Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

Detection and molecular identification of Entamoebaspecies in faecal samples from Duhok province, Kurdistan Region, Iraq.

The study involved the estimation of the prevalence of Entamoeba spp. using microscopy and molecular techniques among symptomatic outpatients during April 2021 to March, 2022. Stool samples were collected from 2592 outpatients with amoebiasis symptoms of both sexes and different ages (≤ l to 60). Also, 107 stool samples were taken randomly from asymptomatic individuals and examined microscopically to detect infection with Entamoeba spp. the positive specimens were used for molecular analysis with positive symptomatic samples targeting the 18S rRNA gene by nested PCR. Microscopically 21.68% (562/2592) were positive, for Entamoeba spp. Males showed highest infection rate than females (67.43% vs 32.56%). Ages from 1-10 years showed the highest rate (54.09%), and urban inhabitant had somewhat a higher rate than rural one (58.54% vs 41.45%) which was statistically non-significant(P>0.05). Among asymptomatic individuals, 57% (61/107) were positive for Entamoeba spp. Nested PCR analysis yielded 73% positive samples for Entamoeba spp. with a fragment size of 897 bp. Three fragment sizes were produced, for E. histolytica, E. dispar and E. moshkovskii which were 439, 174 and 553 bps, respectively. Single infection occurred with, E. histolytica in 46%, of symptomatic and 6% of asymptomatic cases, E. dispar in 38% of asymptomatic and 10% of symptomatic cases, E. moshkovskii, reported at very low rate among both groups.

Survey of Entamoeba infections in schoolchildren and macaques in Kathmandu, Nepal, and analysis of genetic polymorphisms of Entamoeba nuttalli and Entamoeba dispar isolates.

Entamoeba species infect humans and non-human primates, raising concerns associated with potential zoonotic transmission. Therefore, the prevalence of human Entamoeba infections is crucial for its management in areas, where macaques exhibit high infection rates. Previously, we demonstrated prevalent E. nuttalli infections in rhesus macaques in Kathmandu, Nepal. In this study, we surveyed Entamoeba infection among 185 schoolchildren from two schools visited by wild rhesus macaques to assess the risk of transmission. PCR-based screening for Entamoeba species identified E. coli in 13 % and E. dispar in 0.5 % of the human stool samples. However, E. nuttalli and E. chattoni infections, prevalent in macaques, were not detected in human samples. This suggests that Entamoeba spp. are not transmitted through macaques in the school environment. We surveyed the rhesus macaques living in the temple near schools as well as the rhesus and Assam macaques inhabiting Shivapri Nagarjun National Park, Kathmandu. Among the 49 macaque stool samples, E. chattoni, E. coli, E. nuttalli, and E. dispar were detected in 92 %, 86 %, 41 %, and 18 % of the samples, respectively. Notably, E. dispar infections in macaques were mostly prevalent in the temple. A sample isolated from Nagarujun showed an identical genotype at two tRNA-linked short tandem repeat loci to that of E. dispar isolated from humans, suggesting potential transmission from humans to macaques. Genotypic analysis of cultured E. nuttalli strains obtained from the macaques colonizing three locations demonstrated that the geographical distance rather than differences in macaque species played a crucial role in the genetic diversity of the parasites. The phylogenetic tree of E. nuttalli strains, including the previously isolated strains, reflected the geographical distribution of the isolation sites. This study sheds light on the intricate dynamics of Entamoeba transmission and genetic diversity in macaques and humans.

The macrophage polarization in Entamoeba histolytica infection modulation by the C fragment of the intermediate subunit of Gal/GalNAc-inhibitable lectin.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, with clinical outcomes ranging from asymptomatic infections to severe invasive diseases. The innate immune system, particularly macrophages, is of paramount importance in resisting the invasion of host tissues and organs by the trophozoites of E. histolytica. Parasite-derived pathogenic factors, such as lectins, play a pivotal role in the promotion of macrophage polarization phenotypes that have undergone alteration. Nevertheless, the precise mechanisms by which E. histolytica modulates immune polarization remain largely unknown. The current study focused on the immunomodulatory effects of the Igl-C fragment of E. histolytica Gal/GalNAc lectin on macrophage polarization. These results demonstrated that Igl-C could induce the secretion of IL-1ß, IL-6, and other cytokines, activating a mixed M1/M2 polarization state. M1 polarization of macrophages occurs in the early stages and gradually transitions to M2 polarization in the later stages, which may contribute to the persistence of the infection. Igl-C induces the macrophage M1 phenotype and causes the release of immune effector molecules, including iNOS and cytokines, by activating the NF-κB p65 and JAK-STAT1 transcription factor signaling pathways. Furthermore, Igl-C supports the macrophage M2 phenotype via JAK-STAT3 and IL-4-STAT6 pathways, which activate arginase expression in later stages, contributing to the tissue regeneration and persistence of the parasite. The involvement of distinct signaling pathways in mediating this response highlights the complex interplay between the parasite and the host immune system. These findings enhance our understanding of the Igl-C-mediated pathogenic mechanisms during E. histolytica infection.

