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

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

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.

Amoebic colitis and liver abscess: A rare case of autochthonous invasive infection due to Entamoeba histolytica.

We report an unusual and confirmed case of invasive amebiasis in a non-endemic area where the source of infection remains unknown. During her admission, the patient developed amebic colitis and extraintestinal liver abscess with a favorable outcome following the antiparasitic therapy.

Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

Novel diagnostic approach for amoebic liver abscess using cell free (cf) DNA: a prospective study.

BACKGROUND: Amoebic liver abscess (ALA) is commonly seen in tropical countries and diagnosis of ALA relies mainly on non-specific serological and imaging techniques as well as PCR from pus. OBJECTIVE: This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA. METHODS: We prospectively evaluated quantitative PCR (qPCR) for detection of cf DNA in serum and urine sample in all liver abscess patients. The samples were collected from patients reporting to emergency ward of Postgraduate Institute of Medical Education and Research, Chandigarh, India with symptoms suggestive of liver abscess. Real time PCR was done to detect cf DNA in serum and urine by targeting 99-bp unit of small subunit rRNA of Entamoeba histolytica and conventional PCR for pus. RESULTS: A total 113 samples (serum and urine) and 100 pus samples were analysed. A total of 62 ALA patients were confirmed; with maximum 57 patients detected by qPCR for cfDNA in the serum, 55 patients by PCR on pus aspirate and 50 ALA patients by qPCR for cfDNA in urine sample. Therefore, the sensitivity of qPCR for detection of cf DNA in serum was 91.94% and for urine was 80.65%. CONCLUSION: A total of 11.2% of ALA patients were diagnosed only through detection of E. histolytica cf DNA in their serum and urine. Detection of cfDNA from serum, urine of ALA has a potential role in future especially for developing countries as it is a rapid, sensitive and patient friendly diagnostic approach.

Preoperative Transcatheter Arterial Embolization for Spontaneous Rupture of Huge Amebic Liver Abscess with Massive Intraperitoneal Hemorrhage.

An 18-year-old male developed a huge liver abscess with severe anemia over the course of 2 weeks. Abdominal contrast enhanced computed tomography showed ruptured huge liver abscess in the right liver lobe with signs of active hemorrhage (contrast extravasation). Serology examination confirmed amoeba as the suspected pathogen of cause. Angiography was performed followed by transcatheter arterial embolization to localize and control the hemorrhage. Embolization using a combination of polyvinyl alcohol and gelfoam successfully controlled the active hemorrhage. Exploratory laparotomy was performed to evacuate and debride the huge abscess. Metronidazole was given and showed good results. Huge liver abscess size is a predictor of conservative management failure and requires a gradual step-up intervention. The purpose of this paper is to explain the importance of imaging in detecting liver abscess and active hemorrhage along with the role of interventional radiology in this case.

Massive pleural empyema secondary to amoebic liver abscess in a child.

BACKGROUND: Pleural empyema secondary to a ruptured amoebic liver abscess is a rare complication in the pediatric population. CASE REPORT: We report the case of a 13-year-old male with right flank abdominal pain, productive cough with foul-smelling sputum, fever, and respiratory distress. Physical examination revealed breathlessness, decreased vesicular murmur in the right hemithorax, abdominal distension, hepatomegaly, and lower limb edema. Laboratory tests revealed mild anemia, leukocytosis without eosinophilia, elevated alkaline phosphatase, hypoalbuminemia, and positive immunoglobulin G antibodies against Entamoeba histolytica in pleural fluid. He required a chest tube and treatment with metronidazole. After 2 months of follow-up, the abscesses disappeared, and the empyema decreased. CONCLUSIONS: Massive pleural empyema secondary to a ruptured liver abscess is a rare complication. The epidemiological link associated with the symptoms and serological tests can help in the diagnosis.

INTRODUCCIÓN: El empiema pleural secundario a ruptura de absceso amebiano hepático es una complicación poco frecuente en la población pediátrica. CASO CLÍNICO: Se reporta el caso de un paciente de sexo masculino de 13 años que presentó dolor abdominal en flanco derecho, tos productiva con esputo de mal olor, fiebre y dificultad respiratoria. Al examen físico se encontró amplexación y murmullo vesicular disminuido en hemitórax derecho, distensión abdominal, hepatomegalia y edema de miembros inferiores. Los resultados del laboratorio evidenciaron anemia leve, leucocitosis sin eosinofilia, elevación de fosfatasa alcalina, hipoalbuminemia y anticuerpos IgG contra Entamoeba histolytica positivo en líquido pleural. Requirió tubo de drenaje torácico y tratamiento con metronidazol. A los dos meses de seguimiento los abscesos desaparecieron y el empiema disminuyó. CONCLUSIONES: El empiema pleural masivo secundario a ruptura de absceso hepático es una complicación poco frecuente. El nexo epidemiológico asociado con la sintomatología y pruebas serológicas pueden ser de ayuda en el diagnóstico.

