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).

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.

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.

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.

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.

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.

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.

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 specific solid-phase assay for the detection of immune complexes containing Entamoeba histolytica antigens.

An assay specific for immune complexes containing Entamoeba histolytica antigens has been developed. Rabbit antibody specific for human gamma globulin is attached to a solid phase (nitrocellulose). During the first incubation step polyethylene glycol-precipitated immune complexes are bound to the rabbit antibody attached to the solid phase. In the second incubation step a radiolabelled rabbit antibody specific for Entamoeba histolytica combines with E. histolytica antigens in the complexes. Quantitation of the radiolabel provides a direct measurement of the level of specific immune complexes. The principle of the method has been verified using artificial soluble immune complexes prepared by mixing a pool of serum containing a high titre of anti-E. histolytica antibodies (greater than 1:2048 by hemagglutination assay) with the E. histolytica antigen. This antigen was prepared from axenically cultured NIH:200 strains of Entamoeba histolytica. Using sera from patients with clinical amoebic disease this method detected immune complexes containing E. histolytica in a high proportion of cases.

Localization and identification of an Entamoeba histolytica adhesin.

The adherence of Entamoeba histolytica trophozoites to target cells was studied by using monoclonal antibodies (MAbs) and adhesion-deficient mutants of the parasite. MAbs Adh-1 and Adh-2 reacted with a surface protein of approximately 112 kDa of the total proteins of trophozoites from the wild type strain, clone A, strain HM1:IMSS. Both MAbs reacted weakly with the adhesion-deficient mutant clones, C-98, C-919 and C-923, all derived from HM1:IMSS. MAbs Adh-1 and Adh-2 incubated with trophozoites from clone A inhibited adherence to red blood cells, erythrophagocytosis and cytopathic effect on cell culture monolayers. Antibodies against a approximately 112 kDa polypeptide were found in the sera from patients with hepatic abscess. These results demonstrate that the adherence of trophozoites to target cells is a necessary event in order for cytopathogenicity to occur.

Specific antibodies in serum of patients with hydatidosis recognised by immunoblotting.

Hydatid fluids from sheep, goat, pig and man, after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions, revealed at least 15 discrete polypeptide bands of 8-116 Kda. By ELISA, sera from all 20 cases of hydatidosis showed anti-hydatid antibody, but so did 11 (73%) of 15 sera samples from cysticercosis patients, eight (67%) of 12 sera from patients with other parasitic infections (amoebic liver abscess or hymenolepiasis) and one (4%) of 25 sera from healthy controls. Antibody to cysticercus antigen was found in 14 (93%) of 15 sera from cysticercosis patients, 17 (85%) of 20 sera from hydatid patients, six (50%) of 12 sera from patients with other parasitic infections and one (4%) of 25 sera from healthy controls. Sera from 17 (85%) of 20 hydatid patients, 11 (73%) of 15 cysticercosis patients and five (42%) of 12 patients with other parasitic infections had antibodies to both hydatid and cysticercus antigens. Sera from 20 surgically confirmed cases of hydatidosis reacted with 12 polypeptides of 8-116 Kda in Western immunoblot with hydatid antigens. Polypeptides of 16, 24, 38, 45 and 58 Kda were recognised by all hydatidosis sera but also by many sera from patients with other infections. However, polypeptides of 8 and 116 Kda were recognised by all hydatidosis sera but not by any sera from patients with cysticercosis, other parasitic infections or viral hepatitis, or from healthy controls. Thus, recognition of 8- and 116-Kda hydatid antigens by a patient's serum appears to be a specific test confirming a clinical diagnosis in an individual case of hydatidosis.

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.

Serum ferritin in Nigerian patients with amoebic liver abscess and other tropical infections with liver involvement.

Serum ferritin was measured by immunoradiometric assay in 46 Nigerian patients with amoebic liver abscess and other tropical infections involving the liver, and the values were compared with those in 23 control subjects. Serum ferritin was markedly elevated in 100% of the patients with amoebic liver abscess, acute viral hepatitis and liver tuberculosis. Elevated values were observed in about 77% of patients with cirrhosis, 80% of malaria patients, and only about 30% of patients with early infection of schistosomiasis mansoni. The results support previous data indicating that significant changes in serum ferritin occur in acute and chronic liver disease. Assay of serum ferritin may be a useful complimentary liver function test for the diagnosis and monitoring the treatment provided in amoebic liver abscess.

IgA reactivity in patients with intestinal or hepatic amebiasis.

The antigens of E. histolytica recognized by IgA antibodies in sera from patients with either amebic liver abscess or intestinal amebiasis, as well as saliva from the latter, were determined in immunoblots of whole native trophozoite proteins. Results show that sera of patients with amebic liver abscess reacted mainly with amebic proteins of more than 200 kDa, whereas those of individuals with intestinal amebiasis recognized a protein of 145 kDa. The saliva of the latter detected a protein of 210 kDa which is not always seen when using the sera of patients with intestinal or hepatic amebiasis.

