Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Complement is a rat natural resistance factor to amoebic liver infection.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica This illness is prevalent in poor countries causing 100,000 deaths worldwide. Knowledge of the natural resistance mechanisms of rats to amoebic liver abscess (ALA) development may help to discover new pathogenic factors and to design novel therapeutic strategies against amoebiasis. In this work, histologic analyses suggested that the complement system may play a central role in rat natural resistance to ALA. E. histolytica trophozoites disappeared from rat liver within 6 h post-infection with minimal or no inflammatory infiltrate. In vitro findings indicate that rat complement was lethal for the parasite. Furthermore, hamsters became resistant to ALA by intravenous administration of fresh rat serum before infection. The amoebicidal potency of rat complement was 10 times higher than hamster complement and was not related to their respective CH50 levels. The alternative pathway of complement plays a central role in its toxicity to E. histolytica since trypan blue, which is a C3b receptor inhibitor, blocks its amoebicidal activity. These results suggest that amoebic membrane affinity, high for C3b and/or low for Factor H, in comparison with the hamster ones, may result in higher deposition of membrane complex attack on parasite surface and death.

Parallel ELISAs using crude soluble antigen and excretory-secretory antigen for improved serodiagnosis of amoebic liver abscess.

Crude soluble antigen (CSA) produced from Entamoeba histolytica trophozoite is conventionally used for serodiagnosis of invasive amoebiasis. However, high background seropositivities by CSA-assay in endemic areas complicate the interpretation of positive result in clinical settings. Instead, incorporating a second assay which indicates active or recent infection into the routine amoebic serology could possibly complement the limitations of CSA-assay. Hence, the present study aimed to evaluate the diagnostic efficacies of indirect ELISAs using CSA and excretory-secretory antigen (ESA) for serodiagnosis of amoebic liver abscess (ALA). Reference standard for diagnosis of ALA at Hospital Universiti Sains Malaysia is based on clinical presentation, radiological imaging and positive indirect haemagglutination assay (titer ≥256). Five groups of human serum samples collected from the hospital included Group I - ALA diagnosed by the reference standard and pus aspirate analysis using real-time PCR (n=10), Group II - ALA diagnosed by the reference standard only (n=41), Group III - healthy control (n=45), Group IV - other diseases control (n=51) and Group V - other infectious diseases control (n=31). For serodiagnosis of ALA serum samples (Group I and II), CSA-ELISA showed sensitivities of 100% for both groups, while ESA-ELISA showed sensitivities of 100% and 88%, respectively. For serodiagnosis of non-ALA serum samples (Group III, IV and V), CSA-ELISA showed specificities of 91%, 75% and 100%, respectively; while ESA-ELISA showed specificities of 96%, 98% and 100%, respectively. Indirect ELISAs using CSA and ESA have shown distinct strength for serodiagnosis of ALA, in terms of sensitivity and specificity, respectively. In conclusion, parallel analysis by both assays improved the overall efficacies of amoebic serology as compared to either single assay.

Efficacy of aspiration in amebic liver abscess.

BACKGROUND: Amebic liver abscess (ALA) is a common and serious problem in our country. There are only a few controlled trials on the efficacy and advantages of combination therapy with percutaneous needle aspiration and pharmacotherapy, over pharmacotherapy alone for amebic liver abscess. MATERIAL AND METHODS: This study was conducted to compare the efficacy of two different treatment modalities i.e. drug treatment alone vs. drug treatment and aspiration of abscess cavity in patients with small (up to 5 cm) and large (5 cm to 10 cm) size ALA. This is one of the largest single center, prospective, randomized studies comparing the efficacy of aspiration in ALA. RESULTS: (i) Mean body temperature, liver tenderness, total leukocyte count (TLC), serum alanine aminotransferase (ALT) and liver span were significantly decreased in the aspiration group on days 8 and 15 as compared to non-aspiration group especially in large abscess (5 cm to 10 cm). (ii) Abscess cavity maximum diameter decreased significantly in aspiration group on days 8 and 15, and 1 month & 3 months in large abscess (5cm to 10 cm). CONCLUSIONS: (i) Needle aspiration along with metronidazole hastens clinical improvement especially in large (5 cm up to 10 cm) cavities in patients with ALA. (ii) Aspiration is safe and no major complications occurred. (iii) Hence, combination therapy should be the first choice especially in large ALA (5 cm to 10 cm).

