Mixed malaria infections: A critical review and Detect-Characterise-Act surveillance framework

Mixed malaria infections, involving concurrent Plasmodium species and/or multiple genetically distinct parasite clones, remain under-measured in routine malaria programmes despite implications for diagnosis, treatment, drug-resistance surveillance, and elimination planning. This critical narrative review synthesises recent evidence, prioritising 2020-2026 literature, and proposes a Detect-Characterise-Act framework linking point-of-care detection, molecular or genomic confirmation, and clinical or surveillance responses. The review argues that mixed infections should be treated as operational signals of diagnostic sensitivity, species-specific treatment adequacy, parasite population connectivity, diagnostic escape, and antimalarial resistance. Microscopy and many rapid diagnostic tests often underestimate mixed infections, particularly when secondary species are low-density, HRP2-based tests are affected by pfhrp2/pfhrp3 deletions, or non-P. falciparum species are not assayed. Genomic approaches, including targeted amplicon sequencing, nanopore sequencing, and whole-genome sequencing, can resolve species identity, multiplicity of infection, minority resistance variants, and diagnostic-target integrity, but require rigorous thresholds for read depth, allele frequency, contamination control, and sample representativeness. The review contributes four clarifications: distinguishing mixed-species infection, within-species multiplicity, and within-host resistance-associated variation; integrating pfhrp2/pfhrp3-mediated diagnostic escape; clarifying the staged interpretation of minority resistance-associated variants in Africa; and reframing mixed infection as a marker of programme maturity. Countries able to detect, genotype, report, and respond to mixed infections are better positioned to protect case management, contain resistance, and sustain malaria-elimination gains.