Molecular Innovations in Malaria Diagnostics: A Critical Review of Multiplex PCR Approaches for Human and Vector Surveillance

Molecular methods are transforming malaria diagnosis and surveillance by enabling highly sensitive and specific identification of Plasmodium species, including mixed infections that microscopy often misses. Multiplex PCR assays offer additional advantages by detecting several species simultaneously in a single reaction, reducing reagent use and processing time. This review synthesizes current advances in multiplex PCR detection of Plasmodium falciparum, P. ovale, and P. malariae in human and mosquito samples, drawing on published literature and recent assay development efforts. We highlight methodological strengths, including species-specific primer design, improved detection of mixed infections, and applicability to entomological surveillance. We also critically examine persistent challenges small validation sample sizes, lack of analytical sensitivity data, absence of quantified parasite loads, and limitations in mosquito specimen characterization. Finally, we propose a framework for future assay validation that includes parasite quantification, rigorous analytical performance metrics, tissue-specific mosquito sampling, and expanded epidemiological studies. This synthesis demonstrates that multiplex PCR holds strong promise for integrated malaria surveillance but requires standardized validation pipelines before widespread adoption.