Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

The actin cytoskeleton orchestra in Entamoeba histolytica.

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.

A Cross-Sectional Study of Entamoeba histolytica/dispar/moshkovskii Complex in Salvador, Bahia, Brazil.

Epidemiological studies on species-specific Entamoeba infections are scarce due to the morphological similarity of pathogenic Entamoeba histolytica and nonpathogenic E. dispar and E. moshkovskii. The diagnosis of E. histolytica is frequently based on coproantigen (E. histolytica-Gal/GalNAc lectin specific) detection by immunoassays. However, specific E. histolytica-lectin is not expressed in cysts, which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation of amebiasis prevalence. Molecular techniques based on the amplification of parasite DNA have been shown to be a highly sensitive and specific method that allows the detection of different Entamoeba species. This study aimed to assess the frequency of the species from E. histolytica/dispar/moshkovskii complex by molecular and immunological techniques in individuals attended at a public health system in Salvador-Bahia, Brazil. A cross-sectional study involving 55,218 individuals was carried out. The diagnosis was based on microscopy revealing E. histolytica/dispar/moshkovskii complex. The species differentiation was performed by E. histolytica-specific antigen, serological evaluation and by molecular technique. The overall prevalence of E. histolytica/dispar/moshkovskii complex determined by microscopy was approximately 0.49% (273/55,218). E. histolytica-specific antigen detection and molecular characterization returned 100% negativity for E. histolytica. However, serological evaluation returned an 8.9% positivity (8/90). In the stool samples analysed by PCR, it was not possible to identify E. histolytica and E. moshkovskii, although circulating IgG anti-E. histolytica has been detected.

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.

Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals.

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens.

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

Amoebiasis in Iran: a systematic review and meta-analysis.

A comprehensive meta-analysis study was performed to estimate the reliable national prevalence and molecular epidemiology of amoebiasis in Iran. Nine English and Persian databases were searched to achieve the relevant studies. Pooled estimates were generated and meta-regression was performed. We identified 71 eligible articles involving 330 930 subjects from 25 provinces to be included in the final analysis. Moreover, 17 studies compromising 462 polymerase chain reaction (PCR)-positive isolates performed molecular analysis to inter-species differentiation. The pooled prevalence of Entamoeba infection among Iranian population was about 1% (95% CI 0.8-2.0%). Moreover, regarding Human Development Index (HDI), a higher prevalence was observed in undeveloped provinces. Out of 462 PCR-positive isolates, 83% (95% CI 69-94%) and 12% (95% CI 3-24%) were Entamoeba dispar, Entamoeba histolytica, respectively. In subgroup analysis based on molecular results, in general, population prevalence of Entamoeba dispar and E. histolytica were 91% (95% CI 80-99%) and 7%, (95% CI 0-19%), respectively, while prevalence of these species in patients with gastrointestinal disorders were 75% (95% CI 45-96%) and 18% (95% CI 1-43%), respectively. Our findings indicate the low burden of amoebiasis in Iran. E. dispar, that is mostly non-pathogenic, was identified as most prevalent species. Nevertheless, we suggest more public health interventions in areas with lower HDI.

SINE polymorphism reveals distinct strains of Entamoeba histolytica from North India.

Entamoeba histolytica is an intestinal parasite causing significant morbidity and mortality in the developing world. More tools are needed to understand the epidemiology and molecular pathogenesis of amebiasis. Virulence pattern of E. histolytica could be linked with the genotype of a strain. Several loci showing insertion polymorphism of retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2 have been reported among laboratory strains of E. histolytica. The present study was undertaken to validate this observation in clinical isolates from north India. Our results indicate that the Indian samples show a different propensity of SINE retention or loss at two of the polymorphic loci compared with non-Indian samples. Statistical analysis of different loci revealed Locus 17 of EhSINE1as a potential geographical marker for distinguishing Indian isolates from non Indian isolates.

Gene migration for re-emerging amebiasis in Iran's northwest-Iraq borders: a microevolutionary scale for reflecting epidemiological drift of Entamoeba histolytica metapopulations.

In the microevolutionary scales of Entamoeba isolates, the gene migration shows how Entamoeba spp. has epidemiologically drifted among border countries. Five hundred fecal samples were taken from patients suffering gastrointestinal disorders, abdominal pain, and diarrhea at Saggez, northwest Iran located within the border Iraq country. Following parasitological techniques, DNA samples were extracted and amplified by polymerase chain reaction (PCR) of 18S rRNA region to identify Entamoeba infections. To distinguish the Entamoeba spp., a multiplex PCR was conducted. Amplicons were sequenced to reconfirm their heterogeneity traits and phylogenetic analysis. Additionally, Entamoeba histolytica sequences of Iraq were retrieved from GenBank database. The suspected isolates were diagnosed as E. histolytica (2.2 %), Entamoeba moshkovskii (1 %), and Entamoeba dispar (0.4 %). Mixed Entamoeba infections did not detect among isolates. A parsimonious network of the sequence haplotypes displayed star-like features in the overall isolates containing E.h1, E.d2, and E.m3 as the most common haplotypes. According to analysis of molecular variance (AMOVA) test, high partial value of haplotype diversity (0.700 to 0.800) of E. histolytica was shown the total genetic variability within populations while nucleotide diversity was low among Iranian and Iraqi metapopulations. Neutrality indices of the 18S rRNA were shown negative values in E. histolytica populations which indicating significant deviations from neutrality. A pairwise fixation index (F-statistics [Fst]) as a degree of gene flow had a low value for all populations (0.001) while the number of migrants was 2.48. The statistically Fst value indicates that E. histolytica isolates are not genetically differentiated among shared isolates of Iran and Iraq. Occurrence of E.h1 between two regional populations indicates that there is dawn of Entamoeba flow due to transfer of alleles from one population to another population through host mobility and ecological alterations. To evaluate the hypothetical evolutionary scenario, further study is required to analyze Entamoeba spp. in the neighboring Middle East countries.

