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.

A Case Of Entamoeba Histolytica Infection Among Men Who Have Sex With Men.

Entamoeba histolytica is a parasite responsible for intestinal amebiasis and possible extraintestinal manifestations, such as liver abscesses. India, Africa, Mexico, and Central and South America have some of the highest rates of infection due to poor socioeconomic and environmental conditions. The infection has become more common in the United States due to an increase in emigration. There has been a rising incidence of sexual transmission of the infection, most commonly seen in men who have sex with men. Here, we present a case of a symptomatic extraintestinal E histolytica infection in a young Hispanic bisexual man.

Molecular diagnosis of amoebiasis.

Amoebiasis is an intestinal parasitosis caused by the protozoan Entamoeba histolytica that represents the third leading cause of mortality due to parasitosis. It is a prevalent disease in tropical climate regions with poor or absent sanitary services. Microscopy and antigen detection techniques are routinely used to diagnose amoebiasis because of their low cost and ease of application. However, these techniques do not differentiate E. histolytica infections and other potentially pathogenic species such as Entamoeba moshkovskii or Entamoeba bangladeshi. Therefore, in the last decades, molecular tests that allow correct identification of the causal agent of amoebiasis and the establishment of the prevalence of the infecting species have been developed. Techniques based on nucleic acids, such as conventional, multiplex, or real-time polymerase chain reaction (PCR), are being seriously considered in clinical laboratories, because they detect the etiologic agent directly from the sample without the need for previous prolonged culture, thus reducing diagnostic time. Also, the nested PCR test and the sequencing of ribosomal markers have allowed the identification of new parasitic species in humans, such as E. moshkovskii and E. bangladeshi, and an improved characterization of the known infecting species. The application of multiplex platforms allows the simultaneous identification of infecting species, increasing the sensitivity and specificity of these techniques. Therefore, the molecular diagnosis of amoebiasis is projected as an innovative tool in the fight against this parasitosis.

La amebiasis es una parasitosis intestinal causada por el protozoario Entamoeba histolytica y representa la tercera causa de mortalidad por parasitosis. Es una enfermedad prevalente en regiones de clima tropical con deficientes o nulos servicios sanitarios. Las técnicas de microscopía y detección de antígenos se emplean sistemáticamente para el diagnóstico de la amebiasis por su bajo costo y fácil aplicación. Sin embargo, no permiten diferenciar entre infecciones por E. histolytica y otras especies de potencial patogenicidad como Entamoeba moshkovskii o Entamoeba bangladeshi. Ante ello, en las últimas décadas se han desarrollado pruebas moleculares que permiten una correcta identificación del agente causal de la amebiasis y el establecimiento de la prevalencia de la especie infectante. Las técnicas basadas en ácidos nucleicos, como la reacción en cadena de la polimerasa (PCR) convencional, múltiple o en tiempo real, están siendo seriamente consideradas en los laboratorios clínicos, porque detectan al agente etiológico de manera directa en la muestra sin necesidad de cultivo prolongado previo, disminuyendo de esta forma el tiempo del diagnóstico. Asimismo, la PCR anidada sumada a la secuenciación de marcadores ribosomales ha permitido la identificación de nuevas especies parasitarias, como E. moshkovskii y E. bangladeshi en humanos, y una mejor caracterización de las especies infectantes ya conocidas. La aplicación de las plataformas multiplex permite la identificación simultánea de especies infectantes, aumentando la sensibilidad y la especificidad de estas técnicas. Por esto, el diagnóstico molecular de la amebiasis se proyecta como una verdadera herramienta innovadora en la lucha contra las parasitosis.

Socio-environmental and parasitological aspects of waste pickers in Minas Gerais, Brazil

The increase in the generation of Solid Urban Waste causes social, environmental problems and damages to the population's health. Professionals who work in the collection of recyclable waste are exposed to risks of contamination either by toxic elements or pathogenic organisms. The objective of the work was to estimate the types and prevalence of intestinal parasites inwaste pickers. A field research was carried out from December 2017 to April 2018 with the voluntary participation of 26 waste pickers belonging to three associations in the municipality of Conselheiro Lafaiete, Minas Gerais, Brazil (CAAE: nº 79937817.7.0000.8122). In addition to the application a socio-environmental questionnaire, each volunteer provided a stool sample for laboratory testing the parasitological examination. Of the 26 survey participants, four (15.4%) had a positive result and were infected by the parasites Giardia lamblia, Entamoebacoliand Iodamoeba butschlii. Among the main factors that can contribute to the infection these waste pickersare the ingestion of untreated water for consumption in addition to reduced access to Personal Protective Equipment(PPE) during waste management. One way to control the presence of parasites would be through health and environmental education actions, periodic parasitological examinations and permanent use of PPE.

