Molecular Screening of Plasmodium Parasites in Wild Macaques

Ninety-three blood samples were collected from wild macaques by Dr. Wirasak Fungfuang in three areas of Thailand from July 2017–July 2019, including samples from wild M. arctoides at the Pa La U waterfall, Huahin district, Prachuap Kiri Khan Province, wild M. leonina at the Khao Yai National Park, Nakornratchasima Province, and wild M. fascicularis at Chang Island, Mu Ko Ranong National Park, Ranong Province. The gDNA was extracted directly from samples using GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, USA) and stored at −20°C until use. In our previous study, all gDNA samples showed negative results for dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), Leptospira spp., and Burkholderia pseudomallei using molecular detection [17 (link),18 (link)]. To analyze Plasmodium species, a nested PCR approach was employed, following the previously described method [19 (link)]. Plasmodium-positive samples underwent further examination using species-specific primers to identify P. knowlesi, P. coatneyi, P. cynomolgi, P. inui, and P. fieldi, as outlined in a previous study [20 (link)] (Table 2) and P. cynomolgi RPA-LFD assay.