Hypocholesterolemia in patients with an amebic liver abscess.

BACKGROUND/AIMS: Many parasites induce changes in the lipid profiles of the host. Cholesterol increases the virulence of Entamoeba histolytica in animal models and in vitro culture. This study aimed to determine, in patients with an amebic liver abscess, the correlation between cholesterol and other features, such as the size and number of abscesses, standard hematological and serum chemistry profiles, liver tests, and duration of hospital stay. METHODS: A total of 108 patients with an amebic liver abscess and 140 clinically healthy volunteers were investigated. Cholesterol and triglycerides were measured in the sera. The data from medical observations and laboratory tests were obtained from the clinical records. RESULTS: A total of 93% of patients with an amebic liver abscess showed hypocholesterolemia not related to any of the studied parameters. Liver function tests correlated with the size of the abscess. The most severe cases of amebic liver disease or death were found in patients whose cholesterol levels continued to decrease despite receiving antiamebic treatment and hospital care. CONCLUSIONS: Our results show that the hypocholesterolemia observed in patients with an amebic liver abscess is not related to any of the clinical and laboratory features analyzed. This is the first study relating hypocholesterolemia to severity of hepatic amebiasis.

Hypocholesterolemia in Patients with an Amebic Liver Abscess

BACKGROUND/AIMS: Many parasites induce changes in the lipid profiles of the host. Cholesterol increases the virulence of Entamoeba histolytica in animal models and in vitro culture. This study aimed to determine, in patients with an amebic liver abscess, the correlation between cholesterol and other features, such as the size and number of abscesses, standard hematological and serum chemistry profiles, liver tests, and duration of hospital stay. METHODS: A total of 108 patients with an amebic liver abscess and 140 clinically healthy volunteers were investigated. Cholesterol and triglycerides were measured in the sera. The data from medical observations and laboratory tests were obtained from the clinical records. RESULTS: A total of 93% of patients with an amebic liver abscess showed hypocholesterolemia not related to any of the studied parameters. Liver function tests correlated with the size of the abscess. The most severe cases of amebic liver disease or death were found in patients whose cholesterol levels continued to decrease despite receiving antiamebic treatment and hospital care. CONCLUSIONS: Our results show that the hypocholesterolemia observed in patients with an amebic liver abscess is not related to any of the clinical and laboratory features analyzed. This is the first study relating hypocholesterolemia to severity of hepatic amebiasis.

Evaluation of Entamoeba histolytica recombinant phosphoglucomutase protein for serodiagnosis of amoebic liver abscess.

BACKGROUND: Amoebic liver abscess (ALA) is the most frequent clinical presentation of extra-intestinal amoebiasis. The diagnosis of ALA is typically based on the developing clinical symptoms, characteristic changes on radiological imaging and serology. Numerous serological tests have been introduced for the diagnosis of ALA, either detecting circulating amoebic antigens or antibodies. However those tests show some pitfalls in their efficacy and/or the preparation of the tests are costly and tedious. The commercial IHA kit that used crude antigen was reported to be useful in diagnosis of ALA, however high antibody background in endemic areas may cause problems in its interpretation. Thus, discovery of well-defined antigen(s) is urgently needed to improve the weaknesses of current serodiagnostic tests. METHODS: Crude antigen of E. histolytica was analysed by 2-DE and Western blot to identify a protein of diagnostic potential for ALA. The corresponding gene of the antigenic protein was then cloned, expressed and the purified recombinant protein was subsequently evaluated for serodiagnosis of ALA in an indirect ELISA format. RESULTS: Analysis of crude antigen showed that phosphoglucomutase (PGM) has the diagnostic potential. Recombinant PGM (rPGM) showed 79.17% (19/24) sensitivity and 86.67% (195/225) specificity in diagnosis of ALA based on the COV of mean +1SD. There was no significant difference between rPGM-ELISA and IHA diagnostic kit in the diagnosis of ALA in terms of sensitivity and specificity at p-value < 0.05. CONCLUSION: In conclusion, rPGM-ELISA is found to be useful for serodiagnosis of ALA. Future studies will determine whether rPGM-ELISA also detects antibodies produced in amoebic dysentery and asymptomatic cases.

[Amebic colitis and liver abscess complicated by high serum procalcitonin in acute myeloid leukemia].