An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth.

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Epidemiology of clinically relevant Entamoeba spp. (E. histolytica/dispar/moshkovskii/bangladeshi): A cross sectional study from North India.

BACKGROUND: Entamoeba infections have major impact on millions of the people worldwide. Entamoeba histolytica has long been accepted as the only pathogenic species. However, recent reports of other Entamoeba spp. in symptomatic cases have raised questions on their pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: Total 474 stool samples and 125 liver aspirates from patients with intestinal and extra intestinal manifestations and from community were included. Sewage samples from the hospital and the city were also included. Microscopic examination and molecular detection were performed to detect presence of E. histolytica/ dispar/ moshkovskii/ bangladeshi. The associated demographic and socioeconomic factors were statistically analyzed with the presence of Entamoeba. Microscopy detected Entamoeba spp. in 5.4% stool and 6.4% liver aspirate samples. Through nested multiplex PCR, prevalence of Entamoeba spp. in intestinal and extra-intestinal cases was 6.6% (20/301) and 86.4% (108/125) respectively and in asymptomatic population was 10.5% (13/123). Sewage samples did not show presence of any Entamoeba spp. Uneducated subjects, low economic conditions, untreated drinking water, consumption of raw vegetables and habit of not washing hands before meals were significantly associated with presence of Entamoeba spp. CONCLUSIONS: E. histolytica still remains the only Entamoeba spp. in invasive extra intestinal infections. E. dispar was detected in both asymptomatic and symptomatic intestinal infections. Routine identification of Entamoeba spp. should incorporate PCR based detection methods.

Prevalence of Intestinal Parasite Infections and Their Associated Factors among Food Handlers Working in Selected Catering Establishments from Bule Hora, Ethiopia.

Intestinal parasites are responsible for one of the major health problems like food contamination with socioeconomic effects in the world with a prevalence rate of 30-60%, in developing countries that lie within tropical and subtropical areas. They pose a reasonable public health burden, particularly in low- and middle-income countries, including Ethiopia. Globally, due to intestinal parasitic infections, around 3.5 billion people are affected and more than 200,000 deaths are reported annually. Around 50000 deaths yearly are caused by intestinal parasites in Ethiopia. As such, intestinal parasites perceived global and local burdens to various countries. The risk of food contamination depends largely on the health status of the food handlers, their hygiene, knowledge, and practice of food hygiene. Food handlers with poor personal hygiene and sanitation conditions are the major potential sources of intestinal helminthes and protozoa worldwide. The proposed study was aimed at evaluating prevalence of intestinal parasitic infections and their associated factors among food handlers working in selected catering establishments. A cross-sectional study was conducted in Bule Hora Town from March to April 2020. A total of 136 catering establishments were selected using a systematic sampling technique. Data analysis was done using SPSS version 20. The prevalence of intestinal parasites in this study was 46.3%. Entamoeba histolytica was the most predominant parasite (33.3%, i.e., 21/63) while Giardia lamblia was the least (11.1%, i.e., 7/63). Consumption of vended or borehole water and hygienic practices such as hand washing before eating, after using toilet, before cooking and trimming of finger nail and wearing proper working clothes and shoes were statistically significant with intestinal parasitic infection (P < 0.05). Generally, the prevalence of intestinal parasitic infection in this study was high and contributed by low socioeconomic status and poor environmental and personal hygiene. Measures including education on personal hygiene, environmental sanitation, drinking water supply, regular medical checkups, and treatment should be taken into account to reduce the prevalence of intestinal parasites.

Feeding sites promoting wildlife-related tourism might highly expose the endangered Yunnan snub-nosed monkey (Rhinopithecus bieti) to parasite transmission.

