Entamoeba species are commonly detected in stool samples of Orang Asli due to their substandard living conditions and poor hygiene. Among the Entamoeba spp., Entamoeba histolytica is the only known primary pathogenic species. This study determined the prevalence and distribution of anti-amoebic IgG antibody among Orang Asli in Peninsular Malaysia. The results would reflect the prevalence of amoebiasis in the population. This study analysed a total of 375 serum samples from archives of two Orang Asli projects conducted between 2011 and 2014. They were from six different states in Malaysia, namely Johor, Kedah, Kelantan, Pahang, Perak, and Selangor. Anti-amoebic IgG antibody was detected using an enzymelinked immunosorbent assay (ELISA) with crude soluble antigen produced from axenically grown E. histolytica trophozoites. From the analysis, the overall seropositivity was approximately 71% (266/375), while the seropositivity rates for each of the three Orang Asli tribes i.e. Senoi, Negrito and Proto-Malay, were 66% (137/208), 92% (103/112), and 43% (17/ 41) respectively. Orang Asli from Kedah [95% (52/55)] showed the highest seropositivity, followed by Kelantan [79% (54/68)], Perak [73% (78/107)], Pahang [60% (57/95)], Selangor [56% (14/25)], and Johor [48% (10/21)]. Orang Asli from rural [76% (192/254)] and peripheral urban [65% (69/106)] areas showed significantly higher seropositivity (p=0.002) than those from urban areas [36% (4/11)]. The high prevalences of anti-amoebic IgG antibody in these Orang Asli populations comprised both active and past infections. This study provides current insights of amoebiasis in selected Orang Asli settlements in Peninsular Malaysia. The high seropositivity of anti-amoebic IgG antibody suggests that the settlements are endemic for amoebiasis and there is a high risk of acquiring E. histolytica infection among the dwellers.

Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.