Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

Molecular characterization of Entamoeba, Blastocystis and Cryptosporidium species in stool samples collected from Jordanian patients suffering from gastroenteritis.

Little is known about the prevalence of intestinal protozoa in patients suffering from diarrhea in Jordan. The present study aimed to detect and speciate Entamoeba, Blastocystis, and Cryptosporidium species in a total of 159 human patients with diarrhea from November 2014 to October 2016. The overall prevalence for the three parasites was 19.5% (31/159). Entamoeba spp. (Entamoeba. dispar and/or Entamoeba histolytica), Blastocystis hominis, and Cryptosporidium parvum subtype IIaA15G2R1 were detected in 12.6%, 6%, and 0.6 of samples, respectively. This is the first molecular study in Jordan to confirm the diagnosis of Entamoeba species and to discriminate between E. histolytica and E. dispar.

South American Entamoeba dispar strains produce amoebic liver abscesses with different pathogenicities and evolutionary kinetics.

Amoebiasis is a protozoan disease caused by Entamoeba histolytica, and presents a geographic distribution of worldwide amplitude, high incidence, sometimes accompanied by severe clinical manifestations such as amoebic colitis and Amoebic Liver Abscess (ALA), remaining as a public health problem in developing countries. Entamoeba dispar is another species of amoeba that infects approximately 12% of the world's population, and it has previously been classified as noninvasive. However, E. dispar has already been isolated from patients with symptomatic non-dysenteric colitis, as well as its DNA sequences were detected and genotyped in samples from patients with dysenteric colitis, and patients with ALA, suggesting that this species could also be involved in the development of lesions in the large intestine and liver of human beings. In this context, this study aims to evaluate the ability of isolated strains of Entamoeba dispar in South America to cause liver damage, and to better characterize histopathological findings in 3, 8, 12 and 16 days after infection (DAI). Firstly, we assessed whether trophozoites from MCR, ACFN, ICS, ADO and VEJ E. dispar strains, and EGG Entamoeba histolytica strain differed in their in vitro phagocytosis ability, being related to greater ability to phagocyte with greater virulence. Then, we investigate and characterize histopathological changes present in the liver of mice induced by different strains of E. dispar. Our results demonstrated that trophozoites from E. dispar strains are capable of phagocyting human erythrocytes, but in lower amounts than Entamoeba histolytica. In addition, we described and characterized the lesions in different periods after infection by different E. dispar strains, and identified ACFN as the most pathogenic strain, followed by MCR. The large areas of necrosis produced by the ACFN strain as the eighth DAI, which also show high parasitism, led to 100% mortality. On the other hand, the ICS, ADO and VEJ strains did not produce mortality, and this was correlated with the presence of well-developed chronic granulomatous inflammation, necrosis absorption throughout the infection, and regeneration of the liver parenchyma. The greater pathogenicity of the ACFN strain strongly suggests that this strain could be producing higher levels of virulence factors. As the experimental infection, the heterogeneity of biological behavior of different Entamoeba dispar strains could be involved in the development of undiagnosed human clinical conditions.

Analysis of Human Gut Microbiota Composition Associated to the Presence of Commensal and Pathogen Microorganisms in Côte d'Ivoire.

BACKGROUND: The human gut microbiota is a microbial ecosystem contributing to the maintenance of host health with functions related to immune and metabolic aspects. Relations between microbiota and enteric pathogens in sub-Saharan Africa are scarcely investigated. The present study explored gut microbiota composition associated to the presence of common enteric pathogens and commensal microorganisms, e.g., Blastocystis and Entamoeba species, in children and adults from semi-urban and non-urban localities in Côte d'Ivoire. METHODS: Seventy-six stool samples were analyzed for microbiota composition by 16S rRDNA sequencing. The presence of adeno-, entero-, parechoviruses, bacterial and protozoal pathogens, Blastocystis, and commensal Entamoeba species, was analyzed by different molecular assays. RESULTS: Twelve individuals resulted negative for any tested microorganisms, 64 subjects were positive for one or more microorganisms. Adenovirus, enterovirus, enterotoxigenic Escherichia coli (ETEC), and Blastocystis were frequently detected. CONCLUSIONS: The bacterial composition driven by Prevotellaceae and Ruminococcaceae confirmed the biotype related to the traditional dietary and cooking practices in low-income countries. Clear separation in UniFrac distance in subjects co-harboring Entamoeba hartmanni and Blastocystis was evidenced. Alpha diversity variation in negative control group versus only Blastocystis positive suggested its possible regulatory contribution on intestinal microbiota. Pathogenic bacteria and virus did not affect the positive outcome of co-harbored Blastocystis.

