The 26 S proteasome in Entamoeba histolytica: divergence of the substrate binding pockets from host proteasomes.

OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.

Overcoming Geographical Constraints: A Diagnostic and Therapeutic Challenge of Amebic Liver Abscess in a Non-Endemic Region.

This case report provides data on unique challenges related to amoebiasis diagnostics and treatment in non-endemic regions. The presented case report is focused a 28-year-old male patient of Indian origin, temporarily living in Poland, who was diagnosed with an amoebic liver abscess. The patient presented with a range of non-specific symptoms including shortness of breath, chest pain, and fever. The differential diagnosis included cardio-pulmonary diseases, a range of tropical diseases such as malaria or typhoid fever, bacterial abscesses, and malignancies, necessitating a comprehensive, multi-modal diagnostic approach. This approach included an extensive review of patient history, physical examination, and various laboratory and imaging investigations. A further challenge in this case was the unavailability of standard cysticidal treatments in Poland, which required individualized therapeutic strategy. Despite these obstacles, the patient was successfully treated using an alternative regimen of intravenous metronidazole, ceftriaxone, doxycycline, chloroquine, and finally, trimethoprim/sulfamethoxazole (treatment with metronidazole was used as a base drug, due to the lack of typical cysticidal treatment, an alternative treatment was added: chloroquine is a recommended drug used in the treatment of pregnant patients, in addition, doxycycline showed in vitro activity against Entamoeba histolytica). This therapeutic journey underscored the value of adaptability in treatment protocols, particularly in regions where certain resources may not be readily available. This case report underlines the importance of broadening the differential diagnosis in non-endemic regions to include tropical diseases, particularly in the context of increasing global travel and migration. It also highlights the significance of employing comprehensive diagnostic strategies and adaptable treatment protocols in such scenarios. In addition, the report reiterates the need for global collaboration and education among healthcare providers to effectively manage tropical diseases, especially in non-endemic regions. Through its exploration of the complexities associated with diagnosing and managing amebiasis in a non-endemic region, this report offers valuable insights to clinicians worldwide.

Introducing the NUATEI Consortium: A Mexican Research Program for the Identification of Natural and Synthetic Antimicrobial Compounds for Prevalent Infectious Diseases.

The need for new drugs to treat human infections is a global health concern. Diseases like tuberculosis, trypanosomiasis, amoebiasis, and AIDS remain significant problems, especially in developing countries like Mexico. Despite existing treatments, issues such as resistance and adverse effects drive the search for new alternatives. Herein, we introduce the NUATEI research consortium, made up of experts from the Institute of Biomedical Research at UNAM, who identify and obtain natural and synthetic compounds and test their effects against human pathogens using in vitro and in vivo models. The consortium has evaluated hundreds of natural extracts and compounds against the pathogens causing tuberculosis, trypanosomiasis, amoebiasis, and AIDS, rendering promising results, including a patent with potential for preclinical studies. This paper presents the rationale behind the formation of this consortium, as well as its objectives and strategies, emphasizing the importance of natural and synthetic products as sources of antimicrobial compounds and the relevance of the diseases studied. Finally, we briefly describe the methods of the evaluation of the compounds in each biological model and the main achievements. The potential of the consortium to screen numerous compounds and identify new therapeutic agents is highlighted, demonstrating its significant contribution to addressing these infectious diseases.

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.

Effect of Stevioside (Stevia rebaudiana) on Entamoeba histolytica Trophozoites.

Human amoebiasis still represents a major health problem worldwide. Metronidazole has been used as the most common drug to treat the disease; however, it is also known that the drug causes undesirable side effects. This has led to the search for new pharmacological alternatives which include phytochemical compounds with antiamoebic effects. We analyzed the amoebicidal activity of stevioside (STV), a diterpene glycoside present in Stevia rebaudiana, on trophozoites of E. histolytica. Different concentrations of STV were tested, and an inhibitory concentration of 50% of cell viability (IC50) was determined with an exposition of 9.53 mM for 24 h. Trophozoites exposed to STV showed morphological changes evidenced by the decrease in the basic structures related to the movement and adherence to the substrate, as well as ultrastructural features characterized by a loss of regularity on the cell membrane, an increase in cytoplasmic granularity, and an increase in apparent autophagic vacuoles. Also, the decrease in cysteine protease expression and the proteolytic activity of trophozoites to degrade the cell monolayer were analyzed. A histological analysis of hamster livers inoculated with trophozoites and treated with STV showed changes related to the granulomatous reaction of the liver parenchymal tissue. Our results constitute the first report related to the possible use of STV as a therapeutic alternative in amoebiasis.

