Summary/Abstract
Malaria control and elimination face significant challenges due to drug resistance, particularly to
the current front-line artemisinin-based combination therapy in the Greater Mekong Region, to
artemisinin (ART) derivatives in Myanmar and now disturbingly in eastern Africa where most
serious falciparum malaria cases are concentrated. To ensure the continued effectiveness of ART
and its partner drugs, early detection and characterization of resistance, robust surveillance and
subsequent isolation are required. World-wide clinical and molecular surveillance of artemisinin
resistance (ART-R) relies on southeast Asian characteristics of longer parasite clearance times
from patients and genotyping for mutations in the most common ART resistant gene, k13 (a gene
on chromosome 13 coding for a kelch domain protein, K13). The Chittagong Hill Tracts (CHTs) in
Bangladesh borders Myanmar, and is a forested, hilly and remote region that is endemic to 90%
of the country’s malaria. Recent clinical clearance and in vitro assays not only in the CHTs but also
in Africa demonstrate that although combination treatment remains efficacious in infected
patients, a cohort of the field isolates display in vitro low-moderate ART-R with no K13 mutations.
This suggests that clones with partial ART-R exist and are going undetected by patient parasite
clearance studies and molecular surveillance. Proactive analysis of the impact of these molecular
determinants will be critical to characterize resistance before the problem is widespread in these
regions in the pre-elimination era. We hypothesize that 1) Clinical ART resistant mutations can
be predicted from in vitro generated mutants in isolates from the same spatiotemporal space. 2)
ART sensitivity is changing in the CHTs with continued use of artemisinin combination therapy
since 2004. The initial low-moderate ART-R mediated by causal K13 independent mutations will
be an optimal platform for highly resistant and highly fit K13 mutations to arise and spread. We
will test our hypotheses with two specific aims. In Aim 1, we will perform whole-genome
sequencing of in vitro evolved K13 independent ART-R in recent CHT isolates; in parallel
determine the drug sensitivities and genomic sequences of parasites recently isolated from CHT
patients and functionally validate top candidate genes by CRISPR-Cas9 editing. In Aim 2, we will
perform preemptive resistance and fitness cost analysis of major K13 mutations in the K13
independent ART resistant and ART sensitive CHT genetic backgrounds, to determine if these
“unknown factors” assist the resistance potential and sustainability of these K13 mutations.
Together, this study will provide a comprehensive view of the complex emerging ART-R with a
broader applicability to similar scenarios in Eastern Africa.