The epidemiology of amoebiasis in Thi-Qar Province, Iraq (2015-2020): differentiation of Entamoeba histolytica and Entamoeba dispar using nested and real-time polymerase chain reaction.

OBJECTIVES: The objective of this study was to evaluate the present status of amoebiasis in Thi-Qar Province in southern Iraq, and to determine the presence of Entamoeba histolytica and Entamoeba dispar with nested and real-time polymerase chain reaction (PCR). METHODS: Epidemiological data were obtained from the public health department of the Thi-Qar Health Office (2015-2020). Eighty stool samples were also randomly collected from patients ≤12 year of age with diarrhea at 2 hospitals between the beginning of February 2020 and the end of October 2020. These samples were selected after microscopy to identify the 18S rRNA gene in Entamoeba DNA. RESULTS: Of the 341,554 cases of intestinal parasitic infections, 38,004 (11.1%) individuals were recorded as having amoebiasis, which accounted for the highest proportion of infections in 2015 (26.1%) and the lowest in 2020 (8.1%). Amoebiasis was distributed among all age groups, with the age group of 5-14 years accounting for the highest proportion (27.3%). In molecular testing, 42 (52.5%) out of 80 samples were positive for the 18S rRNA gene (888 bp). Using nested PCR, E. histolytica (439 bp) was detected in 25 (31.3%) samples and E. dispar (174 bp) in 14 (17.5%), while using real-time PCR, E. histolytica and E. dispar were detected in 28 (35.0%) and 15 (18.8%) samples, respectively. CONCLUSIONS: Epidemiological data confirmed that amoebiasis is endemic in this province, and is not limited to certain months. Our study confirms the applicability of molecular identification to detect pathogenic and non-pathogenic Entamoeba to prescribe the appropriate drug.

A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans.

There are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

The Entamoeba lysine and glutamic acid rich protein (KERP1) virulence factor gene is present in the genomes of Entamoeba nuttalli, Entamoeba dispar and Entamoeba moshkovskii.

The lysine and glutamic acid rich protein KERP1 is a cell surface-expressed virulence factor in the human pathogen Entamoeba histolytica. It was originally suggested that the gene was absent from the related, avirulent human commensal Entamoeba dispar, an absence which would be relevant to the differential virulence of these species. Here, the gene is shown to be present in E. dispar, and its sequence is presented, as well as in a virulent parasite of macaques, Entamoeba nuttalli, and the primarily free living, opportunistically parasitic Entamoeba moshkovskii.

Weissella paramesenteroides WpK4 ameliorate the experimental amoebic colitis by increasing the expression of MUC-2 and the intestinal epithelial regeneration.

AIMS: This study evaluates the action of Weissella paramesenteroides WpK4 on amoebic colitis. METHODS AND RESULTS: Weissella paramesenteroides WpK4 was administered in Entamoeba dispar infected and noninfected mice and clinical parameters were evaluated. Following 7 days, the caeca were collected for histopathology, morphometry and immunohistochemical staining of MUC-2, CDC-47 and IgA. The treatment reduced diarrhoea and the presence of blood in the faeces and diminished the area of necrosis, also causing weight gain. Also, the addition of this bacterium enhanced the expression of the mucin (MUC-2). The reduction in necrosis and increased CDC-47 expression indicates significant epithelial regeneration. The negative correlation between CDC-47 and the necrosis area reveals that the bacterium favoured the recovery of the necrotic regions and the positive correlation found between the expression of MUC-2 and CDC-47 indicates that the epithelial regeneration also supports the synthesis of MUC-2. CONCLUSIONS: Weissella paramesenteroides WpK4 was able to increase the protection of the intestinal mucosa against experimental amoebic colitis through the increase of MUC-2 and epithelial regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: Weissella paramesenteroides WpK4 presents the potential to become a complementary tool in the treatment of amoebic colitis.

