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Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite's interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles-derived from the plasma membrane) and small (exosomes-derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h.
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Giardia intestinalis is a pollutant of food and water, resistant to conventional disinfection treatments and its elimination requires effective methods action. Herein, mid-high-frequency ultrasound (375 kHz), which produces HO⢠and H2O2, was used as an alternative method of treatment to inactivate Giardia intestinalis cysts in water. The effect of ultrasound power (4.0, 11.2, 24.4 W) on the sonogeneration of radicals was tested, showing that 24.4 W was the condition most favorable to treat the parasite. The viability of the protozoan cysts was evaluated using the immunofluorescence technique and vital stains, showing this protocol was useful to quantify the parasite. The sonochemical method (at 375 kHz and 24.4 W) was applied at different treatment times (10, 20, and 40 min). A significant decrease in the protozoan concentration (reduction of 52.4% of viable cysts) was observed after 20 min of treatment. However, the extension of treatment time up to 40 min did not increase the inactivation. Disinfecting action was associated with attacks on the Giardia intestinalis cyst by sonogenerated HO⢠and H2O2 (which may induce structural damage, even the cell lysis). For future work is recommended to test combinations with UVC or Fenton process to enhance the inactivating action of this method.â¢Mid-high-frequency ultrasound produces HO⢠and H2O2 profitable to inactivate Giardia intestinalis.â¢Immunofluorescence technique and vital stains allowed us to quantify the parasite viability.â¢Giardia intestinalis cysts concentration decreased by 52.4% after only 20 min of sonication.
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Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a protozoa parasite that produces one of the most frequent waterborne causes of diarrhea worldwide. This protozoan infects most mammals, including humans, and colonizes the small intestine, adhering to intestinal cells. The mechanism by which G. intestinalis causes diarrhea is multifactorial, causing intestinal malabsorption. The treatment of giardiasis uses chemotherapeutic drugs such as nitroimidazoles, furazolidone, paromomycin, and benzimidazole compounds. However, they are toxic, refractory, and may generate resistance. To increase efficacy, a current treatment strategy is to combine these drugs with other compounds, such as polysaccharides. Several studies have shown that polysaccharides have gastroprotective effects. Polysaccharides are high-molecular weight polymers, and they differ in structure and functions, being widely extracted from vegetables and fruits. In the present study, we show that polysaccharides found in chamomile tea (called MRW), in contact with antiparasitic agents, potentially inhibit the adhesion of parasites to intestinal cells. Moreover, at 500 µg/mL, they act synergistically with nitazoxanide (NTZ), increasing its effectiveness and decreasing the drug dose needed for giardiasis treatment.
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Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.
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Giardiasis is an intestinal disease caused by the parasite protozoan Giardia intestinalis. For more than five decades, the treatment of this disease has been based on compounds such as nitroimidazoles and benzimidazoles. The parasite's adverse effects and therapeutic failure are largely recognized. Therefore, it is necessary to develop new forms of chemotherapy treatment against giardiasis. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins as tubulin, are found in the Giardia genome and can become an interesting option for giardiasis treatment. In the present study, we evaluated the effects of 4-[(10H-phenothiazin-10-yl)methyl]-N-hydroxybenzamide, a new class I/II KDAC inhibitor, on G. intestinalis growth, cytoskeleton, and ultrastructure organization. This compound decreased parasite proliferation and viability and displayed an IC50 value of 179 nM. Scanning electron microscopy revealed the presence of protrusions on the cell surface after treatment. In addition, the vacuoles containing concentric membranous lamella and glycogen granules were observed in treated trophozoites. The cell membrane appeared deformed just above these vacuoles. Alterations on the microtubular cytoskeleton of the parasite were not observed after drug exposure. The number of diving cells with incomplete cytokinesis increased after treatment, indicating that the compound can interfere in the late steps of cell division. Our results indicate that 4-[(10H-phenothiazin-10-yl)methyl]-N-hydroxybenzamide should be explored to develop new therapeutic compounds for treating giardiasis.
