CONTRIBUTIONS TO UNRAVELLING THE NATURE OF THE FALSE POSITIVITY IN CIRCUMSPOROZOITE Plasmodium falciparum ELISA

INTRODUCTION: Plasmodium falciparum malaria, a vector-borne disease caused by the bite of Anopheles mosquitoes is a public health problem worldwide. Vector control remains as an efficient method to block malaria transmission. Vector incrimination, being a crucial factor for efficient vector control, nowadays mainly relies on detection of the parasite’s circumsporozoite protein in ELISA. However, false positivity in P. falciparum Circumsporozoite ELISA has been reported in Africa and Southeast Asia. The principal objective of this research is to determine the nature of the agent that provokes false positivity in Pf CSP ELISA through in silico analysis of Pf CSP, Next Generation Sequencing of bacterial 16s rDNA, detection of Microsporidia parasites in mosquitoes and isolation of the protein responsible for false positivity. MATERIALS AND METHODS: In silico analysis of Pf CSP was performed through BLAST search. Primers Bakt_341F/Bakt_805R attached to Illumina® adaptors were used to amplify bacterial 16s rDNA in 10 false positive samples from Vietnam and Cambodia, 1 laboratory reared An. stephensi and 2 true negative mosquitoes. Samples were identified into 16s rDNA database from GenBank with the Galaxy workflow modified for metagenomic studies. Cross-reaction of Microsporidia in Pf CSP ELISA was evaluated with Aedes aegypti infected with Vavraia culicis and Edhazardia aedis. Microsporidia were detected by specific amplification of 18s rDNA. Protein capture from 6 false positive samples was carried out using Dynabeads® M270 Epoxy antibody coupling kit and capture monoclonal antibodies 2A10. Isolated protein was separated through SDS-PAGE and identified by MALDI TOF/TOF analysis. RESULTS: In silico analysis of Pf CSP demonstrates some degree of identity with the domain Bacteria and a disordered NANPL region present in the Microsporidia E. aedis. Amplification and sequencing of 16s rDNA of false positive samples did not indicate a single species of bacteria present in the majority of false positive samples analyzed and absent in negative controls. Microsporidia did not show cross reactivity in Pf CSP ELISA and only one sample was found positive in head/thorax and abdomen for detection of Microsporidia by PCR. MALDI TOF/TOF analysis showed that actin cross- reacted with capture monoclonal antibodies in Pf CSP ELISA. DISCUSSION: In silico and laboratory analysis can not confirm Microsporidia, nor one single bacterial
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species as the cross-reactive protein. The remarkable diversity of species supports the hypothesis that more than one bacterial species could cross react with monoclonal antibodies in Pf CSP ELISA. The finding that the actin protein can be recognized by anti-Pf CSP monoclonal antibodies supports the animal origin theory of the causative agent of false positivity. Identification of this agent remains unclear, nevertheless important insights are provided towards a feasible explanation of false positivity in Pf CSP ELISA.