By 2024, Malaysia achieved a significant public health milestone by marking its seventh consecutive year without a single local transmission of indigenous human Plasmodium species. The country has sustained elimination of non-zoonotic malaria, primarily caused by Plasmodium (P.) falciparum and P. vivax, while remaining vigilant against imported and introduced cases driven by crossborder movement and international travel. However, despite this achievement, Malaysia has emerged as the global epicentre of zoonotic for P. knowlesi. In 2021 alone, 3 575 cases of P. knowlesi and 13 deaths were reported1, underscoring the shifting epidemiology of malaria in the country. The growing convergence of habitats shared by humans, non-human primates and mosquito vectors, together with ongoing land-use changes, has contributed to the rising incidence of zoonotic malaria1. The World Health Organization (WHO) further indicates that countries such as Malaysia, are unlikely to achieve malaria elimination if the burden of P. knowlesi cases remains persistently high2. The rise of P. knowlesi has necessitated a strategic transition in national malaria control efforts, with greater emphasis on surveillance and diagnostic precision. A key challenge lies in the continued reliance on microscopy as the gold standard for rapid clinical diagnosis. Although microscopy is widely accessible and cost-effective, distinguishing P. knowlesi morphologically remains difficult, as it closely mimics the early stages of P. falciparum and the later stages of P. malariae3. Consequently, definitive species identification increasingly depends on molecular methods. In response, the Ministry of Health Malaysia established the National Malaria Molecular Surveillance Programme (NMMSP), which integrated polymerase chain reaction (PCR), genotyping and gene sequencing to improve species confirmation, classifying cases by origin, monitoring drug resistance and supporting outbreak investigations4. This diagnostic transition is reflected in the evolving landscape of malaria epidemiological research in Malaysia. There were 31 published malaria epidemiological studies in Malaysia indexed in PubMed and Scopus from 2011 to 2025 (Figure 1). Most studies adopted combined diagnostic approaches, particularly microscopy with PCR (n=16), followed by microscopy with PCR and serology (n=5), and microscopy with PCR and rapid diagnostic tests (RDTs) (n=4). In contrast, reliance on single diagnostic methods was limited (Figure 1). This trend highlights the complementary strengths of microscopy and molecular tools, particularly in the context of malaria elimination, where parasite densities are often low and submicroscopic infections are common5,6.Figure 1: Number of published malaria epidemiological studies in Malaysia indexed in PubMed and Scopus from 2011 to 2025. A systematic search of both database using relevant keywords (“malaria” OR “malari*” OR “plasmodium” OR “malaria transmission” OR “malaria exposure”) AND (“malaysia” OR “sabah” OR “sarawak”) AND (“epidemiolog*” OR “prevalence” OR “incidence” OR “cross-section*” OR “cohort” OR “surveillance” OR “distribution” OR “transmission” OR “exposure”) identified a total of 31 studies conducted in malaria-affected communities, employing various malaria diagnostic approaches, including single methods and combinations of methods. PCR: polymerase chain reaction; RDT: rapid diagnostic test.Microscopy remains indispensable for routine surveillance, especially in rural and resource-limited settings. It enables visualisation of parasite morphology and quantification of parasitaemia, making it practical, portable and cost-effective for field use. However, its diagnostic accuracy depends heavily on operator expertise and is constrained by reduced sensitivity at low parasite densities7. Moreover, morphological similarities among Plasmodium species, particularly between P. knowlesi and P. malariae, pose significant challenges for accurate identification2. This limitation is further illustrated by the first reported human infection of P. cynomolgi in Malaysia, which was initially misidentified as P. vivax by microscopy but later confirmed by PCR8. In contrast, molecular methods such as PCR offer superior sensitivity and specificity. They can detect low-level and submicroscopic infections, identify mixed-species infections and accurately differentiate morphologically similar parasites. These capabilities are critical for surveillance in low-transmission settings and for understanding the true burden of zoonotic malaria. However, PCR-based methods are resource-intensive, require specialized laboratory infrastructure, and do not provide direct information on parasite density or morphology9. As such, they are less suited for immediate clinical decision-making in field settings. The increasing preference for integrated diagnostic approaches in Malaysia reflects the need to balance these strengths and limitations. Combining microscopy and PCR enables both quantitative and qualitative assessment of malaria infections, thereby improving diagnostic accuracy and surveillance sensitivity7. The addition of serological tools, for example, can further enhance epidemiological insights by capturing historical and recent exposure, which is particularly valuable in near-elimination settings where transmission is heterogeneous and often cryptic10. Furthermore, maintaining Malaysia’s malaria-free status for human-only species while addressing the growing threat of zoonotic malaria requires a comprehensive and adaptive strategy. Sustained investment in microscopy training and competency assessment is essential, particularly in low-transmission settings where diagnostic skills may decline due to reduced case exposure. At the same time, molecular diagnostics should be institutionalized as a reference standard in regions with emerging zoonotic transmission to ensure accurate species identification and detection of mixed infections. In conclusion, Malaysia’s experience underscores the importance of integrating conventional and advanced diagnostic tools in malaria surveillance. The synergy between microscopy, molecular methods, and potential use of serology not only strengthens diagnostic accuracy but also provides a robust, data-driven framework for malaria control in the context of zoonotic transmission. As the country moves beyond elimination towards sustained prevention of reintroduction, such integrated approaches will be critical in addressing the evolving challenges of malaria epidemiology. Conflict of interest statement We declare that there is no conflict of interest. Funding It was supported by the Geran Fundamental Fakulti Perubatan (FF-2025-202), Universiti Kebangsaan Malaysia. Authors’ contributions The conceptualization was done by KS and ZMI. The literature and drafting of the manuscript were conducted by KS, SJG, AAMFI and GMW. The editing and supervision were performed by ZMI. All authors have read and agreed to the final version of the manuscript. Publisher’s note The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Edited by Zhang Q, Lei Y, Pan Y
Sangaran et al. (Fri,) studied this question.