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Giardia lamblia , Giardiasis , Animales , Giardia , Giardiasis/tratamiento farmacológico , Lisina/farmacología , TrofozoítosRESUMEN
BACKGROUND: Intestinal parasites (IPs) are widely distributed worldwide and are one of the major contributors to gastrointestinal disease. Their prevalence is associated with poor access to water, sanitation and hygiene (WASH). The objective of this study was to identify the prevalence of IPs, including soil-transmitted helminths (STH), and their relation to socioeconomic characteristics, as well as a first approach to molecularly characterize the types of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina. METHODS: A cross-sectional study was conducted in the rural settlement of Fortin Mbororé between January and March 2018. Socioeconomic variables, household characteristics, and stool and blood samples were collected. Standard coprological techniques were used to analyze stool samples, and a complete hemogram was performed on the blood samples. Giardia intestinalis microscopy-positive samples were genetically typed by the ß-giardin (bg) gene. Molecular identification of Blastocystis spp. subtypes and E. histolytica were carried out by amplification and sequencing of a partial fragment of the small subunit ribosomal RNA gene (SSU rDNA). RESULTS: The overall prevalence of IPs was 92.7%, with 72.0% specifically for hookworm. IPs were significantly more prevalent in preschool- and school-age children (P < 0.05). No formal education (P = 0.035), the presence of unimproved floors (P = 0.001) and overcrowding (P = 0.005) were significantly associated with IP infection. Hookworm was associated with anemia (P = 0.019). Molecular characterization revealed the presence of E. histolytica sub-assemblages AII (12.5%), AIII (87.5%) and BIV (100%); one case of sub-assemblage D for G. intestinalis; and the presence of subtypes ST1 (14.8%), ST2 (14.8%) and ST3 (70.4%) of Blastocystis spp. CONCLUSIONS: Protozoans detected in this study are transmitted mainly through water contaminated with fecal matter, evidencing the need to improve the quality of water and sanitation for the inhabitants of Fortín Mbororé. Molecular characterization showed that domestic animals can be implicated in the zoonotic transmission of G. intestinalis and Blastocystis spp. to humans. A hyperendemic area for STH was found, with hookworm prevalence greater than 50%. Therefore, improvements in WASH as well as mass deworming programs need to be implemented in this area to control and decrease the prevalence of IPs in general and STH in particular.
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Infecciones por Blastocystis/epidemiología , Blastocystis/genética , Entamoeba histolytica/genética , Entamebiasis/epidemiología , Giardia lamblia/genética , Giardiasis/epidemiología , Parasitosis Intestinales/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Argentina/epidemiología , Infecciones por Blastocystis/sangre , Niño , Preescolar , Estudios Transversales , Entamebiasis/sangre , Heces/parasitología , Femenino , Giardiasis/sangre , Humanos , Lactante , Parasitosis Intestinales/sangre , Masculino , Persona de Mediana Edad , Prevalencia , Población Rural/estadística & datos numéricos , Adulto JovenRESUMEN
Indazole is an important scaffold in medicinal chemistry. At present, the progress on synthetic methodologies has allowed the preparation of several new indazole derivatives with interesting pharmacological properties. Particularly, the antiprotozoal activity of indazole derivatives have been recently reported. Herein, a series of 22 indazole derivatives was synthesized and studied as antiprotozoals. The 2-phenyl-2H-indazole scaffold was accessed by a one-pot procedure, which includes a combination of ultrasound synthesis under neat conditions as well as Cadogan's cyclization. Moreover, some compounds were derivatized to have an appropriate set to provide structure-activity relationships (SAR) information. Whereas the antiprotozoal activity of six of these compounds against E. histolytica, G. intestinalis, and T. vaginalis had been previously reported, the activity of the additional 16 compounds was evaluated against these same protozoa. The biological assays revealed structural features that favor the antiprotozoal activity against the three protozoans tested, e.g., electron withdrawing groups at the 2-phenyl ring. It is important to mention that the indazole derivatives possess strong antiprotozoal activity and are also characterized by a continuous SAR.
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Antiprotozoarios/síntesis química , Antiprotozoarios/farmacología , Quimioinformática , Indazoles/síntesis química , Indazoles/farmacología , Antiprotozoarios/química , Entamoeba histolytica/efectos de los fármacos , Giardia lamblia/efectos de los fármacos , Indazoles/química , Concentración 50 Inhibidora , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Trichomonas vaginalis/efectos de los fármacos , UltrasonidoRESUMEN
According to a few parasitological and epidemiological studies, Giardia is the most prevalent parasitic infection among pet dogs in the city of Medellín, the second-largest city in Colombia. This study determined the assemblages of Giardia in the fecal samples of dogs obtained from 18 veterinary centers of Medellín. One hundred fecal samples of dogs diagnosed with Giardia using microscopy were analyzed via nested polymerase chain reaction (PCR) using three genes (gdh, bg, and tpi). The PCR products were purified and sequenced, and phylogenetic analyses were conducted using the maximum likelihood algorithm of the three loci. From the 100 samples analyzed, 47 were Giardia-positive via PCR. Genotypes C and D were detected in six samples, neither of which were associated with human infection. However, the zoonotic potential of Giardia cannot be ruled out because of the small number of samples that could be sequenced for assemblage assignation.
