SOURCE:

Faculty: Pharmaceutical Sciences
Department: Pharm. Microbio & Biotech

CONTRIBUTORS:

Ikegbunam, M. N.
Esimone, C. O.
Nworu, C. S

ABSTRACT:

Malaria is one of the diseases responsible for high mortality in the world, accounting for over 40% global infection. Children and pregnant women bear the brunt of the disease which accounts for 429 000 deaths annually. It is hoped that the development of vaccine against malaria parasite would reduce the scourge of malaria. However, understanding the antigenic markers of P. falciparum circulating in a particular region is fundamental in designing appropriate vaccine that would boost immunity against the disease.This study was therefore, designed to characterize the species of Plasmodium in Nnewi, Southeast Nigeria, assess their genetic diversity and the prevalence of antimalarial resistance gene in circulation in the region. This was a cross sectional study involving eight hundred and twenty one asymptomatic malaria individuals whichincluded 251 children, 259 pregnant women and 312 from other adult population. Malaria parasites were detected in these individuals by microscopy prior to molecular studies. Preliminary demographic data were captured using semi structured questionnaire. Thin and thick blood smears were made for detection and parasite density determination. DNA was extracted from blood spot on filter paper for molecular species characterization, genetic diversity studies and resistance gene assessment. Nested Polymerase chain reaction (PCR) was used to amplify target genes in DNA extract followed by nucleotide sequencing using ABI Sanger sequencer. Mutations were assessed for Pfcrt,pfmdr1, Pf kelch 13, pfatpase 6, pfdhfr, and pfdhps genes at various codons.The calculated mean parasite density for the entire sampled was 1698.56 ± 35.84 cell/µl. Of the 822 samples, 725 were subjected to molecular genotyping. From the molecular analysis, 103 (14.21 %) were P. falciparum, 170 (23.4 %) were P. malariae, 31(4.27 %) were P.vivax. No parasite was detected in 421 (58.07 %) by the PCR. Genetic diversity studies showed that K 1(50.66 %) families were the most predominant in the region for msp-1 gene alleles, followed by MAD20 (30.67 %) and RO33 (18.67 %). For msp-2 gene alleles, 3D7 (53.4 %) was the predominant followed by FC27 (45.6 %). Prevalence of resistance markers were Pfcrt 76Tmutants (94.54 %), 75E mutants (94.54 %), and 74I mutants (94.54 %). Haplotype distributions were CVMNK (5.45 %), SVMNT (0 %) and CVIET (76.36 %). The Pfmdr1 86Y mutants and 184F mutants were found in 7(8.54 %) and 24 (29.27 %) respectively. Isolates that haboured 1246Y mutants were 3.66 %.Heterozygote alleles were found in 3.66 % at codon 184. None of the established mutations were detected in PfKelch 13 gene. Few isolates harboured Pfatpase6 gene at codon 569K (8.33%) 639D (4.17 %) and 1723V (4.17 %). Pfdhfr mutants were observed at codon 511I (57.14 %), 59R (71.43 %), and 108N (71.43 %), while Pfdhps mutants were observed at codon 437G (50 %), 431V (40 %), 581G (20 %) and 613S (50 %). Heterozygote alleles were observed in codon 613. Genetic diversity studies of P.falciparum in Nnewi showed that K1 family (50.66 %) and 3D7 (53.4 %) were the most predominant for msp1 and msp2 genes respectively. Pfcrt (94.54 %) were predominant in the region. We recommend intensive molecular surveillance of these markers, particularly Artemisinin and partner drugs.