A Non-Synonymous Mutation at DHFR Codon 50 is Preventing the Return of Sulfadoxine-Pyrimethamine Sensitivity in Plasmodium Falciparum after the Drug's Discontinuation

Abstract

Background: Resistance to antimalarials in Plasmodium falciparum spreads if mutations increase parasite survival when encountering the drug. Because such mutations typically reduce parasite growth (metabolic costs), they are believed to spread only if drug pressure is sufficiently high, and drug sensitivity returns after drug use is discontinued, as observed with chloroquine resistance. However, this was not observed with sulfadoxine-pyrimethamine (SP) resistance. For instance, when SP was discontinued in Peru the resistant 50R/51I/108N replaced the highly resistant 51I/108N/164L Pfdhfr variant, after the slightly resistant 108N mutant became temporarily predominant.

Methods: Prevalence data of SP resistant Pfdhfr mutations from Peru and results from published yeast expression systems for antifolate sensitivity are combined with an evolutionary-genetic model tailored towards malaria to explain the spread of the 51I/108N/164L and 50R/51II108N mutants.

Findings: The increased parasite growth rate (metabolic advantage) of the C50R mutation, seen in yeast expression systems, explains empirical observations in Peru, and is consistent with findings in other countries. Drug pressure is necessary for the 50R/51I/108N mutant to emerge. However, it cannot become predominant in the presence of more resistant variants until drug pressure is lifted. Afterwards, the 50R/51I/108N mutant spreads and prevents the re-emergence of sensitive types.

Interpretation: If the C50R mutation spreads after SP is discontinued, its associated increased growth rate can negatively affect the clinical pathogenesis of malaria infection or even lead to an increased likelihood of SP-unrelated treatment failures. Particularly, the mutation potentially excels the spread of artemisinin resistance. Also, novel mutations might be falsely associated with drug resistance if the spread of the advantageous C50R mutation is not accounted for. Therefore, surveillance of the often-disregarded C50R mutation must be reconsidered on a global scale in the context of antimalarial drug resistance.

Funding: This study was funded by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch, ProMIS ID P0134 23 N6. This work was also supported by grants of the German Academic Exchange (DAAD; https://www. daad.de/de/; Project-ID 57417782, Project-ID: 57599539), the S ̈achsisches Staatsministerium f ̈ur Wis- senschaft, Kultur und Tourismus and S ̈achsische Aufbaubank – F ̈orderbank (SMWK-SAB; https://www. smwk.sachsen.de/; https://www.sab.sachsen.de/; project “Innovationsvorhaben zur Profilsch ̈arfung an Hochschulen f ̈ur angewandte Wissenschaften”, Project-ID 100257255; project “Innovationsvorhaben zur Profilsch ̈arfung 2022”, Project-ID: 100613388), the Federal Ministry of Education and Research (BMBF) and the DLR (Project-ID 01DQ20002; https://www.bmbf.de/; https://www.dlr.de/).

Declaration of Interest: The authors have declared that no competing interests exist.

Ethical Approval: The protocol for this study (NAMRU6.2019.0009) was reviewed and approved by the head of the Research Administration Program of the Naval Medical Research Unit-6 (NAMRU-6) and determined it to be a non-human subject research. This study was a secondary data analysis and informed consent was not required for this since it does not meet the definition of research involving human subjects per US Code of Federal Regulations, 32 CFR Part 219 - PROTECTION OF HUMAN SUBJECTS, section 219.104 Exempt research. For samples collected as part of previous NAMRU-6 protocols, we used sample codes from volunteers who provided consent for future use.

Keywords: drug resistance, metabolic cost, population genetic, malaria control, survival advantage, selective pressure, fitness parameters, resistant haplotypes