Genotyping and Characterizing to Reveal Genetic Diversity and Multiplicity of Infection by Merozoite Surface Proteins 1 and 2 ( and ) and Glutamate-Rich Protein () Genes

Resistance to current antimalarial drugs is steadily increasing, and new drugs are required. Drug efficacy trials remain the gold standard to assess the effectiveness of a given drug. The World Health Organization (WHO)'s recommendation for the optimal duration of follow-up for assessing antimalarial efficacy is a minimum of 28 days. However, assessing antimalarial drug efficacy in highly endemic regions can be challenging due to the potential risks of acquiring a new infection in the follow-up period, and thus, it may underestimate the efficacy of the given drugs. A new treatment should be introduced if treatment failure rates exceed 10%. Overestimation occurs as a result of retaining a drug with a clinical efficacy of less than 90% with increases in morbidity and mortality, while underestimation may occur due to a misclassification of new infections as treatment failures with tremendous clinical and economic implications. Therefore, molecular genotyping is necessary to distinguish true new infections from treatment failures to ensure accuracy in determining antimalarial efficacy. There are three genetic markers that are commonly used in antimalarial efficiency trials to discriminate between treatment failures and new infections. These include merozoite surface protein 1 (), merozoite surface protein 2 (), and glutamate-rich protein (). The genotyping of by nested polymerase chain reaction (n-PCR) targeting these markers is discussed with the inherent limitations and uncertainties associated with the PCR technique and limitations enforced by the parasite's biology itself.