Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess.

BACKGROUND: Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA. METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA. RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 µg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 µg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity. CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.

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.

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.

Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis.

BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.

Entamoeba histolytica acetyl-CoA synthetase: biomarker of acute amoebic liver abscess.

OBJECTIVE: To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. METHODS: Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. RESULTS: A ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. CONCLUSIONS: This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

Urinary schistosomiasis and malaria associated anemia in Ethiopia.

OBJECTIVE: To assess the prevalence of anemia in children with urinary schistosomiasis, malaria and concurrent infections by the two diseases. METHODS: Urine and blood samples were collected from 387 children (216 males and 171 females) to examine urinary schistosomiasis and malaria and to determine hemoglobin concentration at Hassoba and Hassoba Buri village in Amibara woreda, Afar region, Ethiopia. RESULTS: The overall prevalence of urinary schistosomiasis and Plasmodium falciparum malaria was 24.54% and 6.20% respectively. Only 2.84% of children carried concurrent infections of both parasites. There was high percentage of anemic patients (81.81%) in the coinfected cases than in either malaria (33.3%) or schistosomiasis (38.94%) cases. There was significantly low mean hemoglobin concentration in concurrently infected children than non-infected and single infected (P<0.05). The mean hemoglobin concentration between Plasmodium falciparum and S. haematobium infected children showed no significant difference (P>0.05). The level of hemoglobin was negatively correlated with the number of S. haematobium eggs/10 mL urine (r=-0.6) and malaria parasitemia (r=-0.53). CONCLUSIONS: The study showed that anemia is higher in concurrently infected children than non-infected and single infected. Furthermore, level of hemoglobin was negatively correlated with the number of S. haematobium eggs and malaria parsitemia. Therefore, examination of hemoglobin status in patients co-infected with malaria and schistosomiasis is important to reduce the risk of anemia and to improve health of the community.

Experimental evaluation of Candonocypris novaezelandiae (Crustacea: Ostracoda) in the biocontrol of Schistosomiasis mansoni transmission.

OBJECTIVE: To test Candonocypris novaezelandiae (Baird) (C. novaezelandiae), sub-class Ostracoda, obtained from the Nile, Egypt for its predatory activity on snail, Biomphalaria alexandrina (B. alexandrina), intermediate host of Schistosoma mansoni (S. mansoni) and on the free-living larval stages of this parasite (miracidia and cercariae). METHODS: The predatory activity of C. novaezelandiae was determined on B. alexandrina snail (several densities of eggs, newly hatched and juveniles). This activity was also determined on S. mansoni miracidia and cercariae using different volumes of water and different numbers of larvae. C. novaezelandiae was also tested for its effect on infection of snails and on the cercarial production. RESULTS: C. novaezelandiae was found to feed on the eggs, newly hatched and juvenile snails, but with significant reduction in the consumption in the presence of other diet like the blue green algae (Nostoc muscorum). This ostracod also showed considerable predatory activity on the free-living larval stages of S. mansoni which was affected by certain environmental factors such as volume of water, density of C. novaezelandiae and number of larvae of the parasite. CONCLUSIONS: The presence of this ostracod in the aquatic habitat led to significant reduction of snail population, infection rate of snails with schistosme miracidia as well as of cercarial production from the infected snails. This may suggest that introducing C. novaezelandiae into the habitat at schistosome risky sites could suppress the transmission of the disease.

Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess: comparison of three staining methods.

OBJECTIVE: 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. METHODS: Amoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite. RESULTS: The three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues. CONCLUSIONS: It can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.

Prevalence and risk factors of intestinal helminth infection among rural malay children.

BACKGROUND: Soil-transmitted intestinal helminth infection is prevalent in rural communities of Malaysia. Risk factors contributing to helminth infections are largely unknown in the country. AIM: To determine the prevalence and risk factors of intestinal helminth infections among children in Beris Lalang, a rural Muslim community of Malaysia. SETTINGS AND DESIGN: In this cross-sectional study, children aged 7-9 years were recruited during the mass Friday prayer at Beris Lalang mosque by trained imams (religious leaders). A standardized questionnaire was used to obtain information on socio-demographic profile, daily hygienic practices, and history of helminth infection. RESULTS: Out of 79 samples, 29 (37%) were positive for helminthic ova, of which 24 were ova of Trichuris trichiura. Poor education of the mother (primary education or less) (P=0.015), eating raw salad (P=0.03), and no physical activities (P=0.03) were found independent risk factors for the child's helminth infections in univariate analysis. A higher proportion of children with helminth infections complained of tiredness and fatigue compared to those without such infections (36% vs. 12%, P=0.019). In a multivariate analysis of predictors of helminth infection, poor education of the mother (P=0.02) and eating raw salad (P=0.04) remained statistically significant, after controlling for several other potential risk factors. CONCLUSIONS: T. trichiura was the most prevalent intestinal helminth infection in children in rural Malaysia. Risk factors of helminth infection included mother's poor education and eating raw salad and vegetables.