Amoebic colitis-A diagnostic challenge on endoscopy: Case report.

Amoebic colitis remains a diagnostic challenge on colonoscopy given that its features resemble that of inflammatory bowel disease. We describe a similar case of a 66 years old male patient with multiple comorbidities including morbid obesity, end-stage renal disease requiring haemodialysis, IHD with PCI, T2DM, HTN and new onset dry cough for which he received a short course of steroids. He presented to the colorectal clinic with bleeding and mucus discharge per-rectum with no other symptoms related to bowel and non-specific colonoscopy findings with amoebic colitis only confirmed on histological diagnosis. The patient was commenced on a course of Metronidazole and followed up on first and sixth month after treatment where he remained well and reported no further symptoms.

Observational Study of Clinical Profiles and Management of Liver Abscess in Hospitalized Patients: A North Indian Tertiary Care Perspective.

Background Liver abscesses are a significant health concern, necessitating prompt diagnosis and appropriate management. Spontaneous liver abscesses are a frequent reason for hospitalizations in India, particularly in the northern part. By analyzing demographics, symptoms, radiological findings, laboratory parameters, and treatment outcomes, this study will contribute valuable insights to enhance the understanding and management of liver abscesses. Aims and objective To evaluate demographic, clinical, laboratory, and radiological parameters and management options in hospitalized patients with liver abscesses at a tertiary care center. Methods This study retrospectively analyzed prospectively collected data from 150 patients diagnosed with liver abscesses who were admitted to our ward for one year. Data on demographic characteristics, clinical presentation, etiology, radiological findings, laboratory investigations, management strategies, and treatment outcomes were collected. Descriptive statistics and relevant statistical tests were employed for data analysis. Results The study population had a mean age of 40.28±12.72 years, with a male preponderance (136 (90.7%)). Amoebic abscesses (94 (62.7%)) were the most common. Hepatomegaly (144 (96%)), fever (140 (93.3%)), abdominal pain (136 (90.7%)), and anorexia (118 (78.7%)) were the most common symptoms. Ultrasonography revealed solitary abscesses (99 (66%)) to be more common than multiple abscesses (24 (16%)), with a predominant location in the right lobe (128 (85.3%)). Laboratory investigations showed leukocytosis in 121 (80.7%), elevated liver enzymes (95 (63.3%) aspartate aminotransferase (AST) and 80 (53.3%) alanine transaminase (ALT)), elevated alkaline phosphatase (ALP) in 133 (88.7%), and low albumin levels (138 (92%)) in a significant proportion of patients. Single-time needle aspiration (95 (63.3%)), percutaneous drain (36 (24%)), and surgical intervention (4 (2.7%)) were the primary treatment modalities. Serum albumin level (p<0.001) and ALP (p<0.001) were significantly low and high, respectively, in patients with hospital stays ≥10 days. Conclusions This study provides insights into patients with liver abscesses' clinical and laboratory parameters and management strategies. The findings highlight the diverse clinical presentation, varied etiologies, and the importance of radiological imaging and laboratory investigations in diagnosis and management. Tailored treatment strategies based on the patient's condition are crucial for optimizing outcomes.

Amoebic Liver Abscess: Potential Application of New Diagnostic Techniques for an Old Pathogen.

Amoebic liver abscess is a severe and potentially life-threatening infection requiring prompt diagnosis and early targeted treatment. Diagnosis is challenging because conventional diagnostic methods such as light microscopy and serology are often unreliable. Molecular techniques have emerged as an additional diagnostic tool, suddenly becoming the new diagnostic reference standard. More recently, commercial multiplex PCR panels, including FilmArray, have been introduced, which permit the simultaneous detection of several enteric pathogens including Entamoeba histolytica in stool samples. We report a case of an amoebic liver abscess promptly diagnosed by FilmArray gastrointestinal panel performed on liver drainage fluid.

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.

Acute fulminant amoebic colitis: A Case report.

Amoebiasis is a parasitic infection that represents a public health problem in developing countries including Asia and Latin America where it is endemic (1000-5000 cases/100,000 habitants/year). The majority of patients have an asymptomatic course; however, 10% of patients develop complications with high morbidity and mortality, such as colonic perforation or fulminant amoebic colitis. We report a case in which a 73-year-old female presented with an acute abdomen that was initially attributed to a bowel obstruction that rapidly progressed to fulminant colitis with bowel perforation requiring total colectomy. Pre-surgical endoscopic histopathological examination revealed findings suggestive of Entamoeba histolytica trophozoites that were later confirmed in the colon post-surgical specimen leading to a diagnosis of fulminant amoebic colitis. This atypical presentation of amoebiasis, further expands the already broad differential diagnosis of acute abdominal pathology in the elderly population. A high index of suspicion is required for its prompt treatment and to prevent life-threatening complications.

