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.

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.

Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.

Recently, Entamoeba histolytica clones derived from isolate HM-1:IMSS that differ in their pathogenicity were identified. Whereas some clones induce amoebic liver abscesses (ALAs) in animal models of amoebiasis, others provoke only minimal liver lesions. Based on transcriptome studies of pathogenic and nonpathogenic clones, differentially expressed genes associated with reduced or increased liver pathology can be identified. Here, to analyze the influence of these genes on ALA formation in more detail, an RNA interference-trigger mediated silencing approach was used. Using newly identified trigger sequences, the expression of 15 genes was silenced. The respective transfectants were analyzed for their ability to induce liver destruction in the murine model for the disease. Silencing of EHI_180390 (encoding an AIG1 protein) increased liver pathology induced by a nonpathogenic parent clone, whereas silencing of EHI_127670 (encoding a hypothetical protein) decreased the pathogenicity of an initially pathogenic parent clone. Additional phenotypical in vitro analyses of EHI_127670 silencing as well as overexpression transfectants indicated that this molecule has an influence on size, growth, and cysteine peptidase activity of E. histolytica. This work describes an example of how the sole operational method for effective gene silencing in E. histolytica can be used for comprehensive analyses of putative pathogenicity factors.-Matthiesen, J., Lender, C., Haferkorn, A., Fehling, H., Meyer, M., Matthies, T., Tannich, E., Roeder, T., Lotter, H., Bruchhaus, I. Trigger-induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

BACKGROUND & OBJECTIVES: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. METHODS: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. RESULTS: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. INTERPRETATION & CONCLUSIONS: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

TNFα-mediated liver destruction by Kupffer cells and Ly6Chi monocytes during Entamoeba histolytica infection.

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.

Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism.

Varied Clinical Manifestations of Amebic Colitis.

Invasive amebiasis is common worldwide, but infrequently observed in the United States. It is associated with considerable morbidity in patients residing in or traveling to endemic areas. We review the clinical and endoscopic manifestations of amebic colitis to alert physicians to the varied clinical manifestations of this potentially life-threatening disease. Copyright ©Most patients present with watery or bloody diarrhea. Less common presentations of amebic colitis include abdominal pain, overt gastrointestinal bleeding, exacerbation of inflammatory bowel disease, or the incidental association with colon cancer. Amebic liver abscesses are the most frequent complication. Rectosigmoid involvement may be found on colonoscopy; however, most case series have reported that the cecum is the most commonly involved site, followed by the ascending colon. Endoscopic evaluation should be used to assist in the diagnosis, with attention to the observation of colonic inflammation, ulceration, and amebic trophozoites on histopathological examination.

The MAK16 gene of Entamoeba histolytica and its identification in isolates from patients.

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher's exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient's symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.

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.

Experimental amoebic liver abscess in hamsters caused by trophozoites of a Brazilian strain of Entamoeba dispar.

It has been claimed that amoebic molecules such as amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteases are responsible for host tissue destruction and are present in both pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar. Some reports have provided evidence that after infection with E. dispar, pathological changes may occur in some humans. The aim of this study was to evaluate E. dispar pathogenicity by comparing it to the pathogenicity of E. histolytica through liver abscesses induced in hamsters. Syrian golden hamsters were challenged by intrahepatic inoculation with the 03C E. dispar strain or with two strains of E. histolytica (HM1:IMSS and EGG) to compare their virulence grades. As control groups, we used bacterial flora and Pavlova's modified medium. Lesions were verified at 1, 3 and 6 days after inoculation. Multiplex Polymerase Chain Reaction was performed to characterize each strain using EdP1/EdP2 and EhP1/EhP2 primers. The EGG and HM1:IMSS E. histolytica strains and 03C E. dispar were able to cause liver lesions. The EGG strain caused extensive hepatic abscesses, and trophozoites were found in the lesions throughout the three periods of study. The HM1:IMSS strain caused smaller abscesses when compared to EGG lesions; however, trophozoites were observed at 1 and 3 days after inoculation. The 03C E. dispar strain caused intermediate abscesses when compared to the others; trophozoites were observed in all periods analyzed. The EGG strain caused progressive evolution of the injury, which differed from the HM1:IMSS and 03C strains. These results strongly suggest that the 03C E. dispar strain is pathogenic in the experimental hamster model. Additional studies are necessary to identify potential factors that regulate the manifestation of virulence of this strain and others.

Entamoeba histolytica: the over expression of a mutated EhRabB protein produces a decrease of in vitro and in vivo virulence.

Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica.

Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis.

Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.

Amoebic liver abscess with negative serologic markers for Entamoeba histolytica: mind the gap!

A 38-year-old male German traveller returning from Asia presented with fever, night sweats and abdominal complaints. Abdominal ultrasonography revealed several fast-growing abscesses of the liver. Three blood cultures as well as serologic investigations for the detection of antibodies to Entamoeba histolytica, performed on day 3 and 7 after the onset of clinical symptoms, remained negative. Stool microscopy revealed the presence of amoeba cysts compatible with E. histolytica infection. Taking both the amoebic and bacterial etiology of the abscesses into consideration, the patient was treated with metronidazole and ciprofloxacin followed by paromomycin. Antibodies to E. histolytica tested positive shortly after anti-amoebic therapy was initiated. The patient fully recovered, and ultrasound follow-up showed complete resolution of the abscesses within 50 days. This case leads to the conclusion that amoebic liver abscess should be considered despite negative amoeba serology and that ultrasonography is an important diagnostic tool for the early diagnosis of extraintestinal amoebiasis.

Amoebic liver abscess production by Entamoeba dispar.

Although Entamoeba dispar displays a similar morphology to Entamoeba histolytica, cellular and molecular studies have revealed significant differences between these two amoebae, including the former being characterized as non-pathogenic and the later as pathogenic. However, recent in vivo and in vitro experiments have shown that E. dispar strains of different origin are capable of causing liver damage and destroying cell culture lines in the presence of common intestinal bacteria. These results suggested that E. dispar may present pathogenic behavior according to the specific E. dispar strain, culture and environmental conditions. To investigate this possibility, we carried out in vivo and in vitro studies using a xenic strain E. dispar (ICB-ADO) isolated from a symptomatic non-dysenteric Brazilian patient. This strain was able to induce liver necrosis in a hamster model that was more severe than that produced by E. histolytica. The ICB-ADO isolate also caused significantly more destruction of cultured MDCK cells and increased loss of transepithelial resistance than did the E. histolytica. Xenic E. dispar exhibited high proteolytic activity, which was partially inhibited by the addition of cysteine-protease inhibitors. Based on our biochemical and molecular characterization of E. dispar (ICB-ADO) xenic culture and its ability to produce liver abscesses, we conclude that this specific strain can indeed produce tissue damage, distinct from the frequently used non- pathogenic E. dispar SAW 760 strain.

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China.

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.

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.