Entamoeba histolytica in Different Water Sources of Nigde Province of Turkey.

PURPOSE: This study was carried out to determine the presence of Entamoeba histolytica in water sources of Nigde province in Turkey, between June and November 2021. METHODS: A total of 90 water samples were taken from 15 different water sources (drinking water, well water, spring water, wastewater and dam water) every month and the presence of E. histolytica antigens in the samples was examined by ELISA. RESULTS: The positivity for E. histolytica was determined in 7 (7.7%) of 90 samples. While no antigens were found in any of the samples in June and September, E. histolytica was positive for three samples (20%) in July, one sample (6.6%) in August and October and two samples in November (13.3%). One of 24 dam samples (4.1%), 1 of 12 wastewater samples (8.3%), 1 of 12 well samples (8.3%), and 4 of 24 fountain samples (16.6%) that examined by ELISA were found positive. On the other hand, none of the examined 18 spring samples were positive. In addition, 4 (8.8%) of 45 samples that examined in summer and 3 (6.6%) of 45 samples that examined in autumn were detected positive by using ELISA. Entamoeba histolytica positivity in samples was statistically insignificant in terms of months, water resources and seasons (P > 0.05). CONCLUSION: As a result, the presence of E. histolytica, which is an important public health problem in water sources, was determined for the first time in Nigde province of Türkiye with this study.

Molecular characterization and zoonotic potential of Entamoeba spp., Enterocytozoon bieneusi and Blastocystis from captive wild animals in northwest China.

BACKGROUND: Parasites Entamoeba spp., Enterocytozoon bieneusi and Blastocystis are prevalent pathogens causing gastrointestinal illnesses in animals and humans. Consequently, researches on their occurrence, distribution and hosts are crucial for the well-being of both animals and humans. Due to the confined spaces and frequent interaction between animals and humans, animal sanctuaries have emerged as potential reservoirs for these parasites. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China is chosen as an ideal site for parasite distribution research, considering its expansive stocking area and high biodiversity. RESULTS: We collected 191 fecal specimens from 37 distinct wildlife species and extracted genomic DNA. We identified these three parasites by amplifying specific gene regions and analyzed their characteristics and evolutionary relationships. All the parasites exhibited a high overall infection rate, reaching 90.05%. Among them, seven Entamoeba species were identified, accounting for a prevalence of 54.97%, with the highest infection observed in Entamoeba bovis. In total, 11 Enterocytozoon bieneusi genotypes were discovered, representing a prevalence of 35.08%, including three genotypes of human-pathogenic Group 1 and two novel genotypes (SXWZ and SXLG). Additionally, 13 Blastocystis subtypes were detected, showing a prevalence of 74.87% and encompassing eight zoonotic subtypes. All of the above suggests significant possibilities of parasite transmission between animals and humans. CONCLUSIONS: This study investigated the occurrence and prevalence of three intestinal parasites, enhancing our understanding of their genetic diversity and host ranges in northwest China. Furthermore, the distribution of these parasites implies significant potential of zoonotic transmission, underscoring the imperative for ongoing surveillance and implementation of control measures. These efforts are essential to mitigate the risk of zoonotic disease outbreaks originating from wildlife sanctuary.

Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.

A high-dimensional platform for observing neutrophil-parasite interactions.

