RESUMEN

BACKGROUND: Entamoeba species harbored by humans have different degrees of pathogenicity. The present study explores the intra- and interspecific diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in different Brazilian regions. METHODS: Cross-sectional studies were performed to collect fecal samples (n = 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis. RESULTS: Light microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6-10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four different species were identified: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n = 32) were characterized; however, new ribosomal lineages were not identified. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histolytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the first containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecific diversity for tetra- and octonucleated Entamoeba spp. (H ± SD = 0.9625 ± 0.0126). The intraspecific diversity varied according to species or subtype: E. dispar and E. histolytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni. CONCLUSIONS: Tetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a field to be further explored and provides information with important implications for public health.

Asunto(s)

Ecosistema , Entamoeba/clasificación , Entamoeba/genética , Entamebiasis/epidemiología , Variación Genética , Adolescente , Brasil/epidemiología , Niño , Preescolar , Estudios Transversales , ADN Protozoario/genética , Entamoeba/citología , Heces/parasitología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Filogenia , Prevalencia , Análisis de Secuencia de ADN

RESUMEN

Rural children are one of the populations that are most vulnerable to gastrointestinal parasite infections. Such diseases decrease the quality of life and result in growth and cognitive delays in the long term. This cross-sectional study was conducted to determine the frequency of intestinal parasite infections among rural schoolchildren in the municipality of Apulo, Colombia. A total of 97 stool samples from children aged between 5 and 15 years were collected and examined via direct light microscopy. Microscopic examination was repeated with sediments obtained using a fecal parasite concentrator, and the Kato-Katz test was performed. Frequency of intestinal parasite infection was 100%. Endolimax nana (77.35%), Blastocystis sp. (71.1%), Giardia intestinalis (39.1%), Entamoeba coli (25.7%), and the Entamoeba histolytica/dispar/moshkovskii complex (9.2%) were the most prevalent protozoa. Trichuris trichiura was the most prevalent helminth (12.3%), followed by Enterobius vermicularis (6.15%) and Ascaris lumbricoides (5.1%). Among the analyzed associated factors, consumption of untreated water increased the risk of acquiring pathogenic intestinal parasites. Finally, because G. intestinalis was the most prevalent pathogenic protozoan, molecular analysis was conducted to establish genetic assemblages and subassemblages of Giardia through sequence-based genotyping of the glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin genes. A total of 14 G. intestinalis-positive samples were genotyped, which revealed the presence of subassemblages AI (n = 1), AII (n = 7), BIII (n = 2), BIV (n = 2), and BIII/BIV (n = 1) as well as a mixed subassemblage AII + BIII (n = 1). Our results indicate that gastrointestinal parasite infections in the tested population were mainly caused by suboptimal water quality. Moreover, molecular typing of G. intestinalis suggested contamination of water by animal- and human-derived cysts.

Asunto(s)

Agua Potable/parasitología , Heces/parasitología , Infecciones por Nematodos/epidemiología , Infecciones por Protozoos/epidemiología , Adolescente , Animales , Ascaris lumbricoides/clasificación , Ascaris lumbricoides/aislamiento & purificación , Blastocystis/clasificación , Blastocystis/aislamiento & purificación , Niño , Preescolar , Colombia/epidemiología , Estudios Transversales , Endolimax/clasificación , Endolimax/aislamiento & purificación , Entamoeba/clasificación , Entamoeba/aislamiento & purificación , Enterobius/clasificación , Enterobius/aislamiento & purificación , Femenino , Giardia lamblia/clasificación , Giardia lamblia/genética , Giardia lamblia/aislamiento & purificación , Humanos , Higiene , Masculino , Infecciones por Nematodos/parasitología , Infecciones por Nematodos/transmisión , Prevalencia , Infecciones por Protozoos/parasitología , Infecciones por Protozoos/transmisión , Calidad de Vida , Población Rural , Trichuris/clasificación , Trichuris/aislamiento & purificación

