Country wide surveillance reveals prevalent artemisinin partial resistance mutations with evidence for multiple origins and expansion of sulphadoxine-pyrimethamine resistance mutations in northwest Tanzania

Background: Emergence of artemisinin partial resistance (ArtR) in Plasmodium falciparum in East Africa is a growing threat to the efficacy of artemisinin combination therapies (ACT) and the global efforts for malaria elimination. The emergence of Pfkelch13 R561H in Rwanda, raised concern about the impact in neighboring Tanzania, despite contemporary surveys suggesting limited 561H in the country. In addition, regional concern over resistance affecting sulfadoxine-pyrimethamine (SP) which is used for chemoprevention strategies is high. Methods: To enhance longitudinal monitoring, the Molecular Surveillance of Malaria in Tanzania (MSMT) project was launched in 2020 with the goal of assessing and mapping the profile of antimalarial resistance across transmission zones. Community and clinic samples were assessed for resistance polymorphisms using a high throughput molecular inversion probe platform. Findings: Genotyping of 6,278 samples collected in 2021 revealed a focus of Pfkelch13 561H mutants in North-western Tanzania with prevalence of 7.7% (50/649) in Kagera. A small number of 561H mutants (about 1%) were found as far as 800 km away in Tabora, Manyara, and Njombe. Genomic analysis suggests some of these parasites are highly related to previous isolates collected in Rwanda in 2015, supporting regional spread of 561H. However, a novel haplotype was also observed, likely indicating a second origin in the region. Other validated resistance polymorphisms (622I and 675V) were also identified. A focus of high sulfadoxine-pyrimethamine drug resistance was also identified in Kagera with a prevalence of dhfr164L of 15% (80/526). Interpretation: These findings demonstrate Pfkelch 561H resistant mutation is entrenched in the region and that multiple origins of ArtR, similar as to what was seen in Southeast Asia, are likely to occur. Mutations associated with high levels of SP resistance are increasing. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported, in whole, by the Bill & Melinda Gates Foundation [grant number 002202]. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. This study was also funded by the National Institute for Allergy and Infectious Diseases (R01AI156267 to JAB, DSI and JJJ; K24AI134990 to JJJ). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Informed consent was obtained for each patient and de-identified DBS samples were processed at NIMR in Tanzania and Brown University and University of North Carolina (USA) according to IRB requirements of the Tanzanian Medical Research Coordinating Committee (MRCC) of NIMR. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All sequencing data has been submitted to SRA (# pending). Other metadata is available from the corresponding author upon reasonable request. Code for analysis is available at: https://github.com/bailey-lab/MSMT\_2021\_DR_analyses.