Diarrheal diseases with infectious etiology remain a major cause of death globally, particularly in low-income countries. Entamoeba histolytica is a pathogenic protozoan parasite that is the causative agent of amebiasis. Amebiasis has a wide presentation in clinical severity with many factors, including the bacterial microbiota, contributing to this variation. The innate immune response also plays a critical role in regulating the severity of E. histolytica infection, with neutrophils reported to have a protective role. Despite this, the precise mechanism of how neutrophils mediate amebic killing is poorly understood. Thus, modern platforms that allow for inquiry of granulocyte-ameba interactions will increase our understanding of this disease. Herein, we describe an assay for neutrophil killing of E. histolytica by utilizing high-dimensional spectral flow cytometry. Neutrophils were isolated from wild-type 5-week-old C57BL/6 mice and co-cultured with E. histolytica at various multiplicity of infections (MOIs). After co-culture, neutrophils and E. histolytica were stained for spectral flow cytometry. Cell populations were identified using surface markers and fluorescence minus one (FMO) controls. We have previously shown that animals colonized with a component of the human microbiota, Clostridium scindens, were protected from E. histolytica. This protection was associated with elevated neutrophil count. Here, we explored amebic killing capacity and observed that neutrophils from animals with C. scindens possessed heightened amebic killing compared with controls. Thus, this study establishes a novel platform that can provide an in-depth analysis of granulocyte-parasite interactions in various contexts, including during alteration of the intestinal microbiota.IMPORTANCEThe tools for studying host immune cell-E. histolytica interactions are limited. Factors, such as parasite heterogeneity, infectivity, and difficulties with culture systems and animal models, make interrogation of these interactions challenging. Thus, Entamoeba researchers can benefit from next-generation models that allow for the analysis of both host and parasite cells. Here, we demonstrate the use of a novel platform that allows for the determination of parasite-host cell interactions and customizable high-dimensional phenotyping of both populations. Indeed, spectral flow cytometry can approach >40 markers on a single panel and can be paired with custom-developed parasite antibodies that can be conjugated to fluorochromes via commercially available kits. This platform affords researchers the capability to test highly precise hypotheses regarding host-parasite interactions.

Entamoeba moshkovskii as a potential model organism for Gal/GalNAc lectin intermediate subunit exhibition and functional identification.

In humans, Entamoeba histolytica is the main pathogen causing various amoebiases, while E. moshkovskii falls between being a pathogen and non-pathogen. The two species have similar behavior patterns but differ significantly in pathogenicity, with previous studies and clinical data indicating that E. moshkovskii has a low level of pathogenicity. Meaningfully, the biological characteristics of E. moshkovskii make it a potential model organism and a protein display platform for studying the functions of important Entamoeba proteins. Here, an Amoeba-pcDNA3.1 vector capable of overexpressing E. histolytica-sourced Igl-C protein was constructed and successfully transfected into E. moshkovskii. High levels of expression of the Igl-C, EGFP, and NeoR genes were identified in Igl-C-transfected trophozoites using qRT-PCR, and they were subsequently confirmed using immunoblotting. Transfection of Igl-C protein improved the adherence and phagocytosis of E. moshkovskii, demonstrating that E. histolytica Igl mediated amoebic adhesion. Moreover, as a manifestation of protein virulence, the ability of post-transfected trophozoites to induce inflammation in host macrophages was also enhanced. In conclusion, this study utilizing the characteristics of E. moshkovskii confirmed its potential to serve as a model organism. E. moshkovskii could replace E. histolytica as the target of gene editing, allowing more efficient study of amoebic pathogenicity.

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.

Epidemiological investigation of Entamoeba in wild rhesus macaques in China: A novel ribosomal lineage and genetic differentiation of Entamoeba nuttalli.

Wild rhesus macaques are a potential source of zoonotic parasites for humans, and Entamoeba spp. are common intestinal parasites. To investigate the prevalence of Entamoeba in wild rhesus macaques in China and explore the genetic differentiation of the potentially pathogenic species Entamoeba nuttalli, a total of 276 fecal samples from five populations at high altitudes (HAG, 2,800-4,100 m above sea level) and four populations at low altitudes (LAG, 5-1,000 m above sea level) were collected. PCR methods based on the ssrRNA gene were used to detect Entamoeba infection. Genotyping of E. nuttalli was performed based on six tRNA-linked short tandem repeat (STR) loci for further genetic analyses. The results revealed that Entamoeba infection (69.2%) was common in wild rhesus macaques in China, especially in LAG which had a significantly higher prevalence rate than that in HAG (P < 0.001). Three zoonotic species were identified: Entamoeba chattoni (60.9%) was the most prevalent species and distributed in all the populations, followed by Entamoeba coli (33.3%) and Entamoeba nuttalli (17.4%). In addition, a novel Entamoeba ribosomal lineage named RL13 (22.8%) was identified, and phylogenetic analysis revealed a close genetic relationship between RL13 and Entamoeba. hartmanni. Genotyping of E. nuttalli obtained 24 genotypes from five populations and further analysis showed E. nuttalli had a high degree of genetic differentiation (FST > 0.25, Nm < 1) between the host populations. The result of analysis of molecular variance (AMOVA) revealed that observed genetic differences mainly originate from differences among populations (FST = 0.91). Meanwhile, the phylogenetic tree showed that these genotypes of E. nuttalli were clustered according to geographical populations, indicating a significant phylogeographic distribution pattern. Considering the potential pathogenicity of E. nuttalli, attention should be paid to its risk of zoonotic transmission.

Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers.

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1­M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.

Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Multi-locus sequence analysis reveals phylogenetically segregated Entamoeba histolytica population.

Amoebiasis, caused by the enteric parasite, Entamoeba histolytica, is one of the major food- and water-borne parasitic diseases in developing countries with improper sanitation and poor hygiene. Infection with E. histolytica has diverse disease outcomes, which are determined by the genetic diversity of the infecting strains. Comparative genetic analysis of infecting E. histolytica strains associated with differential disease outcomes from different geographical regions of the world is important to identify the specific genetic patterns of the pathogen that trigger certain disease outcomes of Amoebiasis. The strategy is able to elucidate the genealogical relation and population structure of infecting E. histolytica strains from different geographical regions. In the present study, we have performed a comparative genetic analysis of circulating E. histolytica strains identified from different parts of the world, including our study region, based on five tRNA-linked short tandem repeat (STR) loci (i.e., D-A, NK2, R-R, STGA-D and A-L) and evaluated their potential associations with differential disease outcomes of Amoebiasis. A number of regional-specific, emerging haplotypes of E. histolytica, significantly associated with specific disease outcomes have been identified. Haplotypes, which have a significant positive association with asymptomatic and amoebic liver abscess outcomes, showed a significant negative association with diarrheal outcome, or vice versa. Comparative multi-locus analysis revealed that E. histolytica isolates from our study region are phylogenetically segregated from the isolates of other geographical regions. This study provides a crucial overview of the population structure and emerging pattern of the enteric parasite, E. histolytica.

Short tandem repeat (STR) based sequence typing of Entamoeba histolytica identifies STGA-D locus as a genetic marker, associated with disease outcomes.

Amoebiasis, caused by the enteric parasite Entamoeba histolytica has differential disease outcomes. The association of parasite genotypes with outcomes of amoebic infection is still a paradox and requires to be explored. The genetic information of infecting strains from endemic settings of different geographical regions is essential to evaluate the relation. Comparative genetics of E. histolytica clinical isolates from different disease outcomes have been explored based on two tRNA-linked STR loci (STGA-D and A-L). All of the repeat patterns in the A-L locus were newly identified and unique to Indian isolates. The majority of newly identified repeat patterns in STGA-D locus have outcome-specific distributions, predicting the emergence of disease-specific mutations in this target locus. Statistical analysis further reinforces this observation, as identified repeat patterns only from STGA-D but not A-L locus were significantly associated with disease outcomes. Phylogenetic analysis indicates independent segregation and divergence of tRNA-linked STR arrays for each STR locus.

Investigating genetic polymorphism in E. histolytica isolates with distinct clinical phenotypes.

Amoebiasis is an infection caused by enteric protozoa, most commonly Entamoeba histolytica, and is globally considered a potentially severe and life-threatening condition. To understand the impact of the parasite genome on disease outcomes, it is important to study the genomes of infecting strains in areas with high disease prevalence. These studies aim to establish correlations between parasite genotypes and the clinical presentation of amoebiasis. We employ a strain typing approach that utilizes multiple loci, including SREHP and three polymorphic non-coding loci (tRNA-linked array N-K2 and loci 1-2 and 5-6), for high-resolution analysis. Distinct clinical phenotype isolates underwent amplification and sequencing of studied loci. The nucleotide sequences were analysed using Tandem Repeats Finder to detect short tandem repeats (STRs). These patterns were combined to assign a genotype, and the correlation between clinical phenotypes and repetitive patterns was statistically evaluated. This study found significant polymorphism in the size and number of PCR fragments at SREHP and 5-6 locus, while the 1-2 locus and NK2 locus showed variations in PCR product sizes. Out of 41 genotypes, two (I6 and I41) were significantly associated with their respective disease outcomes and were found in multiple isolates. We observed that I6 was linked with a symptomatic outcome, with a statistically significant p-value of 0.0183. Additionally, we found that I41 was associated with ALA disease outcome, with a p-value of 0.0089. Our study revealed new repeat units not previously reported, unveiling the genetic composition of E. histolytica strains in India, associated with distinct disease manifestations.