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Enfermedades de los Perros , Giardia lamblia , Giardiasis , Animales , Colombia/epidemiología , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/parasitología , Perros/parasitología , Heces/parasitología , Genotipo , Técnicas de Genotipaje/veterinaria , Giardia lamblia/clasificación , Giardiasis/epidemiología , Giardiasis/veterinaria , Tipificación de Secuencias Multilocus/veterinaria , FilogeniaRESUMEN
Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.
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Actinas/metabolismo , Giardia lamblia/metabolismo , Plaquinas/metabolismo , Tubulina (Proteína)/metabolismo , Actinas/química , Secuencia de Aminoácidos , Animales , Ancirinas/química , Secuencia de Bases , Western Blotting , Biología Computacional , Secuencia de Consenso , Citoplasma/química , Citoesqueleto/química , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Dinaminas/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Giardia lamblia/química , Giardia lamblia/ultraestructura , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Plaquinas/química , Alineación de Secuencia , Tubulina (Proteína)/químicaRESUMEN
Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from Giardia. The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells in vitro. Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis.
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Vesículas Extracelulares , Giardia lamblia , Animales , Interacciones Huésped-Patógeno , Desiminasas de la Arginina Proteica , ProteómicaRESUMEN
El objetivo de la presente investigación fue determinar la prevalencia de parásitos intestinales en niños preescolares usuarios de 12 Centros de Educación Inicial Simoncito (CEIS) del municipio Angostura del Orinoco, estado Bolívar, Venezuela. Se estudiaron las heces de 515 niños de ambos géneros (2 a 5 años), mediante la técnica de sedimentación espontánea. La prevalencia de enteroparásitos fue de 39,4% (n=203). No hubo diferencias estadísticamente significativas (χ2 = 1,59 g.l.: 2 p> 0,05) respecto a la edad, pero si según el género (p<0,05), resultando los niños varones más afectados con 46,9%. Se identificaron 11 taxones de enteroparásitos, destacando el cromista Blastocystis spp. con 27,4% (n=141) y el protozoario Giardia intestinalis con 13,2% (n=68). Se encontró una baja prevalencia de helmintos, donde Ascaris lumbricoides fue el más común con 1,6% (n=8). De los 203 niños parasitados, el 70,9% (n=144) estaba monoparasitado y 29,1% (n=59) poliparasitado. Los principales parásitos asociados fueron Blastocystis spp. (89,8%), G. intestinalis (44,1%) y Endolimax nana (35,3%). En conclusión, se determinó una elevada prevalencia de parásitos intestinales en la población estudiada, por lo que estas infecciones continúan siendo un problema de salud pública en niños de Ciudad Bolívar, Venezuela
The objective of the present investigation was to determine the prevalence of intestinal parasites in preschool children users of 12 Simoncito Initial Education Centers (CEIS) of the Angostura del Orinoco municipality, Bolívar state, Venezuela. The feces of 515 children of both genders (2 to 5 years old) were studied using the spontaneous sedimentation technique. The prevalence of enteroparasites was 39.4% (n = 203). There were no statistically significant differences (χ2 = 1.59 d.f .: 2 p> 0.05) with respect to age but if according to gender (p <0.05), because the most affected were male child with 46.9%. Eleven taxa of enteroparasites were identified, highlighting the chromist Blastocystis spp. (27.4%) and the protozoan Giardia intestinalis (13.2%). A low prevalence of helminths was found, where Ascaris lumbricoides was the most common with 1.6%. Of the 203 parasitized children, 70.9% (n = 144) were monoparasitized and 29.1% (n = 59) polyparasitized. The main associated parasites were Blastocystis spp. (89.8%), G. intestinalis (44.1%) and Endolimax nana (35.3%). In conclusion, a high prevalence of intestinal parasites was determined in the population studied, so these infections continue to be a public health problem in children from Ciudad Bolívar, Venezuela