We present a case of amebic colitis and liver abscess complicated by acute myeloid leukemia (AML) with high serum procalcitonin (PCT). A 61-year-old Japanese man seen at our hospital for severe diarrhea and high fever was found to have multiple ulcers in the transverse and sigmoid colon and rectum by colonoscopy and biopsies were conducted. Immature leukocytes with mild anemia and thrombocytopenia were seen in peripheral blood, necessitating bone marrow aspiration and biopsy that yielded a diagnosis of AML (FAB M4Eo). Serum C-reactive protein and PCT were extremely elevated. Blood cultures for bacteria and fungi were negative. Multiple low-density areas in the liver were found in abdominal computed tomography. Histological colon biopsy findings revealed amebic colitis, strongly suggesting amebic liver abscess. Metronidazole treatment was initiated for amebiasis and subsequent standard chemotherapy for AML was followed after fever was lowered. Hematological and cytogenetic CR was maintained with good clinical condition. Few case reports have been published in Japan to date on amebic colitis and liver abscess complicated by AML and no reports have been made on PCT elevation caused by amebiasis. In conclusion, differential diagnosis of amebiasis is necessary in addition to that of bacterial or fungal infection in serum PCT elevation.

Application of real-time polymerase chain reaction in detection of Entamoeba histolytica in pus aspirates of liver abscess patients.

Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.

Amebic liver abscess: report of three cases.

Amebic abscess is a common manifestation of extraintestinal amebiasis and it is associated with relatively high morbidity and mortality. We present three cases seen in Bari, Southern Italy, one of which was autochthonous and the other two were not. Diagnosis was performed by elevated antibody titre for E. histolytica through immunofluorescence assay and positive antigen determination by ELISA in stools and in abscess aspirate. Fever often accompanied by chills, abdominal pain, weight loss and hepatomegaly were present. Laboratory findings also revealed leukocytosis with neutrophilia. Pleural effusion was observed in two patients. In all our patients multiple abscesses were observed. All the patients were treated with metronidazole and two of them also underwent the aspiration of the amoebic abscess. In all of them there was improvement of the clinical picture, as demonstrated by computerized tomography.

Altered lipid parameters in patients infected with Entamoeba histolytica, Entamoeba dispar and Giardia lamblia.

Information on the effect of parasitic infections on lipid parameters is scarce. Certain parasites induce significant changes in lipid parameters, as demonstrated by the fact that substitution of lipid/cholesterol for serum in axenic culture medium (in vitro) and in experimental models (in vivo) supports vigorous growth of Entamoeba histolytica. Thus, significant changes in lipid parameters may be induced in an infected host. Blood samples are obtained from intestinal amoebiasis patients passing E. histolytica (n=8), E. dispar (n=15) or Giardia lamblia (n=9) cysts, or diagnosed with amoebic liver abscess (ALA; n=50) and from apparently normal healthy individuals (control group; n=30). Levels of total serum cholesterol, high-density lipoprotein and low-density lipoprotein are assessed using commercial kits. E. histolytica and E. dispar isolates are differentiated by hexokinase isoenzyme electrophoresis and by enzyme-linked immunosorbent assay (ELISA; Techlab) tests. Results show that E. histolytica, E. dispar and G. lamblia cyst passers had significantly lower levels of total serum cholesterol (73.42 +/- 2.24 mg/dL), compared to levels in ALA cases (101 +/- 2.85 mg/dL) and in controls (166.26 +/- 2.02 mg/dL). Further study of a greater number of cases is needed to explore the relevance of this finding.

A note on indirect hemagglutination (IHA) antibody titers among hospitalized patients in Thailand with amebic liver abscesses.