RESUMEN

Protozoa have long been considered undesirable residents of the human gut, but recent findings suggest that some of them may positively affect the gut ecosystem. To better understand the role and ecological dynamics of these commensal and potentially beneficial protozoan symbionts, we need efficient methods to detect them, as well as accurate estimates of their prevalence across human populations. Metagenomics provides such an opportunity, allowing simultaneous detection of multiple symbionts in a single analytical procedure. In this study, we collected fecal samples of 68 individuals from three Cameroonian populations with different subsistence modes and compared metagenomics-based and targeted methods of detection for two common protozoan genera: Blastocystis and Entamoeba. In addition, we analyzed our data along with publicly available fecal metagenomes from various worldwide populations to explore the prevalence and association patterns of ten protozoan genera. Regarding the detection method, microscopy was much less sensitive than metagenomics for Entamoeba, whereas qPCR was at least as sensitive as metagenomics for Blastocystis sp. However, metagenomics was more likely to detect co-colonizations by multiple subtypes. Out of the ten examined genera in 127 individuals from Cameroon, Tanzania, Peru, Italy or USA, only three (Blastocystis, Entamoeba and Enteromonas) had an overall prevalence exceeding 10%. All three genera were more common in less industrialized populations and their prevalence differed between continents and subsistence modes, albeit not in a straightforward manner. The majority (72.5%) of colonized individuals carried at least two protozoan species, indicating that mixed-species colonizations are common. In addition, we detected only positive and no negative association patterns between different protozoa. Despite the pitfalls of the metagenomic approach, ranging from the availability of good-quality sequencing data to the lack of standard analytical procedures, we demonstrated its utility in simultaneous detection of multiple protozoan genera, and especially its ability to efficiently detect mixed-species colonizations. Our study corroborates and expands prevalence results previously obtained for Blastocystis sp. and provides novel data for Entamoeba spp. and several other protozoan genera. Furthermore, it indicates that multiple protozoa are common residents of the healthy human gut worldwide.

Asunto(s)

Blastocystis/aislamiento & purificación , Entamoeba/aislamiento & purificación , Tracto Gastrointestinal/parasitología , Metagenómica/métodos , Análisis de Secuencia de ADN/métodos , Adulto , Anciano , Blastocystis/clasificación , Blastocystis/genética , Camerún/epidemiología , ADN Ribosómico/genética , Países Desarrollados , Entamoeba/clasificación , Entamoeba/genética , Heces/parasitología , Femenino , Voluntarios Sanos , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Perú/epidemiología , Prevalencia , ARN Ribosómico 16S/genética , Tanzanía/epidemiología , Estados Unidos/epidemiología

RESUMEN

Microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of intestinal parasites. Recently, polymerase chain reaction (PCR) has been approved by the World Health Organization as the method of choice for the diagnosis of Entamoeba histolytica. Of the 106 stool samples collected from the Esmeraldas and Pichincha provinces of Ecuador, all (100%) were positive for E. histolytica/Entamoeba dispar by light microscopy, whereas using real-time PCR (RT-PCR) DNA amplification, 74 (69.8%) were positive for E. dispar and only three (2.8%) were positive for E. histolytica. Some 29 (27.4%) samples were negative for the presence of either E. histolytica or E. dispar, this may be due the presence of Entamoeba mosksvskii, which is morphologically identical to E. histolytica/E. dispar and not specifically targeted by the RT-PCR used. These results indicate the necessity of reevaluating the epidemiology of amebiasis in Ecuador as the prominent species found are nonpathogenic.

Asunto(s)

Entamoeba histolytica/aislamiento & purificación , Entamoeba/aislamiento & purificación , Entamebiasis/diagnóstico , Parasitosis Intestinales/diagnóstico , Técnicas de Diagnóstico Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Antígenos de Protozoos/análisis , ADN Protozoario/genética , Ecuador/epidemiología , Entamoeba/clasificación , Entamoeba histolytica/patogenicidad , Entamebiasis/epidemiología , Entamebiasis/parasitología , Ensayo de Inmunoadsorción Enzimática , Heces/parasitología , Humanos , Parasitosis Intestinales/epidemiología , Microscopía

