Absolute quantification of gene copy number variation in Plasmodium falciparum novel putative genes associated to drug resistance (the actin-binding coronin protein, cysteine desulfurase and plasmepsin 2), using Eva green-based quantitative PCR

Abstract

Background: Exposure of Plasmodium falciparum to host immune system and antimalarials pressure leads the parasite to exploit genetic factors such as genome sequence variation (structural variation and single nucleotide polymorphism) to withstand environmental conditions. Gene copy number variation (CNVs) is a type of genome sequence polymorphism and it has been shown that CNVs contribute to parasite adaptation to environmental pressure, acquisition of drug resistance and enable the parasite to escape from host immune system. In this study, we explored CNVs in P.falciparum three putative genes related to drug pressure.Method: Blood samples were obtained from asymptomatic school children collected from December 2016 to October 2018 in Mbita sub-county, western Kenya. Genomic DNA was extracted using ISOLATE II Genomic DNA kit (Bioline, UK) following the manufacturer’s instructions. Real-time quantitative PCR was performed and the target genes copy numbers were determined by absolute quantification method.Results: The findings revealed differential level of copy number variation in target genes investigated. One isolate (KE024/18) was found to contain 5.29 copies of Pfcoronin gene, t(2) = 27.91, P = 0.0013, 95% CI [3.62 - 4.95]. In the Pfcysteine desulfurase gene, similar level of copy amplification was observed in the OS0149/18, (5.09 copies) t(2) = 7.85, P = 0.0148, 95% CI [1.85 – 6.33]. These two isolates were found to harbour the highest level of CNVs of the corresponding genes. Pfplasmepsin 2 gene was found to be less polymorphic, however, three isolates showed high copy amplification, OS062/17 (9.63 copies), t(2) = 80.50, P = 0.0002, 95% CI [8.01 – 9.09]; AL106/16 (9.18 copies), t(2) = 28.61, P = 0.0012, 95% CI [6.95 – 9.41], and KM019/18 (9.41 copies), t(2) = 90.51, P = 0.0001, 95% CI [8.01 – 8.81].Conclusion: The results show gene copy number variation in the clinical samples we explored. The findings suggest that P.falciparum may be under drug pressure in the study area, therefore, this calls for close surveillance of drug resistance.