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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
Three new single nucleotide polymorphisms identified in Plasmodium falciparum isolates from Kogi State, Nigeria
Onyekachi Nnaemeka Akoma1, Anthony Chibuogwu Ike1, Ebere Emilia Ayogu2
1Department of Microbiology, Faculty of Biological Sciences, University of Nigeria Nsukka, Enugu, Nigeria.
Background:
High malaria burden remains a remarkable challenge in Nigeria. The high prevalence has been attributed to a number of factors, including the continued evolution of resistance to antimalarial drugs by Plasmodium. However, the actual occurrence of antimalarial drug resistance in the country has not been well-documented. This study was aimed at screening for molecular markers of multidrug resistant Plasmodium species in individuals with parasitaemia in Kogi State, Nigeria.
Method:
Blood samples drawn from 600 symptomatic patients who consented to be recruited for the study were tested by Dipstick antigen test (RDT) for malaria. Genomic DNA from 40 samples, confirmed positive for P. falciparum by the rapid diagnostic test and PCR, were screened for the Pfmdr1 and Kelch-13 genes. Some of the Kelch-13 PCR amplicons were randomly selected and sequenced using Sanger method. Thereafter, the sequences were analysed and compared with wild type for single nucleotide polymorphisms (SNPs).
Results:
Pfmdr1 and K-13 amplicons were successfully obtained from 45 % (18/40) of the malaria-positive blood samples. Bioinformatics analysis of the K-13 sequences showed near 100 % sequence homology and percentage similarity of 99.88 % with the wild type Plasmodium falciparum 3D7 (Accession Number XM_001350122) gene. There were however three non-synonymous mutations (K430E, W706C and Q707K) observed in three samples.
Conclusions:
SNP analysis showed none of these mutations has been previously associated with artemisinin resistance. The significance of the study results was discussed in relation to current national malaria elimination and antimalarial resistance strategic plan or policy.

