Evaluation of three rapid diagnostic tests for the detection of human infections with Plasmodium knowlesi.

BACKGROUND: Plasmodium 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. METHODS: Forty 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. RESULTS: OptiMAL-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. CONCLUSIONS: The 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.

Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques.

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.

Susceptibility of human Plasmodium knowlesi infections to anti-malarials.

BACKGROUND: Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed. METHODS: Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax. RESULTS: Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine. CONCLUSIONS: Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken.

Population genetic analysis of Plasmodium knowlesi reveals differential selection and exchange events between Borneo and Peninsular sub-populations.

The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax malaria has been the focus of control efforts. Understanding of the genetic diversity of P. knowlesi parasites can provide insights into its evolution, population structure, diagnostics, transmission dynamics, and the emergence of drug resistance. Previous work has revealed that P. knowlesi fall into three main sub-populations distinguished by a combination of geographical location and macaque host (Macaca fascicularis and M. nemestrina). It has been shown that Malaysian Borneo groups display profound heterogeneity with long regions of high or low divergence resulting in mosaic patterns between sub-populations, with some evidence of chromosomal-segment exchanges. However, the genetic structure of non-Borneo sub-populations is less clear. By gathering one of the largest collections of P. knowlesi whole-genome sequencing data, we studied structural genomic changes across sub-populations, with the analysis revealing differences in Borneo clusters linked to mosquito-related stages of the parasite cycle, in contrast to differences in host-related stages for the Peninsular group. Our work identifies new genetic exchange events, including introgressions between Malaysian Peninsular and M. nemestrina-associated clusters on various chromosomes, including in parasite invasion genes (DBP[Formula: see text], NBPX[Formula: see text] and NBPX[Formula: see text]), and important proteins expressed in the vertebrate parasite stages. Recombination events appear to have occurred between the Peninsular and M. fascicularis-associated groups, including in the DBP[Formula: see text] and DBP[Formula: see text] invasion associated genes. Overall, our work finds that genetic exchange events have occurred among the recognised contemporary groups of P. knowlesi parasites during their evolutionary history, leading to apparent mosaicism between these sub-populations. These findings generate new hypotheses relevant to parasite evolutionary biology and P. knowlesi epidemiology, which can inform malaria control approaches to containing the impact of zoonotic malaria on human communities.

Zoonotic malaria: Plasmodium knowlesi, an emerging pathogen.

PURPOSE OF REVIEW: The emergence of Plasmodium knowlesi, a parasite of Southeast Asian macaques, into the human population is ongoing and widespread across Southeast Asia. Humans entering P. knowlesi transmission areas are at risk. Patients present with uncomplicated, complicated and fatal disease, therefore prompt accurate diagnosis and treatment are essential. This review focuses on recent descriptions of asymptomatic and symptomatic infections in children, pathophysiology in adults, treatment and diagnosis, and highlights the importance of monitoring transmission and host-switch events. RECENT FINDINGS: New reports on P. knowlesi infections identify regional differences in aetiology and vector species. Parasitaemia is associated with disease severity and specific diagnostic tools are required. Treatment failures have not been reported. The severe form of P. knowlesi malaria can be compared with severe falciparum malaria to inform the pathophysiology of both infections. SUMMARY: P. knowlesi presents new challenges to malaria-control efforts in Southeast Asia. Sensitive and specific diagnostic tools are required for communities and travellers at risk. Currently P. knowlesi transmission appears to occur away from human settlements. However, ongoing host-switch events from macaques to humans cannot be excluded. Changes in P. knowlesi transmission across the region should be monitored to preempt outbreaks of this virulent pathogen.

Laboratory markers of disease severity in Plasmodium knowlesi infection: a case control study.

BACKGROUND: Plasmodium knowlesi malaria causes severe disease in up to 10% of cases in Malaysian Borneo and has a mortality rate of 1 - 2%. However, laboratory markers with the ability to identify patients at risk of developing complications have not yet been assessed as they have for other species of Plasmodium. METHODS: A case control study was undertaken in two hospitals in Sarikei and Sibu, Malaysian Borneo. One hundred and ten patients with uncomplicated (n = 93) and severe (n = 17) P. knowlesi malaria were studied. Standardized pigment-containing neutrophil (PCN) count, parasite density and platelet counts were determined and analysed by logistic regression and receiver operating characteristic (ROC) analysis. RESULTS: The PCN count was strongly associated with risk of disease severity. Patients with high parasite density (≥ 35,000/µl) or with thrombocytopaenia (≤ 45,000/µl) were also more likely to develop complications (odds ratio (OR) = 9.93 and OR = 5.27, respectively). The PCN count yielded the highest area under the ROC curve (AUC) estimate among all markers of severity (AUC = 0.8561, 95% confidence interval: 0.7328, 0.9794). However, the difference between all parameter AUC estimates was not statistically significant (Wald test, p = 0.73). CONCLUSION: Counting PCN is labour-intensive and not superior in predicting severity over parasitaemia and platelet counts. Parasite and platelet counts are simpler tests with an acceptable degree of precision. Any adult patient diagnosed with P. knowlesi malaria and having a parasite count ≥ 35,000/µl or ≥ 1% or a platelet count ≤ 45,000/µl can be regarded at risk of developing complications and should be managed according to current WHO guidelines for the treatment of severe malaria.

