The protein profile of serum samples from patients with amoebic liver abscess (ALA) was compared to those of normal individuals to determine their expression levels and to identify potential surrogate disease markers. Serum samples were resolved by two dimensional electrophoresis (2-DE) followed by image analysis. The up and down-regulated protein spots were excised from the gels and analysed by MS/MS. The concentration of three clusters of proteins i.e. haptoglobin (HP), α1-antitrypsin (AAT) and transferrin in serum samples of ALA patients and healthy controls were compared using competitive ELISA. In addition, serum concentrations of HP and transferrin in samples of patients with ALA and pyogenic liver abscess (PLA) were also compared. The results of the protein 2-DE expression analysis showed that HP cluster, AAT cluster, one spot each from unknown spots no. 1 and 2 were significantly up-regulated and transferrin cluster was significantly down-regulated in ALA patients' sera (p<0.05). The MS/MS analysis identified the unknown protein spot no.1 as human transcript and haptoglobin and spot no. 2 as albumin. Competitive ELISA which compared concentrations of selected proteins in sera of ALA and healthy controls verified the up-regulated expression (p<0.05) of HP and the down-regulated expression (p<0.01) of transferrin in the former, while there was no significant difference in AAT expression (p> 0.05). However, when ALA and PLA samples were compared, competitive ELISA showed significant increased concentration of HP (p<0.05) while transferrin levels were not different. In conclusion, this study showed that HP is a potential surrogate disease marker for ALA.
A 38 year old gentleman presented with fever and right hypochondrial pain. On further evaluation he was detected to have an amoebic liver abscess (ALA) in the right lobe of the liver. The abscess yielded anchovy sauce pus on percutaneous drainage. Following the percutaneous drainage the patient developed tachycardia. Electrocardiogram revealed atrial flutter with rapid ventricular rate and ST elevation in all leads suggestive of pericarditis. The atrial flutter was reverted to sinus rhythm by cardioversion. The patient then had an uncomplicated convalescence. Amoebic pericarditis, though rare, is a serious complication of amoebic liver abscess. Pericardial complications are usually seen with left lobe liver abscess due to its proximity. Both pericarditis and cardiac arrhythmias due to amoebic liver abscess especially from right lobe are very rare.
We reviewed 204 cases of liver abscess seen between 1970 and 1985. Ninety were found to be amoebic, 24 pyogenic and one tuberculous. The cause of the abscesses in the remaining 89 patients was not established. The patients were predominantly male, Indians, and in the 30-60 age group. The majority of patients presented with fever and right hypochondrial pain. The most common laboratory findings were leucocytosis, hypoalbuminaemia and an elevated serum alkaline phosphatase. Amoebic abscesses were mainly solitary while pyogenic abscesses were mainly multiple. Complications were few in our patients and included rupture into the pleural and peritoneal cavities and septicaemic shock. An overall mortality of 2.9% was recorded. The difficulty in diagnosing the abscess type is highlighted. The single most important test in helping us diagnose amoebic abscess, presumably the most common type of abscess in the tropics, is the Entamoeba histolytica antibody assay. This test should be used more frequently in the tropics.
Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.
To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.
Serodiagnosis of amoebiasis remains the preferred method for diagnosis of amoebic liver abscess (ALA). However, the commercially available kits are problematic in areas of endemicity due to the persistently high background antibody titers. Human serum samples (n = 38) from patients with ALA who live in areas of endemicity were collected from Hospital Universiti Sains Malaysia during the period of 2008 to 2010. Western blots using excretory-secretory antigen (ESA) collected from axenically grown Entamoeba histolytica were probed with the above serum samples. Seven antigenic proteins of ESA with various reactivities were identified, i.e., 152 kDa, 131 kDa, 123 kDa, 110 kDa, 100 kDa, 82 kDa, and 76 kDa. However, only the 152-kDa and 110-kDa proteins showed sensitivities above 80% in the Western blot analysis. All the antigenic proteins showed undetectable cross-reactivity when probed with healthy human serum samples (n = 30) and serum samples from other infections (n = 33). From the matrix-assisted laser desorption ionization-two-stage time of flight (MALDI-TOF/TOF) analysis, the proteins were identified as heavy subunits of E. histolytica lectin and E. histolytica pyruvate phosphate dikinase, respectively. Use of the E. histolytica lectin for diagnosis of ALA has been well reported by researchers and is being used in commercialized kits. However, this is the first report on the potential use of pyruvate phosphate dikinase for diagnosis of ALA; thus, this molecule merits further evaluation on its diagnostic value using a larger panel of serum samples.
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.
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.
Amoebic serodiagnosis at Hospital Universiti Sains Malaysia (HUSM), Kelantan employs an indirect haemagglutination assay (IHA) which detects anti-Entamoeba histolytica antibodies in patients' serum samples. In an amoebiasis endemic area such as Kelantan, interpretation of a positive IHA result can be problematic due to the high background antibody levels. The TechLab E. histolytica II ELISA is a commercial kit for detection of specific Gal/GalNAc lectin antigen in stool samples, and has been reported to be able to detect the antigen in serum samples from patients with amoebic liver abscess (ALA). Thus in this study we investigated the usefulness of TechLab E. histolytica II ELISA for diagnosis of ALA by comparing it with IHA. This is a cross sectional study involving 58 suspected ALA patients who were admitted to the surgical ward, HUSM, Kelantan. The diagnosis of ALA was established based on clinical symptoms and signs, ultrasound and/or CT scan results. The serum specimens obtained from the patients were tested with IHA (Dade Behring Diagnostics, Marburg, Germany) and TechLab E. histolytica II ELISA (Techlab, Blacksburg, Virginia, USA) according to the manufacturers' instructions. Of the 58 patients, 72.4% (42) were positive by IHA and only 8.6% (5) were positive by the TechLab E. histolytica II ELISA. Agreement between the IHA and ELISA was poor (kappa value 0.019, p=0.691). There was also no correlation between ELISA results and IHA antibody titers. The TechLab E. histolytica II ELISA was not sensitive in detecting amoebic antigen in samples from ALA patients. In addition the results of the test did not correlate with the IHA anti-E. histolytica antibody titres. Therefore, the TechLab E. histolytica II ELISA was found not to be useful for serological diagnosis of ALA at HUSM.
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.