A case report of colonic Ameboma mimicking colon cancer in an immunocompromised patient.

INTRODUCTION AND IMPORTANCE: Entamoeba histolytica is an anaerobic protozoan. It infects humans causing Amoebiasis. Most infections are asymptomatic; however, clinical manifestations include intestinal or extraintestinal. In rare instances, patients can present with Ameboma: a mass of granulation tissue consisting of a core of inflammation with peripheral fibrosis related to chronic amoebic infection usually found in the cecum/ascending colon. PRESENTATION OF CASE: A 50-year-old patient presented with signs and symptoms of bowel obstruction. He had raised inflammatory markers and was HIV positive on treatment and virally suppressed. At laparotomy a hepatic flexure stricture with desmoid reaction, and wall thickening with induration at the mesenteric border of mid-transverse colon was found, with disseminated nodular disease. Histology confirmed entamoeba histolytica infection. DISCUSSION: The parasite exists in two forms: a cyst stage and a trophozoite stage: the form causing invasive disease. The presentation of intestinal amoebiasis ranges from an asymptomatic carrier state, colitis, to abscess formation and bowel perforation The management principles: to eliminate the invading trophozoites and eradicate the organism's intestinal carriage and manage complications such as obstruction, fulminant colitis, or toxic megacolon with surgery. CONCLUSION: This case identifies a rare cause of bowel obstruction in a patient without a travel history. Even with the lack of significant risk factors other than HIV, clinicians should be aware of the infective causes of colonic mass/stricture outside of malignant causes. Entamoeba histolytica is an infection of impoverished communities in developing countries, it should remain high on our differential for tumour-like granulation tissue in the colon.

The Amoebicidal Activity of Diferrocenyl Derivatives: A Significant Dependence on the Electronic Environment.

Amoebiasis is the second leading cause of death worldwide associated with parasitic disease and is becoming a critical health problem in low-income countries, urging new treatment alternatives. One of the most promising strategies is enhancing the redox imbalance within these susceptible parasites related to their limited antioxidant defense system. Metal-based drugs represent a perfect option due to their extraordinary capacity to stabilize different oxidation states and adopt diverse geometries, allowing their interaction with several molecular targets. This work describes the amoebicidal activity of five 2-(Z-2,3-diferrocenylvinyl)-4X-4,5-dihydrooxazole derivatives (X = H (3a), Me (3b), iPr (3c), Ph (3d), and benzyl (3e)) on Entamoeba histolytica trophozoites and the physicochemical, experimental, and theoretical properties that can be used to describe the antiproliferative activity. The growth inhibition capacity of these organometallic compounds is strongly related to a fine balance between the compounds' redox potential and hydrophilic character. The antiproliferative activity of diferrocenyl derivatives studied herein could be described either with the redox potential, the energy of electronic transitions, logP, or the calculated HOMO-LUMO values. Compound 3d presents the highest antiproliferative activity of the series with an IC50 of 23 µM. However, the results of this work provide a pipeline to improve the amoebicidal activity of these compounds through the directed modification of their electronic environment.

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.

Primary pulmonary amoebiasis mimicking lung tumour in immunocompromised patient: A case report.

Amoebiasis is the most common protozoan disease caused by Entamoeba histolytica. The second most frequent extraintestinal infection, behind amoebic liver abscess, is pulmonary amoebiasis. We present the case of an immunocompromised 40-year-old man. He complained of cough for 1 month, shortness of breath, and fever. Chest x-ray demonstrated left paracardial consolidation, possibly pneumonia or a mass. Chest CT scans with contrast revealed the presence of an abscess-mimicking tumour. CT-guided TTB and histology examinations indicated the presence of trophozoites of E. histolytica. This patient was diagnosed with pulmonary amoebiasis. Diagnostic criteria for pulmonary amoebiasis include clinical manifestations, radiography, and microscopic examination. There was an improvement in clinical response after a 10-day course of antibiotics. Amoebiasis of the lungs is treatable with medicines and drainage when necessary. Early diagnosis and treatment are imperative to decrease mortality and morbidity.

Amoebiasis: Advances in Diagnosis, Treatment, Immunology Features and the Interaction with the Intestinal Ecosystem.