Cytoadherence and virulence - the case of Plasmodium knowlesi malaria.

BACKGROUND: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36. METHODS: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3. RESULTS: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36. CONCLUSIONS: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions.

De Novo Assembly of Plasmodium knowlesi Genomes From Clinical Samples Explains the Counterintuitive Intrachromosomal Organization of Variant SICAvar and kir Multiple Gene Family Members.

Plasmodium knowlesi, a malaria parasite of Old World macaque monkeys, is used extensively to model Plasmodium biology. Recently, P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes uncomplicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such, P. knowlesi presents a unique opportunity to develop experimental translational model systems for malaria pathophysiology informed by clinical data from same-species human infections. Experimental lines of P. knowlesi represent well-characterized genetically stable parasites, and to maximize their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence and de novo assemble P. knowlesi genomes from frozen clinical samples. The sequencing platform and assembly pipelines were designed to facilitate capturing data and describing, for the first time, P. knowlesi schizont-infected cell agglutination (SICA) var and Knowlesi-Interspersed Repeats (kir) multiple gene families in parasites acquired from nature. The SICAvar gene family members code for antigenically variant proteins analogous to the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) multiple var gene family. Evidence presented here suggests that the SICAvar family members have arisen through a process of gene duplication, selection pressure, and variation. Highly evolving genes including PfEMP1family members tend to be restricted to relatively unstable sub-telomeric regions that drive change with core genes protected in genetically stable intrachromosomal locations. The comparable SICAvar and kir gene family members are counter-intuitively located across chromosomes. Here, we demonstrate that, in contrast to conserved core genes, SICAvar and kir genes occupy otherwise gene-sparse chromosomal locations that accommodate rapid evolution and change. The novel methods presented here offer the malaria research community not only new tools to generate comprehensive genome sequence data from small clinical samples but also new insight into the complexity of clinically important real-world parasites.

Plasmodium knowlesi - Clinical Isolate Genome Sequencing to Inform Translational Same-Species Model System for Severe Malaria.

Malaria is responsible for unacceptably high morbidity and mortality, especially in Sub-Saharan African Nations. Malaria is caused by member species' of the genus Plasmodium and despite concerted and at times valiant efforts, the underlying pathophysiological processes leading to severe disease are poorly understood. Here we describe zoonotic malaria caused by Plasmodium knowlesi and the utility of this parasite as a model system for severe malaria. We present a method to generate long-read third-generation Plasmodium genome sequence data from archived clinical samples using the MinION platform. The method and technology are accessible, affordable and data is generated in real-time. We propose that by widely adopting this methodology important information on clinically relevant parasite diversity, including multiple gene family members, from geographically distinct study sites will emerge. Our goal, over time, is to exploit the duality of P. knowlesi as a well-used laboratory model and human pathogen to develop a representative translational model system for severe malaria that is informed by clinically relevant parasite diversity.

Clinical and parasitological response to oral chloroquine and primaquine in uncomplicated human Plasmodium knowlesi infections.