Amebic hepatic abscess is a tropical disease with a wide spectrum of clinical presentations. A retrospective case review was performed on 39 hospitalized patients in Thailand with the diagnosis of amebic liver abscess. A total of 23 men (59%) and 16 women (41%), with a mean age of 44.56 +/- 21.81 years (range, 10 to 88 years), were involved in the study. The average duration of present illness was 7.33 +/- 0.83 days. Abscesses were discovered in the right lobe in 29 patients (74.4%), in the left lobe in 3 patients (7.7%), and in both lobes in 7 patients (17.9%). Thirty patients had single abscesses (76.9%) and 9 patients had multiple abscesses (23.1%). On admission, the average white blood count was 17.37 +/- 6.34 x 1000 WBC/mm3, serum albumin was 2.86 +/- 0.61 g/dL, prothrombin time was 16.52 +/- 5.8 seconds, serum aspartate transaminase (AST) was 92.62 +/- 118.74 U/L, serum alanine transaminase (ALT) was 83.74 +/- 107.84 U/L, serum alkaline phosphatase (ALP) was 407.68 +/- 343.42 U/L, and serum bilirubin was 2.44 +/- 2.08 g/dL. Average indirect hemagglutination (IHA) titer of the cases was 1:1190.35 +/- 895.42 (range, 1:256 to 2048). Concerning the multiple logistic regression analysis, no significant correlation was found between antibody titer and the other parameters. Of interest, pathogenic organisms were detected in stool in only 2 cases. This study shows the usefulness of serologic study in diagnosis of amebic liver abscess.

Evaluation of soluble adhesion molecules in the diagnosis of amoebiasis, giardiasis and toxoplasmosis.

A total of 47 patients with toxoplasmosis (21 cases) with amoebic liver abscess (14 cases) and with giardiasis (12 cases) as well as 14 healthy control were subjected to thorough history taking, clinical examination, stool & urine analysis, complete blood picture, ESR, C-reactive protein, ASO, widal test, blood cultures, liver function tests, serum creatinine, hepatitis viral markers, rheumatoid factor, auto-antibodies, stool culture, rectal snip, chest X-ray, abdominal sonar, level of serum adhesion molecules (sICAM-1, sELAM-1), ELISA detection of Toxoplasma antibodies in serum, liver biopsy, detection and counting of Giardia cysts. In toxoplasmosis group, highly significant increase in serum levels of sICAM-1 (P<0.01) and significant increase in serum levels of sELAM-1 (P<0.05) in comparison to control. However, only sICAM-1 levels were significantly increased in IgM cases more than in IgG cases. In amoebic liver abscess group, both sICAM-1 and sELAM-1 significantly increased when compared with control. In giardiasis group, highly significant increase of serum levels of sELAM-1 was noticed than in control group (P<0.01), while sICAM-1 showed no significant difference (P>0.05). There was no correlation between sELAM-1 and number of cysts in the stool (intensity of infection). Soluble forms of adhesion molecules especially sICAM-1 have the potentiality as good markers of endothelial damage, severity of disease and to less extend load of infection.

[Hemorheological indices in patients with amebic abscesses of the liver and their correction]. / Gemoreologicheskie pokazateli u bol'nykh s amebnymi abstsessami pecheni i ikh korrektsiia.

Hemorheological indices were studied in 89 patients. In 61 patients the authors have revealed somewhat increased viscosity potential of blood and negligible changes in the aggregation activity of erythrocytes which is thought by the authors to be due to the saved compensatory potentialities of the organism. In 28 patients with the complicated course of the disease the rheological parameters of blood became worse because of the suppressed compensatory potentialities of organism. So, in addition to the adequate transfusion-infusion therapy the complex of treatment must include immunostimulating means (Levamisole, Prodigiozan, Tactivin etc.).

Common presentations of amebic liver abscess.

STUDY OBJECTIVE: The most common extraintestinal manifestation of Entamoeba histolytica, the agent of amebiasis, is a hepatic abscess. This infection is common throughout the world and can be associated with life-threatening consequences. Given the often nonspecific nature of the complaints related to an amebic abscess, a retrospective review of patients with confirmed disease was done to recognize the most common patterns of presentation. METHODS: A retrospective case series was conducted of all patients with confirmed amebic liver abscess over a 5-year period. All available emergency department and inpatient records were reviewed. Age, sex, country of origin, chief complaint (including duration), vital signs, and physical and laboratory findings were recorded. The use of ultrasonography, computed tomography scan, chest radiograph, and serum antibodies was noted, as well as the final ED diagnosis. RESULTS: Seventy-five patients were reviewed; mean patient age was 35.5 years, 80% were male, and Mexico was the country of origin for 64%. The most common complaint was fever (77%), followed by abdominal pain (72%), which was most often located in the right upper quadrant. Cough (16%), chest pain (19%), and chest radiographic abnormalities (57%) were also common. The majority of patients (69%) had symptoms for less than 13 days. The WBC count was the most consistent laboratory abnormality (83%), whereas the liver aminotransferase, alkaline phosphatase, and bilirubin levels were often normal. Most patients received their diagnoses on the basis of ultrasonography (85%), followed by a confirmatory serum antibody titer (88%). The diagnosis of amebic liver abscess was correctly made in the ED in 31.5% of the patients, with the most common misdiagnoses being cholecystitis (16.4%), hepatitis (12.3%), and pneumonia (9.6%). CONCLUSION: Patients with amebic liver abscess do present to EDs in the southwestern United States, especially in areas with a high immigrant population from endemic areas. Patients with complaints of fever and right upper quadrant abdominal pain, especially men of Hispanic origin, warrant a high degree of vigilance. Whereas most laboratory studies are unhelpful, the diagnosis can often be made in the ED by means of a bedside ultrasonographic test. Treatment should be initiated with metronidazole with disposition to an inpatient medical service.