RESUMEN

Advances in molecular biology have facilitated analyses of the oral microbiome; however, the parasites role is poorly understood. Periodontal disease is a multifactorial process involving complex interactions among microorganisms, the host, and environmental factors. At present, the precise composition of the mouth parasites microbiota is unclear. Two protozoan species have been detected in the oral microbiota: Trichomonas tenax and Entamoeba gingivalis, and a new variant, E. gingivalis-ST2-kamaktli, was recently identified by us. In this study, both E. gingivalis and the new E. gingivalis-ST2-kamaktli variant were detected in the oral cavities of people with healthy periodontium, individuals undergoing orthodontic treatment, and patients with periodontal disease. In the group with healthy periodontium, the prevalence of E. gingivalis-ST1 was 48.6% and that of E. gingivalis-ST2-kamaktli 29.5%, with a combined prevalence of 54.3%. In patients undergoing orthodontics treatment, 81.2% carried both amoebas, with 47.5% having E. gingivalis-ST1 and 73.8% E. gingivalis-ST2-kamaktli. In people with periodontal disease, the prevalence of E. gingivalis-ST1 was 57.8%, and that of E. gingivalis-ST2-kamaktli 50.0%, with a combined prevalence of 73.5%; hence, E. gingivalis-ST1 and E gingivalis-ST2-kamaktli were detected in all three groups. The question arises, what are E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli doing in the oral cavity? Although, the answer remains unclear, our results suggest that each amoeba subtype is genetically distinct, and they exhibit different patterns of infectious behavior. We hypothesize that E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli may represent separate species. Our data contribute to better understanding of the roles of E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli in the oral microbiota.

Asunto(s)

Entamoeba/clasificación , Entamoeba/aislamiento & purificación , Entamebiasis/epidemiología , Boca/parasitología , Enfermedades Periodontales/parasitología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Entamoeba/genética , Entamebiasis/parasitología , Femenino , Humanos , Masculino , Microbiota , Persona de Mediana Edad , Epidemiología Molecular , Prevalencia , Trichomonas/aislamiento & purificación , Tricomoniasis/parasitología , Adulto Joven

RESUMEN

Entamoeba gingivalis is a protozoan that resides in the oral cavity. Using molecular biology techniques, we identified a novel organism that shares the same ecological niche as E. gingivalis. To differentiate this organism from E. gingivalis, we named it "kamaktli variant." By sequencing the 18S-ITS1-5.8S-ITS2 rRNA region, we demonstrated that kamaktli variant is 89% identical to E. gingivalis. To elucidate the relationship between kamaktli variant and E. gingivalis, we performed a phylogenetic analysis. Both taxa clustered in the same clade with high support, indicating that the amoebas are closely related (98/99/1.00, maximum parsimony/maximum likelihood/MrBayes, respectively). Given this information, we propose that these molecular differences between kamaktli variant and E. gingivalis ST1 are sufficient to distinguish them as independent subtypes, and we name the new subtype "E. gingivalis ST2, kamaktli variant."

Asunto(s)

Entamoeba , Boca/parasitología , Animales , ADN Espaciador Ribosómico/genética , Entamoeba/clasificación , Entamoeba/genética , Entamoeba/aislamiento & purificación , Humanos , Filogenia , ARN Ribosómico 18S/genética , ARN Ribosómico 5.8S/genética

RESUMEN

Our knowledge of the parasite species present in wildlife hosts is incomplete. Protozoans such as amoebae of the genus Entamoeba infect a large variety of vertebrate species, including NHPs. However, traditionally, their identification has been accomplished through microscopic evaluation; therefore, amoeba species have not always been identified correctly. We searched for Entamoeba spp. using a fragment of the small subunit rDNA in free-ranging howler monkeys (Alouatta palliata and A. pigra) from southeast Mexico. One hundred fifty five samples were collected, with 46 from A. palliata and 109 from A. pigra and 8 of the total samples were positive. We detected a new clade of Entamoeba, which was separated from other described species but closer to E. insolita, as well as an unnamed sequence typically found in iguana species with low shared identity values (<90%). We designated this new clade as conditional lineage 8 (CL8) and we have shown that members of this group are not exclusive to reptiles.

Asunto(s)

Alouatta/parasitología , Entamoeba/aislamiento & purificación , Reptiles/parasitología , Animales , Animales Salvajes/genética , Animales Salvajes/parasitología , ADN Ribosómico , Entamoeba/clasificación , Entamoeba/genética , México

