BackgroundPlasmodium knowlesi, a malaria parasite of Southeast Asian macaques, infects humans and can cause fatal malaria. It is difficult to diagnose by microscopy because of morphological similarity to Plasmodium malariae. Nested PCR assay is the most accurate method to distinguish P. knowlesi from other Plasmodium species but is not cost effective in resource-poor settings. Rapid diagnostic tests (RDTs) are recommended for settings where malaria is prevalent. In this study, the effectiveness of three RDTs in detecting P. knowlesi from fresh and frozen patient blood samples was evaluated.MethodsForty malaria patients (28 P. knowlesi, ten P. vivax and two P. falciparum) diagnosed by microscopy were recruited in Sarawak, Malaysian Borneo during a 16-month period. Patient blood samples were used to determine parasitaemia by microscopy, confirm the Plasmodium species present by PCR and evaluate three RDTs: OptiMAL-IT, BinaxNOW® Malaria and Paramax-3. The RDTs were also evaluated using frozen blood samples from 41 knowlesi malaria patients.ResultsOptiMAL-IT was the most sensitive RDT, with a sensitivity of 71% (20/28; 95% CI = 54-88%) for fresh and 73% (30/41; 95% CI = 59-87%) for frozen knowlesi samples. However, it yielded predominantly falciparum-positive results due to cross-reactivity of the P. falciparum test reagent with P. knowlesi. BinaxNOW® Malaria correctly detected non-P. falciparum malaria in P. knowlesi samples but was the least sensitive, detecting only 29% (8/28; 95% CI = 12-46%) of fresh and 24% (10/41; 95% CI = 11-37%) of frozen samples. The Paramax-3 RDT tested positive for P. vivax with PCR-confirmed P. knowlesi samples with sensitivities of 40% (10/25; 95% CI = 21-59%) with fresh and 32% (13/41; 95% CI = 17-46%) with frozen samples. All RDTs correctly identified P. falciparum- and P. vivax-positive controls with parasitaemias above 2,000 parasites/μl blood.ConclusionsThe RDTs detected Plasmodium in P. knowlesi-infected blood samples with poor sensitivity and specificity. Patients with P. knowlesi could be misdiagnosed as P. falciparum with OptiMAL-IT, P. vivax with Paramax-3 and more correctly as non-P. vivax/non-P. falciparum with BinaxNOW® Malaria. There is a need for a sensitive and specific RDT for malaria diagnosis in settings where P. knowlesi infections predominate.

P lasmodium spp. were identified in the late 1800s, and >30 species have been described in primates, including humans, apes, and monkeys (1,2). Of these, humans are natural hosts to 4 species: P. falciparum, P. malariae, P. vivax, and P. ovale. Human infections with simian malaria parasites were thought to be extremely rare until P. knowlesi was identified as a major cause of malaria in humans in Kapit, Malaysian Borneo (3). Subsequent human cases of infection with P. knowlesi have been reported across Southeast Asia (4-9). Most cases are reported in the Malaysian Borneo states of Sarawak and Sabah (4,10,11). The zoonotic capability of this parasite was confirmed with the aid of molecular techniques because P. knowlesi is morphologically

Background Plasmodium knowlesi, a simian malaria parasite, has become the main cause of malaria in Sarawak, Malaysian Borneo. Epidemiological data on malaria for Sarawak has been derived solely from hospitalized patients, and more accurate epidemiological data on malaria is necessary. Therefore, a longitudinal study of communities affected by knowlesi malaria was undertaken.MethodsA total of 3002 blood samples on filter paper were collected from 555 inhabitants of 8 longhouses with recently reported knowlesi malaria cases in the Betong Division of Sarawak, Malaysian Borneo. Each longhouse was visited bimonthly for a total of 10 times during a 21-month study period (Jan 2014–Oct 2015). DNA extracted from blood spots were examined by a nested PCR assay for Plasmodium and positive samples were then examined by nested PCR assays for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium cynomolgi and Plasmodium inui. Blood films of samples positive by PCR were also examined by microscopy.ResultsGenus-specific PCR assay detected Plasmodium DNA in 9 out of 3002 samples. Species-specific PCR identified 7 P. knowlesi and one P. vivax. Malaria parasites were observed in 5 thick blood films of the PCR positive samples. No parasites were observed in blood films from one knowlesi-, one vivax- and the genus-positive samples. Only one of 7 P. knowlesi-infected individual was febrile and had sought medical treatment at Betong Hospital the day after sampling. The 6 knowlesi-, one vivax- and one Plasmodium-infected individuals were afebrile and did not seek any medical treatment.ConclusionsAsymptomatic human P. knowlesi and P. vivax malaria infections, but not P. cynomolgi and P. inui infections, are occurring within communities affected with malaria.

Background Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak. Methods Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis. Results Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo. Conclusions Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

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