Molecular Assessment of Antimalarial Drugs Resistance in Plasmodium Falciparum at Nuba Mountains, South Sudan

Abstract

One of the major obstacles to the treatment of malaria is the emergence and spread of parasite resistant to antimalarial drugs. Both northern and southern Sudan is deploying artemisinin-based combinations against uncomplicated Plasmodium falciparum malaria. In this study, the efficacy of artesunate (AS) in combination with sulfadoxine- pyrimethamine (SP) or amodiaquine (AQ) was evaluated. This study aimed at determining the prevalence of antimalarial drug resistance markers in P. falciparum isolates from Nuba Mountains, which involves the detection of mutations at the Pfcrt and mdr 1- 86 that associates with chloroquine resistance and amodiaquine resistance, and dhfi mutations which are associated with SP resistance. Samples in this study were obtained from Nubian region of Sudan. A total of 1 60 field isolates that were from patients of between 6 months and 10 years were recruited for the study and four positive controls were analyzed, 80 of the patients were of AS/AQ treatment arm and the other 80 were treated with AS/ SP combination. The investigation involved collecting blood samples from malaria patients, extracting the DNA of Plasmodium falciparum from these blood samples, amplifying the extracted DNA using PCR method and finally analysing the amplified products using RFLP for Pfirrt and dhfi gene markers and radioisotopic method to determine the prevalence of ma'r1- 86. Detection for mutations at the P. falciparum chloroquine transporter (Pfcrt) gene (associated with CQ resistance) from pre-treatment blood samples was analysed to correlate the existing use and resistance to Chloroquine. The Pfcrt K76T mutation occurred in 90.0% of infections, suggesting CQ would work poorly in this region. Furthermore, among patients with failure endpoints, genotyping of merozoite surface proteins 1 and 2 (msp-1 and msp-2) was employed to compare the pre- and post-treatment to establish whether failure was due to true recrudescence or a novel infection. The prevalence of the dhfr point mutation among the field isolates of P. falciparum was established, and the result correlated between these mutations and resistance of isolates to antimalarial. Overall, 82.5% carried mutations at dhfi", but triple mutants were rare (3.1%). The dot-bl0t/ probe hybridization, which is more sensitive and specific, was adopted to determine multi-dmg resistance (mdrl -86). Baseline dot-blot hybridisation of the mdrl 86 locus shows 33 (89.l9%) carried parasites harboring the mdr1-86 Tyr resistance allele, while 3 (8.l 1%) carried mdrl-86 Asn sensitivity allele and l (2.7%) were of mixed infection, having both resistance and wild type allele. The research presented here shows that the number of mutations present in dhfi has a significant effect on the type of treatment following SP chemotherapy. SP resistance may propagate rapidly, and AS + AQ is likely to be a better long-term option, provided AQ use is limited to the combination. The significance of the study shows that indeed combination of drugs improves SP therapy at the study site.