Abstract
In Africa, the first Plasmodium falciparum Kelch13 (K13) artemisinin partial resistance mutation 561H was first detected and validated in Rwanda. Surveillance to better define the extent of the emergence in Rwanda and neighboring countries as other mutations arise in East Africa is critical. We employ a novel scheme of liquid blood drop preservation combined with pooled sequencing to provide a cost-effective rapid assessment of resistance mutation frequencies at multiple collection sites across Rwanda and neighboring countries. Malaria-positive samples (n=5,465) were collected from 39 health facilities in Rwanda, Uganda, Tanzania, and the Democratic Republic of the Congo (DRC) between May 2022 and March 2023 and sequenced in 199 pools. In Rwanda, K13 561H and 675V were detected in 90% and 65% of sites with an average frequency of 19.0% (0-54.5%) and 5.0% (0-35.5%), respectively. In Tanzania, 561H had high frequency in multiple sites while it was absent from the DRC although 675V was seen at low frequency. Conceringly candidate mutations were observed: 441L, 449A, and 469F co-occurred with validated mutations suggesting they are arising under the same pressures. Other resistance markers associated with artemether-lumefantrine are common: P. falciparum multidrug resistance protein 1 N86 at 98.0% and 184F at 47.0% (0-94.3%) and P. falciparum chloroquine resistance transporter 76T at 14.7% (0-58.6%). Additionally, sulfadoxine-pyrimethamine-associated mutations show high frequencies.
Overall, K13 mutations are rapidly expanding in the region further endangering control efforts with the potential of engendering partner drug resistance.
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
This project was funded by the National Institute of Allergy and Infectious Diseases (R01AI156267 to JAB, JJJ, DSI, SH, and JBM; 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:
Ethical approval for this study was obtained from the Rwanda National Ethics Committee (reference 12/RNEC/2022), the Medical Coordinating Committee of the National Institute for Medical Research Tanzania, the MUST Research Ethics Committee, Ethics Committee of Ecole de Santé Publique de Kinshasa and the Institutional Review Board at Brown University (RI, USA). University of North Carolina approved this work under a reliance agreement with Brown University. Written informed consent was obtained from participants or parents/guardians of eligible children in the local language which is Kinyarwanda in Rwanda, Kiswahili in Tanzania, French in DRC, and English in Uganda.
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).
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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.
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Data Availability
Illumina data is available from the SRA (Project # pending). All sequenced MIP counts are included within the supplementary materials. MIPWrangler and MIPTools software is available on GitHub https://github.com/bailey-lab. R scripts used to generate analysis and figures are available at https://github.com/nnwyoung/ArtR_Pooled_MIPs.
List of Abbreviations
- (Pf)
- Plasmodium falciparum
- (ACTs)
- artemisinin combination therapies
- (ART)
- artemisinin
- (ART-R)
- ART partial resistance
- (K13)
- Plasmodium falciparum Kelch13
- (AL)
- Artemether-lumefantrine
- (ASAQ)
- artesunate-amodiaquine
- (MDR1)
- P. falciparum multidrug resistance protein 1
- (CRT)
- P. falciparum chloroquine resistance transporter
- (DRC)
- Democratic Republic of the Congo
- (LBD)
- liquid blood drop
- (MIPs)
- molecular inversion probes
- (mRDT)
- malaria rapid diagnostic test
- (CGB)
- Center for Genomic Biology
- (SPRI)
- solid phase reversible immobilization
- (UMI)
- Unique Molecular Identifiers
- (SNPs)
- single nucleotide polymorphisms
- (SP)
- sulfadoxine-pyrimethamine
- (DHFR)
- dihydrofolate reductase
- (DHPS)
- dihydropteroate synthase