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BACKGROUND: Intestinal parasitic protozoa represent a serious problem of public health particularly in developing countries. Protozoa such as Blastocystis, Giardia intestinalis, Entamoeba histolytica and Cryptosporidium spp. are associated with diarrheal symptoms. In Colombia, there is little region-specific data on the frequency and circulating genotypes/species of these microorganisms. Therefore, the main objective of our study was to employ molecular detection and genotyping of G. intestinalis and Blastocystis, Cryptosporidium and Entamoeba spp. in samples from different biogeographical regions of Colombia. METHODS: We collected 649 human fecal samples from five biogeographical regions of Colombia: the Amazon, Andean, Caribbean, Orinoco and Pacific regions. Blastocystis, G. intestinalis, Cryptosporidium spp. and Entamoeba complex were detected by microscopy and conventional PCR. Molecular genotyping was conducted to identify Blastocystis subtypes (STs) (18s), G. intestinalis assemblages (triose phosphate isomerase and glutamate dehydrogenase) and Cryptosporidium species (18s). Genetic diversity indices were determined using dnasp.5. RESULTS: We detected G. intestinalis in 45.4% (n = 280) of samples, Blastocystis in 54.5% (n = 336) of samples, Cryptosporidium spp. in 7.3% (n = 45) of samples, Entamoeba dispar in 1.5% (n = 9) of samples, and Entamoeba moshkovskii in 0.32% (n = 2) of samples. Blastocystis STs 1-4, 8 and 9 and G. intestinalis assemblages AII, BIII, BIV, D and G were identified. The following Cryptosporidium species were identified: C. hominis, C. parvum, C. bovis, C. andersoni, C. muris, C. ubiquitum and C. felis. The Caribbean region had the highest frequency for each of the microorganisms evaluated (91.9% for G. duodenalis, 97.3% for Blastocystis, 10.8% for Cryptosporidium spp., 13.5% for E. dispar and 2.7% for E. moshkovskii). The Orinoco region had a high frequency of Blastocystis (97.2%) and the Andean region had a high frequency of G. intestinalis (69.4%). High and active transmission was apparent in several regions of the country, implying that mechanisms for prevention and control of intestinal parasitosis in different parts of the country must be improved.
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Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.
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Giardia lamblia/citología , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Microscopía ElectrónicaRESUMEN
OBJECTIVE: To evaluate the effectiveness and tolerability of secnidazole combined with high-dose mebendazole for treatment of 5-nitroimidazole-resistant giardiasis. METHOD: Adults with microscopically verified Giardia intestinalis monoinfection attending a secondary level hospital in Matanzas City, Cuba were prospectively included in a cohort. A recently introduced treatment ladder consisting of metronidazole as first-line treatment, followed by secnidazole, tinidazole, secnidazole plus mebendazole and quinacrine as second-to fifth-line treatments, respectively, was used. Adverse events and treatment success were determined by questioning and microscopy on concentrated stool samples, respectively on days 3, 5 and 7 after the end of treatment. If G. intestinalis was detected on day 3, 5 or 7, then the infection was classified as refractory and no further microscopy was performed. RESULTS: A total of 456 individuals were included. Metronidazole, 500 mg three times daily for 5 days, cured 248/456 (54%) patients. A single 2-g secnidazole dose as second-line treatment cured 50/208 (24%) patients. A single 2-g tinidazole dose as third-line treatment cured 43/158 (27%) patients. Three rounds of 5-nitroimidazole therapy therefore cured 341/456 (75%) patients. Secnidazole plus mebendazole (200 mg every 8 hours for 3 days) cured 100/115 (87%) of nitroimidazole refractory infections. Quinacrine cured the remaining 15 patients. All treatments were well tolerated. CONCLUSIONS: 5-Nitroimidazole refractory giardiasis was common, indicating that an alternative first-line treatment may be needed. Retreatment of metronidazole refractory giardiasis with an alternative 5-nitroimidazole was suboptimal, indicating cross-resistance. Mebendazole plus secnidazole were well tolerated and effective for the treatment of 5-nitroimidazole refractory G. intestinalis infection in this setting.