Prevalence of Intestinal Parasitic Infestation Among Expatriate Workers.

Background Parasitic infestations of the gastrointestinal tract remain a common problem in third-world countries. Poverty, illiteracy, poor hygiene, scarcity of potable water, as well as the hot and humid tropical climate, are all contributing factors associated with intestinal parasitic infestation. Objective This cross-sectional study aims to evaluate the prevalence of intestinal parasitic infestation amongst expatriate workers in Benghazi City, Libya. Patients and methods A total of 250 stool samples (200 male and 50 female) were randomly collected between October 2017 to April 2018 from expatriate workers in Benghazi City, Libya. The samples examined were used to detect the presence of intestinal parasitic infestation while the study utilized a pre-tested structure. Cases were matched based on demographic parameters, such as age, gender, and nationality, while the history of diarrhea was recorded using direct smear microscopy for the detection of intestinal parasitic infestation. Results Of the 250 immigrants looking for work, 95 (38%) were found to be infested with two or more intestinal parasites. The protozoa included: Blastocystis hominis, Giardia lamblia, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium parvum (47.4%, 38.9%, 17.9%, 17.9%, and 4.2%, respectively); the non-pathogenic protozoa included the prevalence of Entamoeba coli (E. coli), which is 12.6%, and the helminth Ascaris lumbricoidesis 1.1%. Conclusion The prevalence of parasitic infection was relatively high (38%) and was affected by individual hygiene. Therefore, comprehensive healthcare education aimed at reducing parasitic infestation is needed.

Molecular identification of Entamoeba histolytica from stool samples of Ilam, Iran.

INTRODUCTION: Amoebiasis is a multifactorial, life-threatening public health issue and the third parasitic disease cause of mortality in worldwide, particularly in low- and mid-income countries. The aim of this study was to attempt to explore genetic encoding differences of CP8 (conserved gene) of Entamoeba histolytica/Entamoeba dispar in its various infectious properties isolated from Ilam located at a southwest part of Iran. MATERIALS AND METHODS: A total of 2023 stool samples were collected between 2016 and 2018 from the hospital in Ilam, of which only 30 isolates were identified as E. histolytica/E. dispar. These isolates were collected from the intensive care unit, infectious disease, and surgery settings. The isolates were identified and the polymerase chain reaction (PCR) was performed to detect the CP8 gene. In all stages, Entamoeba histolytica HM1: IMSS was used as a positive control. RESULTS: In genotype confirmation, only two isolates had the CP8 gene found in the PCR technique. The sequencing results confirmed the mentioned gene with 99%-100% specificity. CONCLUSION: It is concluded that PCR is highly sensitive to detect E. histolytica and indicating this important role as screening tools in direct DNA extraction from stool samples and valuable technique in early detection of symptomatic and asymptomatic E. histolytica patients.

Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation.

Amoebiasis is an infection of global importance, caused by the eukaryotic parasite Entamoeba histolytica. Pathogenic E. histolytica is associated worldwide with over a million cases of amoebic dysentery, colitis, and amoebic liver abscess. In contrast, the nonpathogenic Entamoeba dispar does not cause these diseases, although it is commonly found in the same areas as pathogenic amoeba. Entamoeba histolytica infection is usually associated with infiltrating neutrophils. These neutrophils appear to play a defensive role against this parasite, by mechanisms not completely understood. Recently, our group reported that neutrophil extracellular traps (NET) are produced in response to E. histolytica trophozoites. But, there is no information on whether nonpathogenic E. dispar can also induce NET formation. In this report, we explored the possibility that E. dispar leads to NET formation. Neutrophils were stimulated by E. histolytica trophozoites or by E. dispar trophozoites, and NET formation was assessed by video microscopy. NET induced by E. histolytica were important for trapping and killing amoebas. In contrast, E. dispar did not induce NET formation in any condition. Also E. dispar did not induce neutrophil degranulation or reactive oxygen species production. In addition, E. histolytica-induced NET formation required alive amoebas and it was inhibited by galactose, N-acetylgalactosamine, and lactose. These data show that only alive pathogenic E. histolytica activates neutrophils to produce NET, and suggest that recognition of the parasite involves a carbohydrate with an axial HO- group at carbon 4 of a hexose.

