Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine-pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey

Abstract

Background: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs.

Methods: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda.

Findings: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide.

Interpretation: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa.

Funding: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.

Figure 1

Tanzania malaria prevalence and study design (A) Map of Tanzania showing malaria prevalence in children aged 6–59 months in 2017 (National Malaria Control Program data). Health facilities where sampling occurred for this study are shown as circles and cross-sectional community survey collection sites are shown as squares. (B) Study profile showing the number of samples collected at health facilities and cross-sectional community surveys and the success of genotyping.

Figure 2

Antimalarial resistance polymorphisms in Tanzania (A) k13 gene Arg561His mutations in Tanzania by region. White areas represent no sampling, grey areas represent sampled areas where no Arg561His was found, and coloured areas are shaded by the frequency of samples with the Arg561His mutation. (B) k13 gene Arg561His mutations by district in Kagera. The districts with detected mutation all border Rwanda, with no mutations detected in eastern Kagera. The prevalence of dhfr Ile164Leu (C) and dhps Ala581Gly (D) in sampled regions is shown in colour gradients. Regions with no sampling are shown in white and regions where a polymorphism was not detected are shown in grey.

Figure 3

Extended flanking haplotype plot around k13 Arg561His mutations Every SNP, insertion or deletion passing filters between 1 707 537 bp and 1 752 231 on chromosome 13 is presented as a coloured line, the SNP coding for pure mutant Arg561His is shown in red. All samples were jointly called for variation. Grey regions represent loci where a call could not be made due to low coverage. The top panel represents wild-type and mixed genotypes at position 561. The middle panel shows a common haplotype between Rwandan and Tanzanian isolates. The bottom panel shows a novel haplotype flanking Arg561His detected in Kagera. SNP=single-nucleotide polymorphism.

Figure 4

Extended-haplotype homozygosity analysis of Arg561His haplotypes (A) Extended-haplotype homozygosity for the Rwanda and Tanzania haplotype 1 Arg561His (n=12) compared to wild-type parasites. (B) Extended-haplotype homozygosity of the Tanzania haplotype 2 Arg561His (n=4) compared with wild-type parasites. The vertical dotted line represents the position of the single-nucleotide polymorphism for Arg561His. The x axes show the upstream and downstream genomic coordinates from the locus of interest and the homozygosity scale is shown on the y axes, ranging from 0 to 1 (0 representing no homozygosity and 1 representing complete homozygosity).