RESUMEN

INTRODUCTION: A cross-sectional study was carried out to assess the prevalence of E. histolytica and E. dispar by examining stool samples obtained from 1,003 students of public schools in Maceió, Alagoas, Brazil. METHODOLOGY: All stool samples were processed using the spontaneous sedimentation technique and examined microscopically for the presence of Entamoeba species. In order to distinguish infections caused by E. histolytica, fecal samples presenting cysts of Entamoeba were subjected to specific enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). RESULTS: The analysis of the fecal specimens by microscopy identified 6.4% (64/1,003) students positive for E. histolytica/E. dispar/E. moshkovskii cysts. The prevalence of E. histolytica detected by ELISA was 3.0% (30/1,003) and by PCR 2.8% (28/1,003), but the difference is not statistically significant (p > 0.05). The prevalence of E. dispar in schoolchildren was 5.0% (50/1,003). Mixed infections with E. histolytica and E. dispar were also detected by PCR.  Even though immunological and molecular methods have shown similar results for identification of E. histolytica, ELISA is advantageous over the PCR since it is relatively cheaper and easier to perform. CONCLUSIONS: Our study demonstrated the occurrence of E. histolytica in Maceió and highlights the need to introduce a specific diagnostic test to detect amoebiasis cases in public laboratories.

Asunto(s)

Entamoeba/clasificación , Entamoeba/aislamiento & purificación , Entamebiasis/epidemiología , Entamebiasis/parasitología , Adolescente , Brasil/epidemiología , Niño , Preescolar , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Estudios Epidemiológicos , Heces/parasitología , Femenino , Humanos , Masculino , Microscopía , Reacción en Cadena de la Polimerasa , Prevalencia , Instituciones Académicas , Estudiantes

RESUMEN

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

Asunto(s)

ADN Protozoario/análisis , Agua Potable/parasitología , Entamoeba , Entamebiasis/epidemiología , Heces/parasitología , Tipificación Molecular/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Niño , Estudios Transversales , Sequías , Entamoeba/clasificación , Entamoeba/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pobreza , Prevalencia , Pozos de Agua , Adulto Joven

RESUMEN

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

Asunto(s)

Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , ADN Protozoario/análisis , Agua Potable/parasitología , Entamoeba , Entamebiasis/epidemiología , Heces/parasitología , Tipificación Molecular/métodos , Brasil/epidemiología , Estudios Transversales , Sequías , Entamoeba/clasificación , Entamoeba/genética , Reacción en Cadena de la Polimerasa , Pobreza , Prevalencia , Pozos de Agua

RESUMEN

We conducted an observational study to determine the prevalence of Entamoeba spp., in samples collected in a waste water treatment plant that provides water for agricultural irrigation. Samples were collected weekly over a period of 10 weeks at representative contamination stages from within the treatment plant. Protozoan identification was performed via light microscopy and culture. PCR amplification of small subunit rRNA gene sequences of E. histolytica/dispar/moshkovskii was performed in culture positive samples. Light microscopy revealed the presence of Entamoeba spp., in 70% (14/20) of the raw waste water samples and in 80% (8/10) of the treated water samples. PCR amplification after culture at both 24 and 37°C revealed that 100% (29/29) of the raw waste water samples and 78.6% (11/14) of the treated waste water were positive for E. moshkovskii. We report the first isolation of E. moshkovskii in Colombia, confirmed by PCR. Recent reports of E. moshkovskii pathogenic potential suggest this finding could constitute a public health risk for people exposed to this water.

Asunto(s)

Agricultura , Entamoeba/aislamiento & purificación , Entamebiasis/parasitología , Aguas Residuales/parasitología , Colombia , ADN Protozoario/análisis , Entamoeba/clasificación , Reacción en Cadena de la Polimerasa , Prevalencia

RESUMEN

BACKGROUND: Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. histolytica is the only true pathogen. The epidemiology of this infection is complex due to the absence of a routine exam that allows a correct discrimination of the Entamoeba species complex. Therefore, molecular methods appear as the unique epidemiological tool to accomplish the species discrimination. Herein, we conducted a cross-sectional study to determine the frequency of Entamoeba species infections in a group of asymptomatic individuals from a rural area in central Colombia. METHODOLOGY/PRINCIPAL FINDINGS: A total of 181 fecal samples from asymptomatic children under 16 years old from the hamlet La Vírgen, Cundinamarca (Colombia) that voluntarily accepted to participate in the study were collected. The fecal samples were examined by light microscopy and DNA-extracted, subsequently submitted to molecular discrimination of E. dispar/E. histolytica/E. moshkovskii infection based on a multiplex PCR assay targeting the 18S rRNA fragment. To confirm the species description, twenty samples were randomly submitted to DNA sequencing of the aforementioned fragment. By direct microscopic examination, frequency of the complex E. histolytica/E. dispar/E. moshkovskii was 18.8% (34/181). PCR showed a frequency of 49.1% (89/181), discriminated as 23.2% (42/181) that were positive for E. dispar, 25.4% (46/181) for E. moshkovskii and 0.55% (1/ 181) for E. histolytica. Also, mixed infections were detected between E. dispar and E. moshkovskii at 4.42% (8/181) of the samples. Molecular barcoding confirmed the diagnosis depicted by the multiplex PCR assay. CONCLUSIONS/SIGNIFICANCE: This is the first description of E. moshkovskii in Colombia and the second report in South-America to our knowledge. Our results suggest the need to unravel the true epidemiology of Entamoeba infections around the world, including the real pathogenic role that E. moshkovskii may have.