Functionally conserved RNA-binding and protein-protein interaction properties of LINE-ORF1p in an ancient clade of non-LTR retrotransposons of Entamoeba histolytica.

Retrotransposons are mobile genetic elements found in most organisms. Their origin and evolution is not very well understood. Retrotransposons that lack long terminal repeats (non-LTR) have been classified based on their reverse transcriptase (RT) and endonuclease sequences into groups, of which R2 is the most ancient. Its members contain a single open reading frame (ORF) while there are two ORFs in the other groups, of which ORF2 contains the RT and endonuclease sequences. It is thought that ORF1 was added later to the single-ORF-containing elements, and codes for a protein with nucleic acid binding activity. We have examined the non-LTR retrotransposons in Entamoeba histolytica, an early-branching parasitic protist, which belongs to the R2 group. However, unlike other members of R2, E. histolytica contains two ORFs. Here we show that EhLINE1-ORF1p is functionally related to the ORF1p found in the non-R2 groups. Its N-terminal region has RNA-binding activity and its C-terminal has a coiled coil domain which participates in protein-protein interaction. It lacks sequence-specificity of RNA-binding and binds to EhLINE1-RNA fragment and ribosomal RNA with comparable affinities. Our study suggests that ORF1p could have evolved independently to maintain functional conservation.

Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba.

Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria.

Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa.

A PCR-RFLP method for the simultaneous differentiation of three Entamoeba species.

Amoebiasis caused by Entamoeba histolytica continues to be one of the most common parasitic diseases in the developing world. Despite its relevance, due to the lack of accurate diagnostic methods, the true clinical and public health importance of this parasite remains uncertain. The aim of this study was to develop a new diagnostic tool to differentiate E.histolytica from the morphologically undistinguishable E.dispar and E.moshkovskii. We developed a specific, fast and simple PCR-RFLP method that was able to accurately differentiate experimentally-obtained restriction patterns from the three Entamoeba species. This new method could prove useful for clinical and epidemiological purposes.

Copro prevalence and estimated risk of Entamoeba histolytica in Diarrheic patients at Beni-Suef, Egypt.

Amoebiasis diagnosis is usually based on microscopy that cannot differentiate pathogenic E. histolytica from morphologically identical non-pathogenic species. 194 fecal samples were collected from diarrheic &/or dysenteric patients and examined for Entamoeba complex microscopically, E. histolytica/E. dispar coproantigen using ICT and E. histolytica coproantigen using Tech lab E. histolytica II ELISA test. Entamoeba complex trophozoites/cysts, E. histolytica/E. dispar coproantigen and E. histolytica coproantigen were detected in 22.2, 14.4 and 3.6 % of samples, respectively. Microscopy and ICT method had limited sensitivity with poor PPV (9.3 and 7.1 %, respectively) and both slightly agree with ELISA test. The prevalence of E. histolytica was low (3.6 %) in studied individuals and was 14 times lower than non-pathogenic amoebae. E. histolytica detection studied individuals was positively associated with mucoid and bloody stool, which makes them disease predictors. E. histolytica fecal ELISA assay for E. histolytica detection surpassed microscopy and E. histolytica/E. dispar ICT assay. This has highlighted the need for practical non-microscopic detection methods that can differentiate between amoeba infections to avoid unnecessary and possibly harmful therapies and to determine the true prevalence and epidemiology of E. histolytica.

Genetic variability of the serine-rich Entamoeba histolytica protein gene in clinical isolates from the United Arab Emirates.

The genetic diversity of 20 Entamoeba histolytica isolates from asymptomatic individuals from the UAE was investigated by analyzing polymorphism in the serine-rich E. histolytica gene (SREHP) by nested polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) on DNA extracted directly from stool samples. The SREHP gene was successfully amplified in 15 out of 20 E. histolytica-positive samples. Four out of the remaining five isolates did not amplify for the SREHP gene. Despite successful amplification of the SREHP gene in the fifth isolate, AluI digestion of the amplified PCR product revealed no bands. As a result, all five samples were excluded from the study. Twelve different profiles were obtained from the 15 successfully amplified isolates. Thus, demonstrating extensive genetic variability and reinforcing the argument that E. histolytica has an extremely polymorphic genetic structure. Despite the sample size limitation, a finding in the study was the occurrence of one profile common to one Indian isolate while another profile common to one Pakistani isolate; indicating the possibility of clonal infection. Furthermore, we found one isolate from a Bangladeshi expatriate identical to 2 asymptomatic Bangladeshi isolates reported in an earlier study. No clear association between the different genotypes and the study population demographics was noted. The results also indicated the possibility of strains clustering by region.

