,2%) y en los aspirados duodenales predominaron los trofozoitos (47,1%). Todos los niños (100%) curaron con el Metronidazol, observándose efectos colaterales sólo con la dosis máxima, siendo éstos, náuseas 40%, cefalea 10% y presencia de levaduras en el 50% del aspirado duodenal

Preliminary notes on the effect of praziquantel against hymenolepis nana and giardia lamblia

Praziquantel is the most recent anti-bilharzial drug being given orally. This study is an attempt to evaluate the effect of this drug against other parasitic infections, especially Hymenolepis nana and Giardia lamblia which are common among school children in Menia Governorate. For this purpose, sixty patients harboring these parasites were selected for this trial. Forty cases of them harbored Hymenolepis nana only, ten patients harbored Giardia lamblia only, while the third group [ten patients] harbored both infection. Three schedules were applied in this trial; 20 mg/kg bw as one dose, 40 m/kg bw divided into two doses with six hours interval and 40 mg/kg bw divided into three equal doses with six hours intervals. Follow up was carried out weekly for three months duration