An investigation into the the prevalence of point mutations associated with antimalarial drug resistance in Plasmodium Falciparum found in the Zambezi region of Namibia

A research thesis submitted in fulfillment of the requirements for the Degree of Master of Science

Surveillance and monitoring of emerging drug resistance is important as Namibia moves towards malaria elimination. In 2005 artemisinin combination therapies (ACT’s) were introduced as first-line treatment for uncomplicated falciparum malaria in Namibia. However, reduced ACT efficacy has been reported in Asia and in some parts of Western Africa, raising concerns around the efficacy of artemisinin. This study aimed to identify the different plasmodium species found in Zambezi region, Namibia as well as to investigate the prevalence of antimalarial drug resistance polymorphisms in the pfcrt, pfmdr1 and Kelch 13 genes in the region. A QIAamp DNA mini-kit (Qiagen, Germany) was used to extract DNA from malaria positive Dried Blood Spots (DBS). Prior to investigating the prevalence of point mutations, multiplex PCR was performed to investigate the different plasmodium species found in the Zambezi region of Namibia. Seventy-two P. falciparum positive samples out of a total of 143 malaria positive samples were analysed by PCR-RFLP at codon N86Y in the pfmdr1 whereas the prevalence of haplotypes at codons 72-76 in the pfcrt gene were analysed using Quantitative-PCR. Additionally, mutations at 25 codons in the Kelch 13 gene were analysed by n-PCR followed by sequencing. The study found that 76.2% of the cases were caused by P. falciparum which was less than expected. Additionally, 7.1% of the cases were found to be caused by P. vivax followed by 6.0% of P. malariae infections. In the pfcrt gene 92.5% samples contained the wild type haplotype, 3.8% contained the mutant haplotype while 3.8% samples had mixed haplotype. Furthermore, 18.5% mutant and 59.3% wild and type alleles were observed in pfmdr1 gene at N86Y. No mutant alleles were observed in the Kelch 13 gene. This study provides the first data on point mutations in the pfcrt, pfmdr1 and Kelch 13 genes in Namibia. A low prevalence of mutations was observed in this study which shows that ACTs are still effective in Namibia. However, continued surveillance is recommended with similar studies in other endemic regions of Namibia. Additionally, it is recommended that further studies such as in vivo and in vitro responses to drug treatment be done to determine the role of these polymorphisms in drug resistance to support these findings.