Children from a rural region in The Chiapas Highlands, Mexico, show an increased risk of stunting and intestinal parasitoses when compared with urban children / Alto riesgo de desmedro y parasitosis intestinal en niños de una región rural de los Altos de Chiapas, México, en comparación con niños de una región urbana

Abstract Background: The state of Chiapas has held the first place of extreme poverty in Mexico. The majority of Chiapas' municipalities are inhabited by marginalized, indigenous populations, who usually present diarrhea of unknown etiology. We evaluated the nutritional status, intestinal parasites, and common bacterial pathogens, including DEC (diarrheagenic Escherichia coli) strains, in 178 children under five years of age with a high (rural) and a moderate (urban) degree of marginalization. Methods: Z-scores for anthropometric indexes from the children were obtained, whereas intestinal parasites were investigated by using a direct coproparasitoscopic analysis and a concentration method. DEC strains were detected by polymerase chain reaction (PCR). Results: The stunting prevalence in children from the rural and urban regions was 79.8 and 7.5%, respectively. Only children from rural municipalities were parasitized (72.6%), being Ascaris lumbricoides and Entamoeba histolytica/Entamoeba dispar the most prevalent parasites (57.1 and 38.1%, respectively). More than half of the children presented moderated ascariasis. Besides Giardia intestinalis, these parasites were associated with stunting. The prevalence of DEC strains was similar in both regions. Conclusions: Only children from the Chiapas Highlands (rural zone) exhibited high prevalences of stunting and intestinal parasites. A reevaluation of social development programs should be in place to address stunting and intestinal parasitoses, mainly in rural regions of Chiapas, to avoid adverse functional consequences on these children.

Resumen Introducción: El estado de Chiapas ha ostentado el primer lugar de pobreza extrema en México. La mayor parte de la población de los municipios de Chiapas es indígena, vive en condiciones de marginación y padece de diarrea de etiología desconocida. Este trabajo evaluó el estado nutricional, la presencia de parásitos intestinales y patógenos bacterianos comunes, además de cepas DEC (Escherichia coli diarreogénica) en 178 niños menores de cinco años, provenientes de una localidad con alto grado de marginación (rural) y de una con moderada marginación (urbana). Métodos: Se obtuvieron los puntajes Z de los índices antropométricos de los niños. Los parásitos intestinales se investigaron con el método coproparasitoscópico directo y un método de concentración. Las cepas DEC se detectaron mediante reacción en cadena de la polimerasa. Resultados: La prevalencia de desmedro en niños de la zona rural y urbana fue de 79.8 y 7.5%, respectivamente. Únicamente los niños de la zona rural estuvieron parasitados (72.6%), y los más prevalentes fueron Ascaris lumbricoides y Entamoeba histolytica/Entamoeba dispar (57.1 y 38.1%, respectivamente). Más de la mitad de los infantes exhibieron ascariasis moderada. Estos parásitos, además de Giardia intestinalis, se asociaron con el desmedro. En ambas regiones, la prevalencia de DEC fue similar. Conclusiones: Solo los niños de los Altos de Chiapas (zona rural) exhibieron alta prevalencia de desmedro y parásitos intestinales. Para evitar las consecuencias adversas entre los infantes, es necesario reevaluar los programas de desarrollo social para combatir el desmedro y la parasitosis intestinal, principalmente en las regiones rurales de Chiapas.

