|
UPSI Digital Repository (UDRep)
|
|
| ||||||||||||||||||||||||
| Abstract : Universiti Pendidikan Sultan Idris |
| Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries. Intestinal amoebiasis may produce symptoms such as colitis and dysentery; while extraintestinal amoebiasis commonly results in amoebic liver abscess (ALA). The commercial antigen detection tests for intestinal infection vary in their sensitivities and specificities; most of them use native parasite antigen extract which have specificity issues. Thus this study was aimed at improving the laboratory diagnosis of ALA and intestinal amoebiasis. Previously, pyruvate phosphate dikinase (PPDK) had been identified as a marker for detection of extraintestinal amoebiasis, thus the recombinant form of the protein (rPPDK) was used in this study to develop a rapid antibody dipstick test for ALA. Search for new anti-amoebic agents is important due to the side-effects of metronidazole and reports of resistance against it. PPDK is involved in a key pathway in the energy metabolism of the parasite, thus is a promising target for a new anti-amoebic agent. Therefore, the present study was also aimed to investigate in vitro anti-amoebic activity of potential compounds using E. histolytica HM-l: IMSS. The PPDK gene was custom cloned into pET28a(+) expression vector, transformed into E. coli BL21 (DE3), expressed and purified. Diagnostic sensitivity and specificity of the purified rPPDK protein were evaluated by Western blot using individual serum samples. For comparison, the above procedures were also performed to produce recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (rGal/GaINAc lectin). Western blots using patients and control serum samples probed with anti-human IgG4-HRP showed higher diagnostic sensitivity (93.3%) and specificity (100%) as compared to blots using IgG and IgGl as secondary antibodies. rPPDK was found to show better specificity when compared to rGal/GalNAc lectin. A rapid dipstick test using rPPDK was developed and evaluated for ALA serodiagnosis. The results showed 96.7% (n=29/30) diagnostic sensitivity and 100% (n=40/40) specificity. For the development of an antigen detection rapid test for intestinal amoebiasis, polyclonal antibody (PAb) against rPPDK, rGal/GalNAc lectin and E. histolytica excretory-secretory antigens (Eh ESA) were produced. The best detection of E. histolytica in stool samples was shown when anti-rPPDK PAb was lined on the dipstick and gold conjugated anti-Eh ESA PAb was used as the detector reagent. Performance of the developed dipstick test was compared with commercial TechLab E. histolytica II ELISA and real-time PCR, using 70 stool samples from patients and controls. Ten compounds from the National Cancer Institute (NCI) were selected from a previous virtual screening performed against ATP site of a three-dimensional PPDK model. Using an enzymatic assay, seven of the compounds showed inhibitory activities, they include NSC349156 and NSC228137 with MIC values of 25 11M and 50 11M respectively. Using NBT reduction assay, NSC349156 showed the lowest IC50 (14.04 11M), followed by NSC228137 (20.7 11M); while IC50 of metronidazole was 6.15 11M. After 24 hours of treatment, NSC228137 and NSC349156 exhibited 37.61% and 75.73% growth inhibition, respectively. In conclusion, rPPDK was successfully used in this study for development of rapid antibody and antigen detection tests for diagnosis of extraintestinal and intestinal amoebiasis respectively. In addition, two compounds showed potential to serve as anti-amoebic agents. |
| This material may be protected under Copyright Act which governs the making of photocopies or reproductions of copyrighted materials. You may use the digitized material for private study, scholarship, or research. |