Development of Plasmodium knowlesi species specific reagents to help characterise antibody isotype profiles in endemic human populations

Abstract

Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease. Using in silico tools, we designed and expressed a panel of P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools. Antibody prevalence to these antigens was determined by ELISA, Multiplex Bead Assay (MBA) and Protein Microarray for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, Malaysia (n=110 individuals; 298 total samples for all time points), a small community survey in Sabah, Malaysia (~2000), and Palawan, The Philippines (~550), and a large cross sectional survey in Sabah, Malaysia (~10,000). Both ELISA and MBA showed similar results, with higher responses observed for the PkSERA3 antigen 2, both at the clinical level across all time points and at the community level. This antigen is suggested to be a short term marker of exposure as reactivity to it tended to decline by day 28 of diagnosis. It was possible to determine that P. knowlesi is prevalent in Sabah, Malaysia (37.8%, 3,827/10,125). Seropositivity was found to be associated with an increase of age (p<0.0001) as well as seeing macaques (p<0.0001). The protein microarray, which was used to determine isotype reactivity profiles (IgM, IgG and IgA) obtained results concordant with the ELISA and MBA results, with PkSERA3 antigen 2 eliciting the highest response for all isotypes. IgM decreased significantly across time while IgG tended to increase across time. We find it necessary to further develop and expand our current panel of P. knowlesi antigens in order to better dissect the epidemiology of P. knowlesi in Southeast Asia.