Creep in nitroimidazole inhibitory concentration among the Entamoeba histolytica isolates causing amoebic liver abscess and screening of andrographolide as a repurposing drug.

Infections by Entamoeba histolytica (E. histolytica) lead to considerable morbidity and mortality worldwide and treatment is reliant on a single class of drugs, nitroimidazoles. Treatment failures and intermittent reports of relapse from different parts of world indicate towards development of clinical drug resistance. In the present study, susceptibility testing of clinical isolates of E. histolytica was carried against metronidazole and tinidazole. Additionally, anti-amoebic property of active compounds of Andrographis paniculata was also evaluated. Prevalence of metronidazole resistance gene (nim) in patients attending hospital was also done to get comprehensive insight of present situation of drug resistance in E. histolytica. Mean inhibitory concentration 50 (IC50) value of E. histolytica isolates against metronidazole and tinidazole was 20.01 and 16.1 µM respectively. Andrographolide showed minimum mean IC50 value (3.06 µM). Significant percentage inhibition of E. histolytica isolates by andrographolide was seen as compared to metronidazole (p = 0.0495). None of E. histolytica isolates showed presence of nim gene. However, in stool samples from hospital attending population, prevalence of nimE gene was found to be 76.6% (69/90) and 62.2% (56/90) in diarrheal and non-diarrheal samples respectively. Inhibitory concentration of commonly used nitroimidazoles against clinical isolates of E. histolytica are on rise. Percentage inhibition of E. histolytica isolates by andrographolide was significantly higher than control drug metronidazole.

[Clinical and radiological differences between amoebic and pyogenic liver abscess: A case-control study]. / Analyse des différences cliniques et radiologiques entre abcès hépatiques amibiens et à pyogènes à partir d'une étude cas-témoin.

INTRODUCTION: Amoebic liver abscess (ALA) is the fourth cause of mortality by parasitic infection. This study aimed to assess clinical, radiological and therapeutic characteristics of patients admitted for amoebic liver abscess compared to pyogenic abscess in a French digestive tertiary care-centre. MATERIAL AND METHOD: The charts of patients hospitalized for a liver abscess between 2010 and 2020 were retrospectively assessed then separated in two groups: amoebic liver abscess and pyogenic liver abscess from portal underlying cause. Clinical and radiological data were collected for univariate comparison. RESULTS: Twenty-one patients were hospitalized during the time of the study for ALA, and 21 patients for pyogenic liver abscess with a portal mechanism. All patients hospitalized for ALA lived in and/or had travelled recently in an endemic area. In comparison with patients hospitalized for pyogenic abscess, patients admitted for ALA were younger (44years old vs. 63years old, P<0.001), had less comorbidities (5% vs. 43% of patients with at least one comorbidity, P<0.01), a longer median duration of symptoms (10days vs. 3days, P=0.015), abdominal pain (86% vs. 52%, P=0.019), and a slighter leucocytosis (9600G/L vs. 15,500G/L, P=0.041) were more frequent. On the abdominal tomodensitometry, density of ALA was higher (34 vs. 25 UH, P<0.01), associated with a focal intra-hepatic biliary dilatation and less often multiloculated. CONCLUSION: While rare in western countries, amoebic liver abscess care should not be underestimated. The presence of a solitary liver abscess of intermediate density on computed tomography, occurring on a patient returning from an endemic zone should lead the physician to a possible diagnosis of ALA.

Epidemiology of the first seven years of national surveillance of amoebic liver abscesses in Mexico.

The epidemiological behaviour of the main extraintestinal complication due to E. histolytica (amoebic liver abscess, ALA) has been little explored in developing countries. Since Mexico meets the characteristics to be considered as endemic, the aim of this work was to analyze the national surveillance data (seven years) of ALA issued by the General Directorate of Epidemiology. An analysis of cases and incidence of ALA (2014-2020) was performed in the annual reports issued by the GDE in Mexico. Cases and incidence of ALA were classified by year, incidence, age group, sex and seasons. Geographical distribution map for the whole country of ALA was constructed. The cases and mean incidence of ALA did not shown significant variation during the study period. Of the total cases accumulated, Sonora, Sinaloa, Nayarit, Colima and Zacatecas states showed the most incidence by ALA. Male sex of 24-44 years old showed higher ALA cases. No temporal behaviour was identified between the ALA cases. In Mexico, the incidence of ALA remains unchanged, however, it should not be underestimated since the monitoring programs for the search for new cases have not yet been implemented mainly in endemic states. These results summarize the priority in the national ALA report.

Testosterone affects type I/type II interferon response of neutrophils during hepatic amebiasis.

