High-sensitivity detection of asymptomatic malaria in high-risk Thai populations using droplet digital PCR

BACKGROUND: Asymptomatic infections present a major obstacle to malaria elimination efforts, particularly within high-risk populations where low-density parasitemia often escapes detection by conventional diagnostic methods. In such cases, onward transmission remains possible, sustaining the parasite reservoir. Therefore,highly sensitive screening techniques are essential for effective malaria prevention and control. Molecular methods provide greater sensitivity and specificity in detecting low-level infections compared to traditional approaches. In this study, we evaluated droplet digital PCR (ddPCR) for its ability to detect asymptomatic Plasmodium infections, with the aim of supporting malaria transmission reduction and elimination strategies. METHODS: Specimens were collected from individuals residing in malaria-endemic regions of Thailand (n = 403) and from Thai workers returning from Sudan (n = 270). All samples were evaluated for malaria using ddPCR and microscopic examination. The ddPCR assay targeted multicopy genomic regions, including the 18S rRNA gene for genus-level screening, as well as and species-specific markers for the five human Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi). RESULTS: The ddPCR assays identified malaria infections in 0.25 % of samples from Tak, Thailand, and 7.41 % of samples from Sudan. Both dried blood spots (DBS) and venous blood specimens were analyzed using the ddPCR assay. Compared with microscopy, ddPCR demonstrated superior sensitivity by detecting a greater proportion of low-parasitemia infections. Only 2.59 % of samples collected from workers returning from Sudan were positive for Plasmodium falciparum by microscopic examination. The ddPCR method quantified 18S rRNA copy numbers at the genus level, which showed a positive correlation with species-specific quantification. The median concentration of positive samples measured by the genus-specific ddPCR assay was 0.95 copies/μL (IQR: 0.37-29.48), with concentrations ranging from 0.16 to 7033.43 copies/μL. CONCLUSIONS: This study applied the ddPCR assay totwo specimen types for surveillance of asymptomatic malaria, demonstrating its capability to detect hidden parasite reservoirs that are critical targets for malaria control and elimination strategies.