An increasing number of studies have found that the implementation of feeding sites for wildlife-related tourism can affect animal health, behaviour and reproduction. Feeding sites can favour high densities, home range overlap, greater sedentary behaviour and increased interspecific contacts, all of which might promote parasite transmission. In the Yunnan snub-nosed monkey (Rhinopithecus bieti), human interventions via provisioning monkeys at specific feeding sites have led to the sub-structuring of a group into genetically differentiated sub-groups. The fed subgroup is located near human hamlets and interacts with domesticated animals. Using high-throughput sequencing, we investigated Entamoeba species diversity in a local host assemblage strongly influenced by provisioning for wildlife-related tourism. We identified 13 Entamoeba species or lineages in faeces of Yunnan snub-nosed monkeys, humans and domesticated animals (including pigs, cattle, and domestic chicken). In Yunnan snub-nosed monkeys, Entamoeba prevalence and OTU richness were higher in the fed than in the wild subgroup. Entamoeba polecki was found in monkeys, pigs and humans, suggesting that this parasite might circulates between the wild and domestic components of this local social-ecological system. The highest proportion of faeces positive for Entamoeba in monkeys geographically coincided with the presence of livestock and humans. These elements suggest that feeding sites might indirectly play a role on parasite transmission in the Yunnan snub-nosed monkey. The implementation of such sites should carefully consider the risk of creating hotspots of disease transmission, which should be prevented by maintaining a buffer zone between monkeys and livestock/humans. Regular screenings for pathogens in fed subgroup are necessary to monitor transmission risk in order to balance the economic development of human communities dependent on wildlife-related tourism, and the conservation of the endangered Yunnan snub-nosed monkey.

Entamoeba Chitinase is Required for Mature Round Cyst Formation.

Entamoeba histolytica, a protozoan parasite, causes amoebiasis in humans. Amoebiasis transmission is solely mediated by chitin-walled cysts, which are produced in the large intestine of humans from proliferative trophozoites by a cell differentiation process called encystation. Resistance to environmental stresses, an essential characteristic for transmission, is attributed to the cyst wall, which is constructed from chitin and several protein components, including chitinase. Chitinase may play a key role in cyst wall formation; however, this has not been confirmed. Here, to elucidate the physiological role of chitinase during Entamoeba encystation, we identified a new chitinase inhibitor, 2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide, by recombinant-Entamoeba chitinase-based screening of 400 Pathogen Box chemicals. This compound dose dependently inhibited native chitinase associated with Entamoeba invadens encystation, a model for E. histolytica encystation, with an 50% inhibitory concentration (IC50) of ∼0.6 µM, which is comparable to the IC50s (0.2 to 2.5 µM) for recombinant E. histolytica and E. invadens chitinases. Furthermore, the addition of this compound to E. invadens encystation-inducing cultures increased the generation of cyst walls with an abnormal shape, the most characteristic of which was a "pot-like structure." A similar structure also appeared in standard culture, but at a far lower frequency. These results indicate that chitinase inhibition increases the number of abnormal encysting cells, thereby significantly reducing the efficiency of cyst formation. Transmission electron microscopy showed that compound-treated encysting cells formed an abnormally loose cyst wall and an unusual gap between the cyst wall and cell membrane. Hence, Entamoeba chitinase is required for the formation of mature round cysts. IMPORTANCE Amoebiasis is caused by Entamoeba histolytica infection and is transmitted by dormant Entamoeba cells or cysts. Cysts need to be tolerant to severe environmental stresses faced outside and inside a human host. To confer this resistance, Entamoeba parasites synthesize a wall structure around the cell during cyst formation. This cyst wall consists of chitin and several protein components, including chitinase. The physiological roles of these components are not fully understood. Here, to elucidate the role of chitinase during cyst formation, we identified a new chitinase inhibitor by screening a library of 400 compounds. Using this inhibitor, we showed that chitinase inhibition causes the formation of abnormal cyst walls, the most characteristic of which is a "pot-like structure." This results in decreased production of mature cysts. Chitinase is therefore required for Entamoeba to produce mature cysts for transmission to a new host.

Effect of magnetic field on the growth of the cultured Entamoeba histolytica isolated from patients in Palestine.

Static magnetic field (SMF) is generated in vicinity of moving charge or current passing through conductor. In this study, we aimed to investigate the effect of SMF on the growth of the cultured Entamoeba histolytica (E. histolytica) trophozoites. Different SMF strengths with maximum value equals 30 mT (mT) was applied on the E.histolytica for different periods of times: 0 h, 24 h, 48 h, and 72 h. A modified diphasic liver infusion agar medium was used for culturing E. histolytica in vitro. The results showed the successful stabilization of culture of E. histolytica trophozoites. If we kept the sample for longer time, e. g. 14 days, the growth rate decreases to zero. When applying 10 mT and 15 mT SMF on the sample, it is found that the cultivated E. histolytica trophozoites dies after 4 and 2 days respectively. The experiments suggested that the SMF inhibited the growth and the propagation of E. histolytica cells. In addition, it completely killed all the cells in a short time interval which depend on the SMF strength. It is concluded that the SMFs inhibits the growth of E. histolytica and change the morphology of these cells. Thus, we recommend to use SMF as treatment to mitigate the growth of E. histolytica.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.