Differences in immune response between men and women may influence the outcome of infectious diseases. Intestinal infection with Entamoeba histolytica leads to hepatic amebiasis, which is more common in males. Previously, we reported that innate immune cells contribute to liver damage in males in the murine model for hepatic amebiasis. Here, we focused on the influences of sex and androgens on neutrophils in particular. Infection associated with neutrophil accumulation in the liver was higher in male than in female mice and further increased after testosterone treatment in both sexes. Compared with female neutrophils, male neutrophils exhibit a more immature and less activated status, as evidenced by a lower proinflammatory N1-like phenotype and deconvolution, decreased gene expression of type I and type II interferon stimulated genes (ISGs) as well as downregulation of signaling pathways related to neutrophil activation. Neutrophils from females showed higher protein expression of the type I ISG viperin/RSAD2 during infection, which decreased by testosterone substitution. Moreover, ex vivo stimulation of human neutrophils revealed lower production of RSAD2 in neutrophils from men compared with women. These findings indicate that sex-specific effects on neutrophil physiology associated with maturation and type I IFN responsiveness might be important in the outcome of hepatic amebiasis.

Gene expression of RD5 and Psp5 in extra intestinal Entamoeba histolytica isolated from liver abscesses.

Liver abscesses are known to be trophozoites of the amoeba parasite. They are devoured by the neutrophil cells in the liver and become large assemblies because these white cells do not eliminate the parasite and these white cells multiply. In this study, venous blood samples were taken from 61 patients have hepatic amoebosis and 61 healthy individuals as a control group. The patients attended Ghazi Al-Hariri Surgical Specialities Hospital, the Medical City, Baghdad, Iraq during the period from 15th January to 18th September 2021. The results showed that the mean age of patients was (41.84±15.88) years, while the mean age of the control group was (41.84±15.88) years with no significant difference (P>0.05). The prevalence rate of Entamoeba histolytica infection was 27 (44.2%) in males, and 34 (55.8%) in females with no significant difference. The mean anti-Entamoeba antibody IgA in urban areas was (1.95±1.25) and in the rural areas was (2.05±1.10), while the mean anti-Entamoeba antibody IgG in urban areas was (14.86±6.71), and in the rural areas was (13.55±7.43), with no significant differences (P>0.05). The mean anti-Entamoeba antibody IgA was (2.00±1.17) among the patient's group in comparison with the control group which was (0.09±0.17), while the mean anti-Entamoeba antibody IgG was (14.20±7.06) among the patients when compared with the control group which was (0.06±0.11) with highly significant differences (P<0.01). Expression of RD5 gene was investigated in E. histolytica in liver abscess patients and healthy controls by using qRT-PCR and the findings of amplification regarding atypical amplification plot. The amplification reaction had an early threshold cycle that was consistent with high levels of RD5 gene and the healthy controls. Psp5 gene was expression to investigated E. histolytica in liver abscess in 60 patients and (60) individuals as a control group by using qRT-PCR and the findings of amplification regarding atypical amplification plot. The amplification reaction had an early threshold cycle that was consistent with high levels of Psp5 gene and the healthy controls.

Evaluation of total immunoglobulin G and subclass antibodies in an enzyme-linked immunosorbent assay for serodiagnosis of human amebic liver abscess.

Background: Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods: A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276). Results: ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8-100.0] and 97.8% specificity 95% CI [95.5-99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8-100.0]) and a slightly higher specificity (99.1% 95% CI [97.3-99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar's test; p > 0.05), with a concordance of 98.2%, represented by a Cohen's kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion: ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.

Hypothetical proteins play a role in stage conversion, virulence, and the stress response in the Entamoeba species.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and amoebic liver abscess in humans, affecting millions of people worldwide. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. As cysts can be ingested from contaminated food and water, this parasite is prevalent in underdeveloped countries and poses a significant health burden. Until recently there was no reliable method for inducing stage conversion in E. histolytica in vitro. As such, the reptilian pathogen, Entamoeba invadens, has long-served as a surrogate. Much remains unclear about stage conversion in these parasites and current treatments for amoebiasis are lacking, as they cause severe side effects. Therefore, new therapeutic strategies are needed. The genomes of these parasites remain enigmatic as approximately 54% of E. histolytica genes and 66% of E. invadens genes are annotated as hypothetical proteins. In this study, we characterized two hypothetical proteins in the Entamoeba species, EIN_059080, in E. invadens, and its homolog, EHI_056700, in the human pathogen, E. histolytica. EHI_056700 has no homolog in the human host. We used an RNAi-based silencing system to reduce expression of these genes in E. invadens and E. histolytica trophozoites. Loss of EIN_059080 resulted in a decreased rate of encystation and an increased rate of erythrophagocytosis, an important virulence function. Additionally, mutant parasites were more susceptible to oxidative stress. Similarly, loss of EHI_056700 in E. histolytica trophozoites resulted in increased susceptibility to oxidative stress and glucose deprivation, but not to nitrosative stress. Unlike the E. invadens mutants, E. histolytica parasites with decreased reduced expression of EHI_056700 exhibited a decreased rate of erythrophagocytosis of and adhesion to host cells. Taken together, these data suggest that these hypothetical proteins play a role in stage conversion, virulence, and the response to stress in the Entamoebae. Since parasites with reduced expression of EHI_056700 show decreased virulence functions and increased susceptibility to physiologically relevant stressors, EHI_056700 may represent a possible therapeutic target for the treatment of amoebiasis.