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Antiprotozoarios/administración & dosificación , Giardiasis/tratamiento farmacológico , Mebendazol/administración & dosificación , Metronidazol/análogos & derivados , Quinacrina/administración & dosificación , Adulto , Anciano , Antiprotozoarios/farmacología , Cuba , Esquema de Medicación , Resistencia a Medicamentos/efectos de los fármacos , Quimioterapia Combinada , Heces/parasitología , Femenino , Giardia lamblia/efectos de los fármacos , Giardia lamblia/aislamiento & purificación , Humanos , Masculino , Mebendazol/farmacología , Metronidazol/administración & dosificación , Metronidazol/farmacología , Persona de Mediana Edad , Nitroimidazoles/uso terapéutico , Estudios Prospectivos , Quinacrina/farmacología , Resultado del Tratamiento , Adulto JovenRESUMEN
Parasitic agents are a common cause of diarrhea in dogs and cats and, thus, determining their prevalence is essential to establish preventive and control measures. This retrospective study examined the fecal tests records from 1111 dogs and 203 cats with diarrhea submitted to a diagnostic laboratory in the city of Medellin between January and May 2018. The detection of parasites was carried out by direct smears and simple flotation methods. Parasitic organisms were detected in feces from 464 (41.7%) dogs and 96 (47.3%) cats. In order of decreasing prevalence, the parasites detected in dogs were: Giardia intestinalis (13%), ancylostomids (12.6%), Entamoeba spp. (6.1%), coccidian oocysts (5.8%), Toxocara spp. (5.6%) and Dipylidium caninum (1.3%). In cats, the prevalence was: Giardia intestinalis (20%), coccidian oocysts (8.9%), Entamoeba spp. (7.9%), ancylostomids (6.4%), Toxocara spp. (2.5%) and Dipylidium caninum (2%). Age, but not gender, was a predisposing factor, as puppies and kittens had significantly higher infection rates that older age categories. The majority of Giardia intestinalis positive cases occurred in puppies (109/145, 75.2%) and kittens (19/36, 52.8%), making this parasite the most prevalent in amongst animals with diarrhea. Out of 117 positive infections in the adult dog population, ancylostomids accounted for 56 cases (47.9%) and was the most common parasite in this age group. In conclusion, although these results do not imply a cause and effect relationship, they are an estimate of the type of parasites that may be most commonly associated with diarrhea in dogs and cats. The lower diagnostic sensitivity of the traditional methods used here as compared to more contemporary techniques like fecal flotation with centrifugation and PCR, may have underestimated the actual prevalence and diminished the detection of co-infections. Future studies should aim to have diagnostic panels that also screen for other enteric pathogens, including bacterial and viral agents.
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Enfermedades de los Gatos/epidemiología , Diarrea/veterinaria , Enfermedades de los Perros/epidemiología , Giardia lamblia/aislamiento & purificación , Giardiasis/veterinaria , Animales , Enfermedades de los Gatos/parasitología , Gatos , Colombia/epidemiología , Diarrea/epidemiología , Diarrea/parasitología , Enfermedades de los Perros/parasitología , Perros , Heces/parasitología , Femenino , Giardiasis/epidemiología , Giardiasis/parasitología , Masculino , Prevalencia , Estudios RetrospectivosRESUMEN
Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles demonstrated significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest antibody response can be used to measure population-level transmission of diverse enteropathogens in serologic surveillance.
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Anticuerpos Antibacterianos/sangre , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antivirales/sangre , Infecciones Bacterianas/epidemiología , Infecciones por Caliciviridae/epidemiología , Inmunoglobulina G/sangre , Parasitosis Intestinales/epidemiología , Factores de Edad , Niño , Países en Desarrollo , Transmisión de Enfermedad Infecciosa , Monitoreo Epidemiológico , Haití/epidemiología , Humanos , Kenia/epidemiología , Estudios Longitudinales , Estudios Seroepidemiológicos , Tanzanía/epidemiologíaRESUMEN
Giardia lamblia is one of the most important worldwide causes of intestinal infections, yet little is known about its cellular physiology, especially the diversity of ionic channels that this parasite expresses. In this work, we show that injection of mRNA isolated from trophozoites of Giardia, into Xenopus laevis oocytes, induces expression of three types of chloride currents (here referred to as ICl-G1, ICl-G2, and ICl-G3), which have different biophysical and pharmacological properties. ICl-G1 currents show inward rectification and voltage dependence are enhanced by hypotonicity, show a selectivity sequence of (I > Br > Cl > F), and are inhibited by NPPB, DIDS, SITS, 9AC, DPC, and Zinc. These findings suggest that ICl-G1 is the result of expression of chloride channels related to ClC2. ICl-G2 currents show outward rectification and are dependent of intracellular calcium, its selectivity sequence is (Cl > Br > I > F) and are inhibited by NPPB, DIDS, SITS, 9AC, DPC, niflumic acid, tannic acid, and benzbromarone. These findings suggest that they are produced by calcium dependent chloride channels (CaCC). The third type of currents (ICl-G3) appears only after a hypoosmotic challenge, and has similar properties to those described for ICl-swell, such as outward rectification, instant activation, and slow inactivation at large depolarizing voltages. They were blocked by NPPB, DIDS, 9AC, NIf, DCPIB, and tamoxifen. Our results indicate that Giardia intestinalis has at least three types of anion conductances.
