Mutational and Phylogenetic Analyses of Antifolate and Artemisinin Resistance in Plasmodium falciparum Dried Blood Spots Obtained from Patients Attending Three Hospitals of Eritrea

Abstract

Malaria causes approximately 200 million infections and 500,000 deaths yearly. The emergence and spread of antimalarial drug resistance is a major challenge towards control efforts globally. Nonetheless, integrated vector control methods have reduced malaria transmission, resulting in decreased human-parasite reservoirs and lessened spread of antimalarial drug resistance. Eritrea, located in the horn of Africa, has witnessed a considerable reduction in malaria deaths from 405 to 4 between 1998 and 2023. This is attributed to a combination of community health campaigns, prompt case management, and integrated vector management. In Eritrea, Sulfadoxine-Pyrimethamine was previously used as a first-line antifolate treatment, and currently, artesunate is the first-line artemisinin treatment. Due to limited molecular data on these antifolate and artemisinin treatments, the first objective of this study, was to determine antifolate resistance-associated genetic mutations in codon position K540E of Pf-DHPS gene, and codon positions N51I, C59R and S108N of Pf-DHFR gene. The second objective, was to determine artemisinin resistance-associated genetic mutations in codon positions Y493H, R539T, I543T, and C580Y of Pf-K13 gene. The third objective, was to determine the phylogenetic relationships between the genetic markers sampled in this study, and corresponding globally identified genetic markers from other studies. Sample size was determined using Fisher’s formula, and based on patient availability, inclusion and exclusion criteria. Nineteen dried blood spot samples were collected from patients infected with P. falciparum mono-infection, visiting Adi Quala, Keren, and Gash Barka hospitals. Genomic DNA extraction, nested-PCR amplification and Sanger-sequencing of Pf-DHFR, Pf-DHPS, and Pf-K13 partial gene regions was achieved for nine dried blood spots. Sequence contig assembly, genetic mutation visualization, and phylogenetic analyses were performed in CLC main workbench v21.0.4, Jalview v2.11.1.4, and MEGAv7.0. Mutational analyses identified the single-mutant K540E of Pf-DHPS in Adi Quala (n=1), Keren (n=1), and Gash Barka (n=1); double-mutant N51I+S108NI of Pf-DHFR in Adi Quala (n=2); triple-mutant S108N+C59R+N51I of Pf-DHFR in Keren (n=1); mixed-mutant (S108N+N51I+K540E) of Pf-DHFR and Pf-DHPS in Gash Barka (n=1). These findings suggested the likely presence of the quintuple-mutant (S108N, C59R, N51I+A437G, K540E) of Pf-DHFR and Pf-DHPS, associated with full resistance, and used to predict Sulfadoxine-Pyrimethamine treatment failure. Mutational analyses of Pf-K13 identified wild-type haplotypes of Y493Y+R539R+I543I+C580C in Adi Quala (n=2) and wild-types of C580C in Keren (n=1) and Gash Barka (n=3). These findings suggested the likely absence of artemisinin resistance, and predicted artesunate was still effective for malaria treatment. The Dhfr phylogeny predominantly identified the double-mutant haplotype (N51I + S108N) at an estimated distribution ranging from low to high prevalence in Western Kenya (p=0.3%), Myanmar (p=2.5%), India (p=7%) and Sudan (p=80%). The K540E mutation was predominantly identified in the Dhps phylogeny, at an estimated distribution ranging from low to moderate prevalence in Ghana (p=3.4%), Equatorial Guinea (p=5.1%), and Sudan (p=65.7%). These analyses suggested a low to moderate spread of antifolate resistance. The K13 phylogeny predominantly identified wild-type haplotype (Y493Y+R539R +I543I +C580C) at an estimated distribution of high prevalence in Ghana (p=100%), Nigeria (p=96.9%), Niger (p=90%) and Angola (p=>80%). This suggested a limited spread of artemisinin resistance. Future recommendations from this study should estimate the mutational prevalence and phylogenetic distribution of antifolate and artemisinin resistance in a larger sample population.