METHODS: Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in long-tailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations.

RESULTS: Out of 276 samples tested, 177 were Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites (P. knowlesi, P. coatneyi, P. cynomolgi) with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively.

RESULTS: Apart from several named species of malaria parasites, long-tailed macaques were found to be potentially infected with novel species of Plasmodium, namely one we refer to as "P. inui-like." This group of parasites bifurcated into two monophyletic clades indicating the presence of two distinct sub-populations. Further analyses, which relied on the assumption of strict co-phylogeny between hosts and parasites, estimated a population expansion event of between 150,000 to 250,000 years before present of one of these sub-populations that preceded that of the expansion of P. knowlesi. Furthermore, both sub-populations were found to have diverged from a common ancestor of P. inui approximately 1.5 million years ago. In addition, the phylogenetic analyses also demonstrated that long-tailed macaques are new hosts for P. simiovale.

METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.

RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).

METHODS: A total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.

RESULTS: Thirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P. fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.