Polymorphisms in Plasmodium Falciparummalaria Genes in Isolates from Selected Centres in Lagos, Nigeria
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Plasmodium falciparummalaria remains a disease of global andnational health importance due to spread of multi-drug resistance strains of the parasite and insecticide resistance in the mosquito vector resulting in high morbidity and mortality rates. Erythrocyte invasion by P. falciparum is a complex process, of which P. falciparum apical membrane antigen I (PfAMA1) and reticulocyte binding protein homolog 5 (PfRH5) are key proteins, hence, targeted for malaria vaccine development. Immune responses against merozoite attachment and invasion may be associated with genetic variations observed in these vaccine candidate antigens. This study assessed polymorphisms in AMA1 and RH5 among P. falciparum isolates from Nigeria. One thousand eight hundred and eighty-three (1,883) febrile subjects were examined, out of which 384 (20.39%) were microscopically positive for P. falciparum mono-infection. Three hundred of these volunteers were included from three health facilities in Lagos state: Ijede General Hospital, Ikorodu (IJE), Ajeromi General Hospital,Ajegunle (AJE)and St. Kizito Mission Hospital, Lekki, (100 from each site) andEighty age-matched apparently healthy controls from the same communities were included in the study. Giemsastained thick and thin blood films of participants were used for P. falciparum identification and quantitation. Blood collected on Whatmann 3.0 filter paper was used for DNA extraction and assessed for DNA quality and yield. Molecular genotyping was carried out using the block 3 of Merozoite Surface Protein-2 to determine parasite genetic diversity and Multiplicity of Infection (MOI). One hundred and ninety-five samples which were PCR positive for msp2 gene were used for PCR amplification and sequencing of pfama1andPfrh5 genes to determine the genetic structure and polymorphisms on AMA1andRH5. Serum levels of IL-12, TNF-α and IL-1β were determined by Enzyme Linked Immunosorbent Assay (ELISA). Sequence alignment was done usingBioedit and Clustal W in MEGA softwareswhile sequences were analysed using DnaSP. Results revealed that malaria prevalence among the febrile patients was 20.39 %, highest in Ijede and lowest in Lekki. Levels of the pro-inflammatory cytokines studied were higher (p< 0.05) in the infected participants than the controls. Eighteen different alleles were observed for MSP-2 loci, FC27 family being more prevalent. Mean MOI was 1.54 ± 0.2. Heterozygosity (HE) values ranged from 0.77 – 087 and highest for IJE (0.87). Cytokine response was significantly associated with MOI (P < 0.05) but not parasite density. Genetic sequence analysis revealed 93 different haplotypes (H) for AMA1 Domain I. Forty-eight of these haplotypes are new with 34 segregating sites, Haplotype diversity of 0.992 ± 0.004, mean nucleotide diversity of 0.02468 ± 0.00076 and Total number of mutations being 52. Tajima’s D anddN-dS (non-synonymous minus synonymous mutations) were positive showing positive selection on AMA1. Decline in Linkage Disequilibrium was observed showing high recombination events taking place, however, the High Activity Binding Peptide (HABP) sequences of rh5 gene revealed three haplotypes of RH5 with negative Tajima’s D anddN-dS values showing no selection on RH5. A Single Nucleotide Polymorphism (SNP) (G A), on nucleotide position 608 was observed on the RH5 sequences based on 3D7 reference sequence. Phylogenetic analyses of the AMA1 and RH5 sequences from this study showed clustering and evidence of evolutionary relationship with 3D7, Guinean AMA1, PAS-2 and FCB-2 RH5 strains using P. reichenowi as the out-group at 1000 bootstraps replications. The study indicates that the flow of any resistance alleles among P. falciparum isolates in Lagos may be relatively high, with negative impacts on control measures owing to low fstvalues. New AMA1 haplotypes were found suggesting that Nigerian P. falciparum should be given consideration in the design and development of control strategies. The study shows that globally efficacious RH5-based xxii vaccines may be potentially applicable in Nigeria, inspite of the complex genetic diversity of P. falciparum isolates from Nigeria.
University of Lagos School of Postgraduate Studies Phd Thesis and Dissertation
Merozoite , Haplotypes , Synonymous mutations , Polymorphisms
Ajibaye, O (2017) Polymorphisms in Plasmodium Falciparummalaria Genes in Isolates from Selected Centres in Lagos, Nigeria. University of Lagos School of Postgraduate Studies Phd Thesis and Dissertation.