Background About a fifth of malaria cases in 1999 for the Kapit division of Malaysian Borneo had routinely been identified by microscopy as Plasmodium malariae, although these infections appeared atypical and a nested PCR assay failed to identify P malariae DNA. We aimed to investigate whether such infections could be attributable to a variant form of P malariae or a newly emergent Plasmodium species.

Human infection with P. knowlesi, commonly misidentified as the more benign P. malariae, are widely distributed across Malaysian Borneo and extend to Peninsular Malaysia. Because P. knowlesi replicates every 24 h, rapid diagnosis and prompt effective treatment are essential. In the absence of a specific routine diagnostic test for P. knowlesi malaria, we recommend that patients who reside in or have traveled to Southeast Asia and who have received a "P. malariae" hyperparasitemia diagnosis by microscopy receive intensive management as appropriate for severe falciparum malaria.

Abstract. A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus-or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/l of blood using DNA prepared from 25-l blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.Microscopy is the method of choice for the diagnosis of malaria in endemic areas because it is an inexpensive and rapid method of detection. Correct identification of the four species of Plasmodium causing human malaria and the level of detection by microscopy depends on a number of factors, including the experience of the microscopist, proper staining of the slides, appropriate maintenance of microscopes, and the time spent examining each slide. At best, the sensitivity of detection by microscopy is approximately 10-30 parasites/l of blood. 1 However, this level of detection is normally not attained in malaria-endemic areas and particularly during epidemiologic studies when many samples need to be screened in a relatively short time. Thus, incorrect speciation is common and mixed infections and low levels of parasitemia may be missed.To overcome some of the limitations of microscopy for detection of malaria, polymerase chain reaction (PCR)-based assays have been developed for the detection and identification of malaria parasites. These methods have proved to be more specific and sensitive than conventional microscopy and some are reported to be able to detect as few as one parasite/l of blood.2-6 However, to attain such a high sensitivity, blood samples collected from individuals have to be processed immediately or stored at low temperatures and the steps involved in DNA template preparation were multistep, often requiring biohazardous chemicals. Since malaria remains a problem of underdeveloped and often remote areas of the world, it is important to couple PCRbased assays with simple sampling and DNA extraction methods to maximize the value of PCR assays. In a previous study, we coupled the nested PCR assay of Snounou and others 3 to blood collection on filter papers and a simple DNA e...

Knowlesi malaria causes a wide spectrum of disease. Most cases are uncomplicated and respond promptly to treatment, but approximately 1 in 10 patients develop potentially fatal complications.

Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000–40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.