Serum from Entamoeba histolytica-infected gerbils selectively suppresses T cell proliferation by inhibiting interleukin-2 production.

Suppression of T and B cell responses during invasive amebiasis may be serum mediated. The mechanism of serum-mediated suppression of spleen cell lymphoproliferation from gerbils with amebic liver abscess was examined. Compared with uninfected gerbil serum (10%), serum samples collected at days 10, 30, and 60, but not at day 20 after infection, augmented both concanavalin A (Con A; T cell mitogen)- and lipopolysaccharide (LPS; B cell mitogen)-induced proliferation of homologous spleen cells. Only day 20 serum (>5%) inhibited Con A- but not LPS-induced proliferation of spleen cells from uninfected gerbils. The suppressive mechanism was independent of nitric oxide and prostaglandin but involved reduced interleukin (IL)-2 production. Addition of exogenous IL-2 reversed the suppressive effect of day 20 serum on Con A-stimulated proliferation. These results identify a mechanism whereby serum may contribute to transient suppression of T cell responses during Entamoeba histolytica infections.

Multiple liver abscesses: an unusual case which demonstrates the importance of ultrasonography in the detection of liver pathology.

A 48-year-old caucasian male was admitted to hospital with right-sided chest pain, pyrexia and cough. He had no history of dysentery. He was treated with erythromycin and cotrimoxazole for right lower lobe pneumonia but failed to respond. Tender hepatomegaly developed and ultrasound scan demonstrated multiple abscesses in the liver. Entamoeba histolytica was identified in his faeces. He was treated with intravenous metronidazole, chloramphenicol and gentamicin and then oral tinidazole, after which improvement was rapid. He was later transferred to Australia. Subsequent abdominal CAT scan and aspiration of abscesses confirmed the diagnosis of multiple amoebic liver abscesses with secondary bacterial infection. Final treatment was with oral ciprofloxacin and metronidazole for four weeks. Ultrasonography is a noninvasive technique which is invaluable in the diagnosis of abdominal and especially liver pathology. This technique should be available in larger centres in tropical countries. Anyone living in or visiting the tropics should be aware of possible exotic diseases presenting in unusual ways.

Carbohydrate epitopes of Entamoeba histolytica cell surface glycoproteins are major targets of the human humoral response.

The antigens of Entamoeba histolytica recognized by antibodies in 11 individual sera from patients treated for amebic liver abscess were determined both by immunoprecipitation of metabolically-radiolabeled whole trophozoite proteins and by immunoblotting.Collectively, twenty-s even antigens ranging from 167 to 21 kDa were detected in immunoblots of whole trophozoite extracts; eight of these were recognized by all tested patient sera. Immunoprecipitation studies also revealed a complex amebic antigenic profile. Of a total of twenty immunoprecipitated polypeptides (from 200 to 24 kDa), seventeen were uniquely recognized by the patient sera. Eight of these seventeen antigens were immunoprecipitated by most immune sera. The cellular localization of trophozoite antigens was determined by analyzing plasma membrane and soluble cytosol fractions. Plasma membranes contained virtually as many antigenic moieties as the total trophozoite extract; in contrast, the soluble fraction was antigenically less complex. Mild periodate oxidation of plasma membrane antigens indicated that surface glycoproteins are highly immunogenic for the human host and that antibodies to their carbohydrate epitopes are a major component of the total response of most patients.