RESUMEN
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.
Asunto(s)
Antígenos de Protozoos , Entamoeba histolytica/enzimología , Absceso Hepático Amebiano/diagnóstico , Fosfoglucomutasa , Proteínas Protozoarias , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Western Blotting , Electroforesis en Gel Bidimensional , Entamoeba histolytica/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoglobulina G/sangre , Absceso Hepático Amebiano/sangre , Absceso Hepático Amebiano/inmunología , Espectrometría de Masas , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas Serológicas/métodosRESUMEN
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 â¼75 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.