Children from a rural region in The Chiapas Highlands, Mexico, show an increased risk of stunting and intestinal parasitoses when compared with urban children.

Background: The state of Chiapas has held the first place of extreme poverty in Mexico. The majority of Chiapas' municipalities are inhabited by marginalized, indigenous populations, who usually present diarrhea of unknown etiology. We evaluated the nutritional status, intestinal parasites, and common bacterial pathogens, including DEC (diarrheagenic Escherichia coli) strains, in 178 children under five years of age with a high (rural) and a moderate (urban) degree of marginalization. Methods: Z-scores for anthropometric indexes from the children were obtained, whereas intestinal parasites were investigated by using a direct coproparasitoscopic analysis and a concentration method. DEC strains were detected by polymerase chain reaction (PCR). Results: The stunting prevalence in children from the rural and urban regions was 79.8 and 7.5%, respectively. Only children from rural municipalities were parasitized (72.6%), being Ascaris lumbricoides and Entamoeba histolytica/Entamoeba dispar the most prevalent parasites (57.1 and 38.1%, respectively). More than half of the children presented moderated ascariasis. Besides Giardia intestinalis, these parasites were associated with stunting. The prevalence of DEC strains was similar in both regions. Conclusions: Only children from the Chiapas Highlands (rural zone) exhibited high prevalences of stunting and intestinal parasites. A reevaluation of social development programs should be in place to address stunting and intestinal parasitoses, mainly in rural regions of Chiapas, to avoid adverse functional consequences on these children.

Introducción: El estado de Chiapas ha ostentado el primer lugar de pobreza extrema en México. La mayor parte de la población de los municipios de Chiapas es indígena, vive en condiciones de marginación y padece de diarrea de etiología desconocida. Este trabajo evaluó el estado nutricional, la presencia de parásitos intestinales y patógenos bacterianos comunes, además de cepas DEC (Escherichia coli diarreogénica) en 178 niños menores de cinco años, provenientes de una localidad con alto grado de marginación (rural) y de una con moderada marginación (urbana). Métodos: Se obtuvieron los puntajes Z de los índices antropométricos de los niños. Los parásitos intestinales se investigaron con el método coproparasitoscópico directo y un método de concentración. Las cepas DEC se detectaron mediante reacción en cadena de la polimerasa. Resultados: La prevalencia de desmedro en niños de la zona rural y urbana fue de 79.8 y 7.5%, respectivamente. Únicamente los niños de la zona rural estuvieron parasitados (72.6%), y los más prevalentes fueron Ascaris lumbricoides y Entamoeba histolytica/Entamoeba dispar (57.1 y 38.1%, respectivamente). Más de la mitad de los infantes exhibieron ascariasis moderada. Estos parásitos, además de Giardia intestinalis, se asociaron con el desmedro. En ambas regiones, la prevalencia de DEC fue similar. Conclusiones: Solo los niños de los Altos de Chiapas (zona rural) exhibieron alta prevalencia de desmedro y parásitos intestinales. Para evitar las consecuencias adversas entre los infantes, es necesario reevaluar los programas de desarrollo social para combatir el desmedro y la parasitosis intestinal, principalmente en las regiones rurales de Chiapas.

Update on laboratory diagnosis of amoebiasis.

Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries, causing up to 100,000 fatal cases annually. Detection of the pathogenic E. histolytica and its differentiation from the non-pathogenic Entamoeba spp. play a crucial role in the clinical management of patients. Laboratory diagnosis of intestinal amoebiasis in developing countries still relies on labour-intensive and insensitive methods involving staining of stool sample and microscopy. Newer and more sensitive methods include a variety of antigen detection ELISAs and rapid tests; however, their diagnostic sensitivity and specificity seem to vary between studies, and some tests do not distinguish among the Entamoeba species. Molecular detection techniques are highly sensitive and specific and isothermal amplification approaches may be developed into field-applicable tests; however, cost is still a barrier for their use as a routine laboratory test method in most endemic areas. Laboratory diagnosis of extraintestinal amoebiasis faces challenges of lack of definitive detection of current infection and commercially available point-of-care tests. For both types of amoebiasis, there is still a need for highly sensitive and specific tests that are rapid and cost-effective for use in developing countries where the disease is prevalent. In recent years, new molecules of diagnostic value are being discovered and new tests developed. The advances in 'omics' technologies are enabling discoveries of new biomarkers that may help distinguish between different infection stages.