Asunto(s)
Canales de Cloruro/biosíntesis , Giardia lamblia/genética , Oocitos/metabolismo , ARN Mensajero/administración & dosificación , ARN Protozoario/administración & dosificación , Trofozoítos/genética , Xenopus laevis/metabolismo , Animales , Calcio/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Fenómenos Electrofisiológicos , Femenino , Giardia lamblia/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Inyecciones , Potenciales de la Membrana , Oocitos/citología , ARN Mensajero/genética , ARN Protozoario/genética , Trofozoítos/crecimiento & desarrollo , Xenopus laevis/genéticaRESUMEN
Giardia intestinalis is a protozoan of worldwide distribution capable of infecting a large number of species, including humans and domestic animals. Dogs represent a risk to public health due to cross-infections by the zoonotic assemblages. However, there is little information concerning the prevalence and frequency of this parasite and its assemblages in dogs of the central region of Mexico, thus this study aimed to contribute to this matter. A total of 402 feces samples from dogs of different settings (shelter, breeding establishments, domestic and stray) were obtained and direct coproparasitoscopic examination by flotation revealed a prevalence of 25%. PCR was performed for amplification of the ß-Giardin gene, to which 24 samples were positive. Assemblages were obtained through RFLP analysis, using enzymes Hae III to obtain the main genotypes (A-G), and Hha I to subtype assemblage A. All 24 samples were genotyped as assemblage A, with 83% as AI and 17% as AII. Thus, these findings confirm that dogs in the central region of Mexico are a risk for zoonotic transmission of this parasite, emphasizing the importance of a much needed control of the disease in this species.
RESUMEN
Giardiasis is a widespread illness that affects inhabitants of underdeveloped countries, being children and seniors the highest risk population. The several adverse effects produced by current therapies besides its increasing ineffectiveness due to the appearance of resistant strains evidence the urgent need for new therapeutic approaches. We present the antigiardiasic effect of eight Cu(II) coordination compounds, which belong to the family Casiopeínas. Two of them, 4,7-diphenyl-1,10-phenanthroline(acetylacetonato)copper(II) nitrate (CasIII-Ha,36⯵M) and 4,7-diphenyl-1,10-phenanthroline(glycinato)copper(II) nitrate (CasI-gly,36⯵M) have shown the best antiproliferative effect in Giardia intestinalis trophozoite cultures, both with the higher lipophilic character of the series. The antiproliferative effect of these coordination compounds is attributable to its capacity to interact with the cellular membrane and to increase reactive oxygen species (ROS) concentration within the parasite since the first hours of exposure, (2-6â¯h). We found that these compounds mainly induced the cell death of trophozoites by apoptosis, contrary to metronidazole, which induces apoptosis and necrosis in the same ratio. The cytotoxic effects on lymphocytes and macrophages isolated from human peripheral blood allowed us to establish a selectivity index and in turn, identify and propose the best candidates to continue with the assays in animal models. The selected molecules do not include the most active compounds against trophozoites, instead of that, we propose the compounds 4',4'-dimethyl-2,2'-bipyridine(acetylacetonato)copper(II) nitrate (CasIII-ia,IC50â¯=â¯156⯵M) and 4,7-dimethyl-1,10-phenanthroline(acetylacetonato) copper(II) nitrate (CasIII-Ea,IC50â¯=â¯125⯵M), which possess an antiproliferative efficacy comparable with Metronidazole but also are those that produce the lowest effect on the viability of human lymphocytes and macrophages.
Asunto(s)
Antiprotozoarios/farmacología , Membrana Celular/efectos de los fármacos , Complejos de Coordinación/farmacología , Giardia lamblia/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Antiprotozoarios/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Cobre/química , Humanos , Pruebas de Sensibilidad Microbiana , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Trofozoítos/efectos de los fármacosRESUMEN
Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.