Asunto(s)

Entamoeba/genética , Entamebiasis/epidemiología , Entamebiasis/parasitología , Población Rural , Adolescente , Niño , Preescolar , Coinfección , Colombia/epidemiología , Entamoeba/clasificación , Entamebiasis/diagnóstico , Heces/parasitología , Humanos , Lactante , Recién Nacido , Filogenia , Prevalencia , ARN Ribosómico 18S/genética

RESUMEN

Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

Asunto(s)

Entamoeba/aislamiento & purificación , Entamebiasis/diagnóstico , Heces/microbiología , Diagnóstico Diferencial , Entamoeba/clasificación , Humanos , Compuestos Orgánicos , Patología Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

INTRODUCTION: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. METHODS: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. RESULTS: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. CONCLUSIONS: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques.

Asunto(s)

Entamoeba/clasificación , Entamebiasis/epidemiología , Heces/parasitología , Adolescente , Adulto , Brasil/epidemiología , Niño , Preescolar , ADN Protozoario/análisis , Entamoeba/genética , Entamoeba/inmunología , Entamebiasis/diagnóstico , Entamebiasis/parasitología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Adulto Joven

RESUMEN

Introduction: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. Methods: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. Results: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. Conclusions: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques. .

Asunto(s)

Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Adulto Joven , Entamoeba/clasificación , Entamebiasis/epidemiología , Heces/parasitología , Brasil/epidemiología , ADN Protozoario/análisis , Ensayo de Inmunoadsorción Enzimática , Entamoeba/genética , Entamoeba/inmunología , Entamebiasis/diagnóstico , Entamebiasis/parasitología , Reacción en Cadena de la Polimerasa Multiplex

RESUMEN

Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated from HIV patients.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/inmunología , Entamoeba/clasificación , Entamebiasis/inmunología , VIH , Huésped Inmunocomprometido , Filogenia , ARN Ribosómico 18S/clasificación , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/patología , Síndrome de Inmunodeficiencia Adquirida/virología , Adulto , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Coinfección , Entamoeba/genética , Entamoeba/aislamiento & purificación , Entamebiasis/parasitología , Entamebiasis/patología , Femenino , Variación Genética , Humanos , Masculino , Persona de Mediana Edad , Familia de Multigenes , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Carga Viral

RESUMEN

BACKGROUND: Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. METHODS: PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. RESULTS: Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. CONCLUSION: These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Asunto(s)

Entamoeba/aislamiento & purificación , Entamebiasis/diagnóstico , Heces/parasitología , Tamizaje Masivo/métodos , Técnicas de Diagnóstico Molecular/métodos , Parasitología/métodos , ADN Protozoario/genética , Entamoeba/clasificación , Entamoeba/genética , Entamebiasis/parasitología , Antropología Forense , Humanos , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/genética , ARN Ribosómico 18S/genética

