Genetic polymorphism of the N-terminal region in circumsporozoite surface protein of Plasmodium falciparum field isolates from Sudan

BackgroundMalaria caused byPlasmodium falciparumparasite is still known to be one of the most significant public health problems in sub-Saharan Africa. Genetic diversity of the SudaneseP. falciparumbased on the diversity in the circumsporozoite surface protein (PfCSP) has not been previously studied. Therefore, this study aimed to investigate the genetic diversity of the N-terminal region of thepfcspgene.MethodsA cross-sectional molecular study was conducted; 50 blood samples have been analysed from different regions in Sudan. Patients were recruited from the health facilities of Khartoum, New Halfa, Red Sea, White Nile, Al Qadarif, Gezira, River Nile, and Ad Damazin during malaria transmission seasons between June to October and December to February 2017–2018. Microscopic and nested PCR was performed for detection ofP. falciparum. Merozoite surface protein-1 was performed to differentiate single and multiple clonal infections. The N-terminal of thepfcspgene has been sequenced using PCR-Sanger dideoxy method and analysed to sequences polymorphism including the numbers of haplotypes (H), segregating sites (S), haplotypes diversity (Hd) and the average number of nucleotide differences between two sequences (Pi) were obtained using the software DnaSP v5.10. As well as neutrality testing, Tajima’s D test, Fu and Li’s D and F statistics.ResultsPCR amplification resulted in 1200 bp of thepfcspgene. Only 21 PCR products were successfully sequenced while 29 were presenting multiple clonalP. falciparumparasite were not sequenced. The analysis of the N-terminal region of the PfCSP amino acids sequence compared to the reference strains showed five different haplotypes. H1 consisted of 3D7, NF54, HB3 and 13 isolates of the Sudanesepfcsp. H2 comprised of 7G8, Dd2, MAD20, RO33, Wellcome strain, and 5 isolates of the Sudanesepfcsp. H3, H4, and H5 were found in 3 distinct isolates. Hd was 0.594 ± 0.065, and S was 12. The most common polymorphic site was A98G; other sites were D82Y, N83H, N83M, K85L, L86F, R87L, R87F, and A98S. Fu and Li’s D* test value was − 2.70818, Fu and Li’s F* test value was − 2.83907, indicating a role of negative balancing selection in thepfcspN-terminal region. Analysis with the globalpfcspN-terminal regions showed the presence of 13 haplotypes. Haplotypes frequencies were 79.4%, 17.0%, 1.6% and 1.0% for H1, H2, H3 and H4, respectively. Remaining haplotypes frequency was 0.1% for each. Hd was 0.340 ± 0.017 with a Pi of 0.00485, S was 18 sites, and Pi was 0.00030. Amino acid polymorphisms identified in the N-terminal region of globalpfcspwere present at eight positions (D82Y, N83H/M, K85L/T/N, L86F, R87L/F, A98G/V/S, D99G, and G100D).ConclusionsSudanesepfcspN-terminal region was well-conserved with only a few polymorphic sites. Geographical distribution of genetic diversity showed high similarity to the African isolates, and this will help and contribute in the deployment of RTS,S, a PfCSP-based vaccine, in Sudan.