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Background: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised., Methods: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility., Findings: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts., Interpretation: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp., Funding: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Adequate clinical and parasitological response (ACPR) after malaria treatment remains challenging to assess in settings of malaria nonendemicity. Biological evaluation of parasitological clearance relies on microscopic investigation of thick blood smears, which is a specific technique that not all diagnosis laboratories are able to perform. Rapid diagnosis tests (RDTs) and molecular biology techniques are proposed as alternatives to microscope conventional techniques; however, their performance for treatment efficacy evaluation is controversial. We present here a retrospective comparative study for RDT and PCR (nested and high-resolution-melting quantitative PCR [HRM-qPCR]) evaluation of ACPR in a nonendemicity context. Blood samples from 133 patients presenting a Plasmodium falciparum monoinfection were included. Samples obtained at the time of diagnosis and at 3, 7, and 28 days after diagnosis were investigated. Histidine-rich protein 2 (HRP-2)-based RDT results remained positive in 51% of cases 28 days after diagnosis and appropriate therapeutic management. Parasite DNA was detected by the two PCR techniques (nested PCR and HRM-qPCR) in 12% and 10% of samples 28 days after treatment initiation, respectively. No therapeutic failure was recorded in the studied patients. Persistence of positive signal might reflect the presence of circulating asexual parasites or persistence of HRP-2 and parasitic DNA in patient's peripheral blood after parasitic clearance., (Copyright © 2019 American Society for Microbiology.)