RESUMEN

It has been claimed that amoebic molecules such as amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteases are responsible for host tissue destruction and are present in both pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar. Some reports have provided evidence that after infection with E. dispar, pathological changes may occur in some humans. The aim of this study was to evaluate E. dispar pathogenicity by comparing it to the pathogenicity of E. histolytica through liver abscesses induced in hamsters. Syrian golden hamsters were challenged by intrahepatic inoculation with the 03C E. dispar strain or with two strains of E. histolytica (HM1:IMSS and EGG) to compare their virulence grades. As control groups, we used bacterial flora and Pavlova's modified medium. Lesions were verified at 1, 3 and 6 days after inoculation. Multiplex Polymerase Chain Reaction was performed to characterize each strain using EdP1/EdP2 and EhP1/EhP2 primers. The EGG and HM1:IMSS E. histolytica strains and 03C E. dispar were able to cause liver lesions. The EGG strain caused extensive hepatic abscesses, and trophozoites were found in the lesions throughout the three periods of study. The HM1:IMSS strain caused smaller abscesses when compared to EGG lesions; however, trophozoites were observed at 1 and 3 days after inoculation. The 03C E. dispar strain caused intermediate abscesses when compared to the others; trophozoites were observed in all periods analyzed. The EGG strain caused progressive evolution of the injury, which differed from the HM1:IMSS and 03C strains. These results strongly suggest that the 03C E. dispar strain is pathogenic in the experimental hamster model. Additional studies are necessary to identify potential factors that regulate the manifestation of virulence of this strain and others.

Asunto(s)

Entamoeba/patogenicidad , Absceso Hepático Amebiano/parasitología , Animales , Peso Corporal , Brasil , Cricetinae , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Modelos Animales de Enfermedad , Entamoeba/clasificación , Entamoeba/genética , Femenino , Humanos , Hígado/parasitología , Hígado/patología , Absceso Hepático Amebiano/patología , Masculino , Mesocricetus , Reacción en Cadena de la Polimerasa Multiplex , Tamaño de los Órganos

RESUMEN

Few data exist on the parasites of ratites, especially from regions within their natural range. It is only recently that extensive studies on the parasites of ostriches (Struthio camelus) have been published, mainly from European countries where commercial farming has expanded. Two species of ratites are native in South America: the lesser rhea also known as Darwin's rhea (Rhea pennata) and the greater rhea (Rhea americana). Both species are considered near threatened by the IUCN and are included in the CITES' Appendices I and II, respectively. Parasitological studies have conservation implications, as they allow us to assess the risk of transmission of pathogens from farmed ratites to wild populations. In this study 92 faecal samples from greater rheas and 55 faecal samples from lesser rheas from different localities in Argentine were analyzed to determine their gastrointestinal parasites. In greater rheas the protozoa (Balantidium coli-like and Entamoeba spp.) and helminths (Fasciola hepatica and Deletrocephalus spp.). The protozoa had not previously been cited as parasites of greater rheas in South America. Cysts and/or trophozoites of B. coli-like were found in 16.3% of the samples, while the prevalence of the remaining parasites was below 10%. Lesser rheas harbored the protozoa B. coli-like, Entamoeba spp. and Chilomastix spp. as well as F. hepatica and nematode eggs and larvae. B. coli-like cysts were found in 20.0% of the samples, while the prevalence of the other parasites remained below 5%. Some of them had not been cited as infecting lesser rheas yet.

Asunto(s)

Enfermedades de las Aves/parasitología , Tracto Gastrointestinal/parasitología , Helmintiasis Animal/parasitología , Infecciones Protozoarias en Animales/parasitología , Reiformes , Animales , Argentina/epidemiología , Balantidium/clasificación , Balantidium/aislamiento & purificación , Enfermedades de las Aves/epidemiología , Entamoeba/clasificación , Entamoeba/aislamiento & purificación , Heces/parasitología , Helmintiasis Animal/epidemiología , Helmintos/clasificación , Helmintos/aislamiento & purificación , Recuento de Huevos de Parásitos/veterinaria , Prevalencia , Infecciones Protozoarias en Animales/epidemiología , Especificidad de la Especie

RESUMEN

During the last decade Entamoeba moshkovskii has become relevant given its capacity to infect humans, especially when considering that it is morphologically indistinguishable from E. histolytica. For a long time, E. moshkovskii was considered as a free living amoeba, but in the last decade it has been demonstrated that E. moshkovskii can infect humans and can be found more frequently in regions where amebiasis shows high prevalence values, becoming a challenge to differentiate it from the E. histolytica/E. dispar complex. Recently there have been studies that raise the possibility that E. moshkovskii could be a pathogenic species, as there are reports in different countries that associated this infection with gastrointestinal symptoms even though others have described it as a non pathogenic species. For this reasons, both clinical and epidemiological studies are required.

Asunto(s)

Entamoeba/clasificación , Entamoeba/aislamiento & purificación , Entamebiasis/diagnóstico , Entamebiasis/parasitología , Entamoeba/patogenicidad , Entamebiasis/epidemiología , Humanos