Malaria remains one of the most important parasitic diseases worldwide, with more than 240 million cases and over 600,000 deaths reported annually, the vast majority occurring in sub-Saharan Africa [1]. Despite substantial progress over the past two decades, malaria transmission persists in many endemic regions, underscoring the need for accurate, sensitive, and context-appropriate diagnostic tools. Reliable diagnosis is essential not only for individual case management but also for surveillance, stratification of transmission intensity, and evaluation of control and elimination strategies.

Conventional diagnostic methods—light microscopy and rapid diagnostic tests (RDTs)—have played a central role in malaria control. However, both approaches suffer from well-recognised limitations. Microscopy requires skilled personnel, is time-consuming, and has limited sensitivity for low-density infections and mixed-species cases [2]. RDTs, while operationally simple and widely deployed, show variable sensitivity across settings, perform poorly for non-Plasmodiumfalciparum species, and may yield false-positive results due to persistent antigens following parasite clearance [3]. These shortcomings are particularly problematic in low-transmission or pre-elimination settings, where asymptomatic and low-parasitaemia infections contribute disproportionately to residual transmission.

To overcome these constraints, nucleic acid-based diagnostic approaches have gained increasing attention. Techniques such as nested PCR, quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and next-generation sequencing provide substantially higher sensitivity and specificity than conventional methods [4]-[6]. Among these, multiplex PCR—allowing the simultaneous detection of multiple Plasmodium species in a single reaction—offers a practical compromise between analytical performance, cost, and laboratory feasibility. This approach is especially relevant in regions where P.falciparum, P.ovale, and P.malariae co-circulate, and where accurate species identification is critical for appropriate treatment and surveillance.

Beyond clinical diagnosis, multiplex PCR has become increasingly valuable for entomological surveillance. Molecular detection of Plasmodium infections in mosquito vectors enables identification of low-intensity infections that are frequently missed by microscopy-based methods and provides insights into transmission dynamics, vector competence, and intervention impact [7][8]. Such applications are particularly relevant for countries transitioning from control to elimination, where detecting residual transmission foci is a priority.

Despite its promise, the validation and standardisation of multiplex PCR assays remain inconsistent across published studies. Key challenges include the determination of limits of detection, assay reproducibility, and performance across diverse epidemiological settings. In addition, vector sampling strategies, mosquito infection rates, and laboratory capacity vary widely between malaria-endemic countries, influencing the operational utility of molecular tools. Consequently, validation frameworks developed in one context may not be directly transferable to another.

In this review, we synthesise current advances in multiplex PCR assays targeting Plasmodiumfalciparum, P.ovale, and P.malariae in both human and mosquito samples. We highlight progress in species-specific primer design and applications to entomological surveillance, while critically examining remaining technical and operational gaps. Importantly, we contextualise multiplex PCR deployment within heterogeneous malaria control programmes, emphasising the need for locally adapted validation strategies before integration into national malaria control and elimination efforts.

The success of multiplex PCR critically depends on careful primer design to ensure high specificity and avoid cross-reactivity. Padley etal. pioneered species-specific primer sets targeting the 18S rRNA gene, generating amplicons of distinct sizes for P.falciparum, P.vivax, P.malariae, and P.ovale [9]. Key considerations include targeting conserved but species-discriminatory regions, designing primers with compatible melting temperatures, minimizing primer-primer interactions, and ensuring adequate separation of amplicon sizes for gel-based detection. Failure to meet these criteria often results in preferential amplification of dominant templates or loss of sensitivity for minority species. Given the heterogeneity in validation practices across multiplex PCR studies, we propose a standardized minimum reporting and validation checklist (Table 1).

Table 1. Validation checklist for multiplex PCR assays for Plasmodium detection in human and mosquito samples.

Multiplex PCR reduces reagent use and processing time, improves throughput, and enhances detection of mixed infections that are frequently underestimated by microscopy [10]. These features make it attractive for large-scale surveillance in resource-limited settings. However, multiplexing introduces technical challenges, including primer competition, reduced analytical sensitivity compared with nested PCR or qPCR, and difficulties in balancing amplification efficiency across targets [11]. Importantly, many studies fail to report critical analytical metrics such as limits of detection (LOD), DNA input concentrations, or assay reproducibility, limiting cross-study comparison and standardization.

A recurring limitation across the literature is the use of small sample sizes, often involving fewer than 20 clinical samples or a handful of mosquito specimens, which undermines statistical robustness [12]. Parasite densities are rarely quantified prior to molecular testing, despite their strong influence on detection probability. In entomological studies, mosquito species identification, collection methods, tissue specificity (midgut versus salivary glands), and parasite stage are frequently omitted, constraining interpretation of transmission relevance [13]. Additionally, the exclusion of P.vivax from many multiplex panels substantially reduces global applicability, given its major contribution to malaria burden outside Africa [14].

Multiplex PCR represents a powerful diagnostic and surveillance tool for malaria, with clear advantages in sensitivity, species discrimination, and detection of mixed infections. However, most published assays—including recent triplex PCR systems—lack rigorous analytical and epidemiological validation. Establishing standardized guidelines for sample size, parasite quantification, LOD determination, and vector sampling is essential before multiplex PCR can be reliably integrated into national malaria surveillance and elimination programs.

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Available upon reasonable request.

This work was supported by the African Research Initiative for Scientific Excellence (ARISE) grant (ref: ARISE-PP-143) awarded to Dr. Etienne Bilgo. The ARISE project is funded by the European Union and implemented by the African Academy of Sciences, in partnership with the African Union Commission and the European Commission.

DFDSH and EB wrote the first draft of the manuscript. All authors read and approved the final manuscript.