Prevalence of Intestinal Parasites and Associated Risk Factors for Infection among Rural Communities of Chachoengsao Province, Thailand.

Intestinal parasitic infections remain a major public health problem in many parts of Thailand, particularly in rural areas. This study aimed to determine the prevalence of intestinal parasitic infections and associated risk factors among the people living in Huai Sai sub-district, Bang Khla district, Chachoengsao Province, central Thailand. A cross-sectional survey was carried out from June 2017 to August 2017 which included a total of 224 participants. Stool samples were examined using a simple direct smear and formalin ethyl acetate concentration technique. Association between risk factors and intestinal parasitic infections was assessed using multivariate logistic regression analysis. The overall prevalence of intestinal parasitic infections was 16.1%. Soil-transmitted helminth infections (14.3%) were more common than protozoan infections (1.8%). The most common intestinal parasites were hookworms (6.7%) followed by Strongyloides stercoralis, (5.0%), Ascaris lumbricoides (1.3%) and Trichuris trichiura (1.3%). Entamoeba histolytica/dispar (1.0%), Giardia intestinalis (0.4%), and Blastocystis hominis (0.4%) were the protozoans identified. A high prevalence of infections was found in male participants of ≥40 years who raised dogs in the households and did not wear boots while working fields. Multivariate analysis showed a significant association of intestinal parasitic infections with gender with the adjusted odds ratio (OR) of 2.4 and 95% confidence interval (CI) of 1.1-5.2 (P=0.020). The results showed a high prevalence of soil-transmitted helminth infections among adults in rural communities which were particularly apparent regarding the skin-penetrating species of nematodes. A greater focus on intervention is required by improving sanitation and personal hygiene to prevent the spread of intestinal parasitic infections.

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification.

OBJECTIVES: Besides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. METHODS: This evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). RESULTS: This new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. CONCLUSION: Given the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.

Prevalence of Intestinal Parasites and Associated Risk Factors for Infection among Rural Communities of Chachoengsao Province, Thailand

Intestinal parasitic infections remain a major public health problem in many parts of Thailand, particularly in rural areas. This study aimed to determine the prevalence of intestinal parasitic infections and associated risk factors among the people living in Huai Sai sub-district, Bang Khla district, Chachoengsao Province, central Thailand. A cross-sectional survey was carried out from June 2017 to August 2017 which included a total of 224 participants. Stool samples were examined using a simple direct smear and formalin ethyl acetate concentration technique. Association between risk factors and intestinal parasitic infections was assessed using multivariate logistic regression analysis. The overall prevalence of intestinal parasitic infections was 16.1%. Soil-transmitted helminth infections (14.3%) were more common than protozoan infections (1.8%). The most common intestinal parasites were hookworms (6.7%) followed by Strongyloides stercoralis, (5.0%), Ascaris lumbricoides (1.3%) and Trichuris trichiura (1.3%). Entamoeba histolytica/dispar (1.0%), Giardia intestinalis (0.4%), and Blastocystis hominis (0.4%) were the protozoans identified. A high prevalence of infections was found in male participants of ≥40 years who raised dogs in the households and did not wear boots while working fields. Multivariate analysis showed a significant association of intestinal parasitic infections with gender with the adjusted odds ratio (OR) of 2.4 and 95% confidence interval (CI) of 1.1–5.2 (P=0.020). The results showed a high prevalence of soil-transmitted helminth infections among adults in rural communities which were particularly apparent regarding the skin-penetrating species of nematodes. A greater focus on intervention is required by improving sanitation and personal hygiene to prevent the spread of intestinal parasitic infections.