First molecular detection of Entamoeba gingivalis subtypes in individuals from Turkey.

Entamoeba gingivalis is a parasitic protozoan that colonizes the human oral cavity and there are two subtypes (ST1 and ST2) that have been identified to date. However, there are no reports on the molecular detection or characterization of E. gingivalis in Turkey. The objective of this study was to detect the presence of E. gingivalis in Turkish healthy individuals and those with periodontal disease and to subtype the isolates using molecular techniques. Samples from the oral cavity of 94 individuals were taken and the presence of E. gingivalis was determined by PCR using primers for SsrRNA and the amplicons were then confirmed by DNA sequencing. Each participant completed a questionnaire that included demographic data, habits and lifestyle, as well as health status. The presence of E. gingivalis was detected in a total of 19 samples (11 patients and eight healthy individuals). Molecular characterization determined that 12 samples belonged to ST1 and seven samples belonged to ST2. The presence of E. gingivalis was higher in patients with periodontal disease than in healthy individuals, and this association was statistically significant (P < .05). This study constitutes the first report of molecular detection and subtyping of E. gingivalis in Turkey.

COMPARISON BETWEEN PCR STUDY AND ELISA STUDY AMONG PATIENTS WITH DIARRHEA.

Amoebic dysentery is a common infectious disease that is acquired through contaminated food and water harboring the infective stage of the parasite. Entamoeba histolytica is a parasite that is spread globally causing increasing morbidity and mortality in developing countries. To Identify the infection of Entamoeba histolytica by PCR and other lab methods among patients attending Kirkuk hospitals. Methods: The current study involved the examination of 220 fecal specimens from children under 15 years during the period of 1st January 2023 until 5th of June 2023. It involved microscopic examination of fecal samples confirmation of diagnosis with two different ELISA tests that capture E. histolytic. Also, microscopic positive samples were submitted to nucleic acid detection of E. histolytic via Real-Time PCR. The percentage of positive specimens that were tested with E. histolytica / dispar ELISA (DRG ELISA), out of 93 stool specimens, 59 (63.44%) were positive, while the remaining specimens 34 (36.56%) were negative despite being tested positive by microscopy. The DRG stool ELISA revealed sensitivity and specificity (69.28% and 97.91%) respectively and a predictive value of (97%). The sample that was the positive result with DRG ELISA was discriminated via Tech Lab E. histolytica ELISA which detects the presence of only E. histolytica alone in fecal samples. Out of 93 examined specimens, only 24 (25.81%) were positive while the remaining 69 (74.19%) were negative. DRG ELISA for E. histolytica/dispar positive results were 63.44%, while TechLab ELISA has produced 25.81% positive E. histolytica. Whereases, RT PCR results were only 20.44%. Qi square analysis was applied and yielded a significant difference v=between the method of diagnosis with P=<0.0001. Microscopy-positive Entamoeba complex is a primitive means of detection of Entamoeba complex and diagnosis should always be confirmed with superior method like ELISA or PCR.

A landscape of gene regulation in the parasitic amoebozoa Entamoeba spp.

Entamoeba are amoeboid extracellular parasites that represent an important group of organisms for which the regulatory networks must be examined to better understand how genes and functional processes are interrelated. In this work, we inferred the gene regulatory networks (GRNs) in four Entamoeba species, E. histolytica, E. dispar, E. nuttalli, and E. invadens, and the GRN topological properties and the corresponding biological functions were evaluated. From these analyses, we determined that transcription factors (TFs) of E. histolytica, E. dispar, and E. nuttalli are associated mainly with the LIM family, while the TFs in E. invadens are associated with the RRM_1 family. In addition, we identified that EHI_044890 regulates 121 genes in E. histolytica, EDI_297980 regulates 284 genes in E. dispar, ENU1_120230 regulates 195 genes in E. nuttalli, and EIN_249270 regulates 257 genes in E. invadens. Finally, we identified that three types of processes, Macromolecule metabolic process, Cellular macromolecule metabolic process, and Cellular nitrogen compound metabolic process, are the main biological processes for each network. The results described in this work can be used as a basis for the study of gene regulation in these organisms.