Perforación intestinal secundaria a amebiasis / Amebiasis intestinal perforation secondary

La amebiasis intestinal es una enfermedad frecuente en países en desarrollo, que es común en regiones tropicales y subtropicales, así como en regiones con servicios sanitarios deficientes. Presentamos el caso de un paciente de 74 años de edad, sexo masculino, originario y residente de la ciudad de Guatemala, quién cursó una diarrea y dolor abdominal. Fue intervenido quirúrgicamente por abdomen agudo, con resección intestinal extensa por perforaciones. En el estudio de anatomía patológica se realizó el diagnóstico de colitis amebiana con perforaciones y peritonitis.

Intestinal amoebiasis is a disease common indeveloping countries, which is common in tropicaland subtropical regions, as well as in regions withpoor sanitation. We report the case of a 74-year-oldmale, resident of Guatemala City, who presented withdiarrhea and abdominal pain. He had a laparatomy foracute abdomen, undergoing wide intestinal resectiondue to perforations. The pathology diag-nosis wasamoebic colitis with perforations and peritonitis.

Prevalence and genetic diversity of Entamoeba species infecting macaques in southwest China.

Many colonies of macaques (Macaca fascicularis and Macaca mulatta) are maintained in China, especially in Guangxi and Guizhou. A total of 803 fresh stool samples infected with Entamoeba were obtained from three big colonies of macaques located in southwest China. The samples were examined for the presence of five Entamoeba species using PCR. Entamoeba nuttalli, Entamoeba dispar, Entamoeba coli, and Entamoeba chattoni infections were detected, but Entamoeba histolytica infection was not. This study is the first to report on the prevalence of E. nuttalli in wild macaques from China. Eighteen E. nuttalli isolates and five E. dispar isolates were obtained by culturing the samples in Tanabe-Chiba medium. The serine-rich protein (SRP), ribosomal RNA (rRNA), hexokinase (HXK), glucose-6-phosphate isomerase (GPI), and phosphoglucomutase (PGM) genes of E. nuttalli isolates were compared with other reported isolates. The results showed clear differences among the Chinese E. nuttalli isolates and other isolates based on the SRP gene sequences. However, HXK, GPI, and PGM genes of these strains were similar to those of other isolates. The rRNA genes of E. coli and E. chattoni were also amplified and analyzed from these samples. The results suggested that host species might be a more important factor than geographic location in amebic genetic diversity.

Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

A Multilocus Sequence Typing System (MLST) reveals a high level of diversity and a genetic component to Entamoeba histolytica virulence.

BACKGROUND: The outcome of an Entamoeba histolytica infection is variable and can result in either asymptomatic carriage, immediate or latent disease (diarrhea/dysentery/amebic liver abscess). An E. histolytica multilocus genotyping system based on tRNA gene-linked arrays has shown that genetic differences exist among parasites isolated from patients with different symptoms however, the tRNA gene-linked arrays cannot be located in the current assembly of the E. histolytica Reference genome (strain HM-1:IMSS) and are highly variable. RESULTS: To probe the population structure of E. histolytica and identify genetic markers associated with clinical outcome we identified in E. histolytica positive samples selected single nucleotide polymorphisms (SNPs) by multiplexed massive parallel sequencing. Profile SNPs were selected which, compared to the reference strain HM-1:IMSS sequence, changed an encoded amino acid at the SNP position, and were present in independent E. histolytica isolates from different geographical origins. The samples used in this study contained DNA isolated from either xenic strains of E. histolytica trophozoites established in culture or E. histolytica positive clinical specimens (stool and amebic liver abscess aspirates). A record of the SNPs present at 16 loci out of the original 21 candidate targets was obtained for 63 of the initial 84 samples (63% of asymptomatically colonized stool samples, 80% of diarrheal stool, 73% of xenic cultures and 84% of amebic liver aspirates). The sequences in all the 63 samples both passed sequence quality control metrics and also had the required greater than 8X sequence coverage for all 16 SNPs in order to confidently identify variants. CONCLUSIONS: Our work is in agreement with previous findings of extensive diversity among E. histolytica isolates from the same geographic origin. In phylogenetic trees, only four of the 63 samples were able to group in two sets of two with greater than 50% confidence. Two SNPs in the cylicin-2 gene (EHI_080100/XM_001914351) were associated with disease (asymptomatic/diarrhea p = 0.0162 or dysentery/amebic liver abscess p = 0.0003). This study demonstrated that there are genetic differences between virulent and avirulent E. histolytica strains and that this approach has the potential to define genetic changes that influence infection outcomes.