Entamoeba histolytica: electrophoretic analysis of isolated caps induced by several ligands.

Caps induced by several ligands including sera from patients with amebic liver abscess (ALA) were isolated by differential centrifugation. Purification was verified by scanning and transmission electron microscopy. Electrophoretic profiles obtained by SDS-PAGE and Western blots were compared when probed with the ligand used to induce capping. It was confirmed that sera from different patients recognize different proteins. Indeed, electrophoretic profiles reveal that capped membrane proteins vary according to the ligand used to induce capping. This would agree with the supposition that E. histolytica might use this process as a mechanism to evade the host's immune response, by clearing the cell surface from recognized antigens.

Profile of amebic liver abscess.

Two hundred cases of amebic liver abscess diagnosed between 1989 and 1991 at the Kasturba Medical College, Manipal were analyzed in this retrospective study. The clinical features and investigation reports were studied and the treatment and its response were analyzed. Amebic liver abscess constituted 0.6% of total hospital admissions during the study period. The male to female ratio was 13:1 with the most common age group of presentation between the fourth and fifth decades of life. Abdominal pain was the most common symptom (92%) and hepatomegaly was observed in 94% of the cases. Ultrasonogram of the abdomen served as the most useful diagnostic aid. Right lobe abscess was observed in 87% of the cases. Abscess was single in 81.5% of cases. Abscess size of more than 4 cm was observed in 46.5% of the cases. Metronidazole and chloroquine were found to be effective in most cases. Aspiration was done in 35.5% of cases. The complications encountered in this study were pleural effusion (two cases), pneumonic consolidation (four cases), pericardial effusion (one case) and ascitis (two cases).

Amebiasis in Nicaragua: class specific serum antibody responses.

With the aid of the indirect hemagglutination (IHA) test and IgG ELISA the antibody profile against E. histolytica in León, Nicaragua was investigated in 562 sera from individuals belonging to various age groups. The highest reactivity was invariably recorded in the age group 6-15 years where 48% were seropositive. Several sera reactive by either one of IHA and IgG ELISA were negative by the other test. The main reason for this seems to be reactivity in different Ig classes. Treatment with 2-mercaptoethanol reduced the titre level in 63 of the 66 sera tested. Immunofluorescence using an anti-IgM conjugate showed that 26 of 43 sera contained specific IgM-antibodies, indicating that also unspecific reactions are involved in the IHA test. A comparison was made between class-specific reactivity in three population groups: healthy residents, healthy cyst carriers and patients with recent or acute liver abscess. No significant difference in the prevalence of reactions above the diagnostic significance level was recorded between cyst carriers and healthy residents. However, among the cyst carriers 33% had IgA and/or IgM antibodies but no demonstrable specific IgG. Most patients with recent and all with acute liver abscess reacted significantly above the diagnostic limit in all three tests.

Early detection of complications in amebic liver abscess.

A retrospective analysis of 140 cases with amebic liver abscess (ALA) seen at the AUNL University Hospital was done to see if patients with complications can be identified earlier in order to decrease morbidity and mortality. Sixteen patients (11.4%) presented complications and six patients died (4.2%). Patients with complications presented jaundice, large or multiple abscesses, acute abdomen, liver failure and sepsis more often than patients without complications. Hemoglobin, hematocrit, prothrombin time, total proteins, albumin, LDH, and BUN were more altered in patients who presented complications. The titer of antibodies against E. histolytica was higher in this group of patients. The six patients who died had been operated on. The causes of death were septic shock in two, sepsis in one, peritonitis in one, liver failure in one and colon perforation in one patient. Pleural effusion, jaundice and acute abdomen were seen in three patients, respectively (50%), two cases had multiple abscesses (33.3%), one patient had a ruptured abscess (16.7%). Patients who died exhibited more alterations in six laboratory examinations at admission: partial prothrombin time, total bilirubin, albumin, BUN, LDH, and leukocytes. Clinical data together with the severe alterations in laboratory examinations at admission for patients with ALA should alert the clinician to suspect complications earlier in order to decrease morbidity and mortality.

Plasma concentrations of fibronectin and C3d in patients with amoebic liver abscess.

By means of simple and specific ELISA techniques, the plasma concentrations of soluble fibronectin and C3d, a breakdown product of C3 complement, were determined in patients with amoebic liver abscesses (ALA) and in healthy controls. The mean plasma fibronectin concentrations in 23 patients with ALA and in 20 controls were found to be 441 +/- 89 mg/l and 442 +/- 66 mg/l, respectively. The difference between these two values was not statistically significant. The mean C3d value in 21 patients with ALA, however, was found to be 84 +/- 14 AU/l which was significantly different from the value of 12 +/- 4.7 AU/l noted in 20 healthy persons. Plasma concentrations of these two proteins are discussed in relation to their possible implications in the immunopathogenesis of amoebic liver abscess.