This review of human amoebiasis is based on the most current knowledge of pathogenesis, diagnosis, treatment, and Entamoeba/microbiota interactions. The most relevant findings during this last decade about the Entamoeba parasite and the disease are related to the possibility of culturing trophozoites of different isolates from infected individuals that allowed the characterization of the multiple pathogenic mechanisms of the parasite and the understanding of the host-parasite relationship in the human. Second, the considerable advances in molecular biology and genetics help us to analyze the genome of Entamoeba, their genetic diversity, and the association of specific genotypes with the different amoebic forms of human amoebiasis. Based on this knowledge, culture and/or molecular diagnostic strategies are now available to determine the Entamoeba species and genotype responsible for invasive intestinal or extraintestinal amoebiasis cases. Likewise, the extensive knowledge of the immune response in amoebiasis with the appearance of new technologies made it possible to design diagnostic tools now available worldwide. Finally, the understanding of the interaction between the Entamoeba species and the intestinal microbiota aids the understanding of the ecology of this parasite in the human environment. These relevant findings will be discussed in this review.

Amoebic Dysentery Complicated by Hypovolemic Shock and Sepsis in an Infant with Severe Acute Malnutrition: A Case Report.

Diarrheal disease continues to be a leading cause of death in children under five years old in developing countries, where it is responsible for the death of approximately half a million children each year. Establishing the cause of diarrheal disease can be difficult in developing areas due to the lack of diagnostic tests, and thus empirical therapies are often required. In these settings, the choice of antibiotic (or the choice to not give it) depends on suspected agents, host conditions and local epidemiology. Herein, we report a representative case of a ten-month-old male patient with severe acute malnutrition (SAM) admitted to the Emergency Paediatric Clinic in Port Sudan for amoebic dysentery complicated by hypovolemic shock and sepsis, treated by target therapy for Entamoeba histolytica infection associated with empiric antibiotic therapy. Due to the absence of clinical improvement, Ciprofloxacin was added to the first-line treatment. This case highlights that in low-income countries amoebiasis, especially in children with SAM, may result in life-threatening complications. Although stool microscopy remains the most used diagnostic test in these settings, a novel inexpensive, easy to use and rapid diagnostic test would be warranted to reach a microbiological diagnosis and guide clinical decision. Further studies will be necessary to identify the patterns of antimicrobial resistance in order to appropriately manage these complicated cases.

Development of a simple PCR-RFLP technique for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii.

Epidemiological studies on amoebic infections are complicated by morphological overlap between the pathogenic E. histolytica, the commensal E. dispar and the amphizoic E. moshkovskii, necessitating molecular identification. The present study developed a simple and economical 18S PCR-RFLP method for the simultaneous detection and differentiation of the three species. PCR products were differentiated by Tat1 restriction digestion generating three different RFLP patterns. Validation was conducted by screening 382 faecal samples from human patients from Kolkata, India, hospitalized for diarrhoea. Analysis indicated that the PCR-RFLP could successfully differentiate between the three species and was confirmed by sequence analysis. This method could prove useful for clinical and epidemiological studies of amoebiasis.

Effect of Entamoeba histolytica infection on gut microbial diversity and composition in diarrheal patients from New Delhi.

During amoebiasis, colonization of the gut by Entamoeba histolytica can lead to alterations of the host microbiota. In this study, we have compared the gut microbiota of patients of amoebiasis with healthy controls using 16S rRNA gene variable regions, (V1-V3) and (V3-V5), of the bacterial genome. From this 16S rRNA gene amplicon data, one paired-end and two single-end datasets were selected and compared by the number of OTUs obtained, sequence count, and diversity analysis. Our results showed that the V1-V3-paired-end dataset gave the maximum number of OTUs in comparison to the two single-end datasets studied. The amoebiasis samples showed a significant drop in richness in the alpha diversity measurements and lower intra group similarity compared to the healthy controls. Bacteria of genus Prevotella, Sutterella, and Collinsella were more abundant in healthy controls whereas Escherichia, Klebsiella, and Ruminococcus were more abundant in the E. histolytica-positive patients. All the healthy controls harbored bacteria belonging to Faecalibacterium, Prevotella, Ruminococcus, Subdoligranulum, and Escherichia genera while all the E. histolytica-positive patient samples contained genus Enterobacter. The compositional changes in the gut microbiome observed in our study indicated a higher prevalence of pathogenic bacteria along with a depletion of beneficial bacteria in E. histolytica-infected individuals when compared with healthy controls. These results underline the interplay between E. histolytica and the human gut microbiome, giving important inputs for future studies and treatments.