Chitinase-gene-based analysis of the genetic variability among the clinical isolates of Entamoeba dispar from Puducherry, India.

INTRODUCTION: Amebiasis is known to be caused by the protozoan parasite Entamoeba histolytica. Entamoeba dispar is considered to be a sibling species of E. histolytica, as the two are phylogenetically closest. There are reports that certain strains of E. dispar isolated were capable of causing hepatic lesions in the experimental animal models. The intra-/inter-species genetic variation has been found to have profound implication in the invasiveness of the disease. Thus, studying polymorphism in E. dispar aids to improve our perspective related to the variability in the genome of the parasite. MATERIALS AND METHODS: The highly polymorphic region of the gene encoding the enzyme chitinase was targeted for the strain variation analysis in E. dispar. Isolates from the stool and liver abscess aspirate were subjected to the polymerase chain reaction (PCR) for the amplification of the targeted polymorphic loci. The PCR products were sequenced, and genetic variability analysis was carried out. RESULTS: A total of 23 samples in the stool and 1 sample from liver abscess pus were positive for E. dispar by nested multiplex PCR which was confirmed by sequencing. Of these positive samples, 13 amplified for chitinase gene by PCR. We observed seven genotypes in our study isolates, of which four were found to be distinct. CONCLUSION: This study shows that high degree of genetic variation exists among the clinical isolates of E. dispar in our location. The future studies including the analysis of other genetic makers such as serine-rich E. dispar protein or other loci have to be carried out to get an idea about the distribution of the different strains of E. dispar.

Prevalence, molecular epidemiology, and zoonotic potential of Entamoeba spp. in nonhuman primates in China.

Amebiasis is a major public-health concern. It has a global distribution, and is listed as the third leading parasitic cause of human mortality. To survey the prevalence and assess the potential zoonotic transmission of Entamoeba spp. in nonhuman primates (NHPs) in China, 2688 fresh fecal specimens were collected from NHPs reared in farms or zoos/parks or free ranging in 13 districts. The overall prevalence of Entamoeba spp. infection determined with microscopy analysis was 39.4% (1059/2688). Higher infection rates were detected in the free ranging group (41.1%, 169/411) and in animals <1year old (58.7%, 556/947). Gene fragments were successfully amplified 463 (87.2%) out of the 531 selected specimens (approximately half of the total microscopy-positive specimens). Polymerase chain reaction (PCR) amplification identified 386 (83.4%) Entamoeba dispar and 287 (62.0%) E. coli infections, and among these, 210 (45.4%) were mixed infections. And that the E. dispar and E. coli had also been detected in humans. In phylogenetic analysis, the E. dispar and E. coli sequences clustered with reference E. dispar and E. coli, respectively. In conclusion, nonhuman primates infected with Entamoeba species, with high prevalence and zoonotic potential, should be considered when evaluating the maintenance of Entamoeba spp. and its transmission between animal reservoirs and humans.

Molecular prevalence of Entamoeba histolytica/dispar infection among patients attending four health centres in north-west Ethiopia.

The prevalence of amoebiasis is often overestimated owing to its epidemiological overlap with the non-pathogenic Entamoeba dispar To provide evidence for this conjecture, a cross-sectional study was conducted from November 2013 to January 2015. A range of 180-200 µg of semi-solid and formed stools and 200 µL of diarrhoeic stool samples were used for DNA extraction from microscopically E. histolytica/dispar positive samples using the QIAamp® DNA Stool Mini Kit according to manufacturers' instructions. Nested PCR targeting 18S ribosomal RNA gene was used. In 422 microscopically positive E. histolytica/dispar stools, molecular prevalence revealed that E. histolytica infestation was present in only 1.7% (95% confidence interval [CI], 0.47-2.93) and E. dispar was found in 42.2% (95% CI, 37.49-46.91), while 56.2% (95% CI, 51.47-60.93) had neither E. histolytica nor E. dispar (P < 0.001). We conclude that infestation with E. histolytica is rarer in our study areas than was previously believed. Hence, accurate differentiation of E. histolytica and E. dispar is crucial.