BACKGROUND: Plasmodium knowlesi is a cause of symptomatic and potentially fatal infections in humans. There are no studies assessing the detailed parasitological response to treatment of knowlesi malaria infections in man and whether antimalarial resistance occurs. METHODS: A prospective observational study of oral chloroquine and primaquine therapy was conducted in consecutive patients admitted to Kapit Hospital, Sarawak, Malaysian Borneo with PCR-confirmed single P. knowlesi infections. These patients were given oral chloroquine for three days, and at 24 hours oral primaquine was administered for two consecutive days, primarily as a gametocidal agent. Clinical and parasitological responses were recorded at 6-hourly intervals during the first 24 hours, daily until discharge and then weekly to day 28. Vivax malaria patients were studied as a comparator group. RESULTS: Of 96 knowlesi malaria patients who met the study criteria, 73 were recruited to an assessment of the acute response to treatment and 60 completed follow-up over 28 days. On admission, the mean parasite stage distributions were 49.5%, 41.5%, 4.0% and 5.6% for early trophozoites, late trophozoites, schizonts and gametocytes respectively. The median fever clearance time was 26.5 [inter-quartile range 16-34] hours. The mean times to 50% (PCT50) and 90% (PCT90) parasite clearance were 3.1 (95% confidence intervals [CI] 2.8-3.4) hours and 10.3 (9.4-11.4) hours. These were more rapid than in a group of 23 patients with vivax malaria 6.3 (5.3-7.8) hours and 20.9 (17.6-25.9) hours; P = 0.02). It was difficult to assess the effect of primaquine on P. knowlesi parasites, due to the rapid anti-malarial properties of chloroquine and since primaquine was administered 24 hours after chloroquine. No P. knowlesi recrudescences or re-infections were detected by PCR. CONCLUSIONS: Chloroquine plus primaqine is an inexpensive and highly effective treatment for uncomplicated knowlesi malaria infections in humans and there is no evidence of drug resistance. Further studies using alternative anti-malarial drugs, including artemisinin derivatives, would be desirable to define optimal management strategies for P. knowlesi.

Severe malaria - a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report.

BACKGROUND: Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here. CASE PRESENTATION: A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent. CONCLUSIONS: The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans.

Clinical and laboratory features of human Plasmodium knowlesi infection.

BACKGROUND: Plasmodium knowlesi is increasingly recognized as a cause of human malaria in Southeast Asia but there are no detailed prospective clinical studies of naturally acquired infections. METHODS: In a systematic study of the presentation and course of patients with acute P. knowlesi infection, clinical and laboratory data were collected from previously untreated, nonpregnant adults admitted to the hospital with polymerase chain reaction-confirmed acute malaria at Kapit Hospital (Sarawak, Malaysia) from July 2006 through February 2008. RESULTS: Of 152 patients recruited, 107 (70%) had P. knowlesi infection, 24 (16%) had Plasmodium falciparum infection, and 21 (14%) had Plasmodium vivax. Patients with P. knowlesi infection presented with a nonspecific febrile illness, had a baseline median parasitemia value at hospital admission of 1387 parasites/microL (interquartile range, 6-222,570 parasites/microL), and all were thrombocytopenic at hospital admission or on the following day. Most (93.5%) of the patients with P. knowlesi infection had uncomplicated malaria that responded to chloroquine and primaquine treatment. Based on World Health Organization criteria for falciparum malaria, 7 patients with P. knowlesi infection (6.5%) had severe infections at hospital admission. The most frequent complication was respiratory distress, which was present at hospital admission in 4 patients and developed after admission in an additional 3 patients. P. knowlesi parasitemia at hospital admission was an independent determinant of respiratory distress, as were serum creatinine level, serum bilirubin, and platelet count at admission (p < .002 for each). Two patients with knowlesi malaria died, representing a case fatality rate of 1.8% (95% confidence interval, 0.2%-6.6%). CONCLUSIONS: 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.

Knowlesi malaria in Vietnam.

The simian malaria parasite Plasmodium knowlesi is transmitted in the forests of Southeast Asia. Symptomatic zoonotic knowlesi malaria in humans is widespread in the region and is associated with a history of spending time in the jungle. However, there are many settings where knowlesi transmission to humans would be expected but is not found. A recent report on the Ra-glai population of southern central Vietnam is taken as an example to help explain why this may be so.

Morphological features and differential counts of Plasmodium knowlesi parasites in naturally acquired human infections.

BACKGROUND: Human infections with Plasmodium knowlesi, a simian malaria parasite, are more common than previously thought. They have been detected by molecular detection methods in various countries in Southeast Asia, where they were initially diagnosed by microscopy mainly as Plasmodium malariae and at times, as Plasmodium falciparum. There is a paucity of information on the morphology of P. knowlesi parasites and proportion of each erythrocytic stage in naturally acquired human infections. Therefore, detailed descriptions of the morphological characteristics and differential counts of the erythrocytic stages of P. knowlesi parasites in human infections were made, photographs were taken, and morphological features were compared with those of P. malariae and P. falciparum. METHODS: Thick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per microl blood, were examined. RESULTS: The P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae. CONCLUSION: Plasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.

Micro-geographic risk factors for malarial infection.

BACKGROUND: Knowledge of geography is integral to the study of insect-borne infectious disease such as malaria. This study was designed to evaluate whether geographic parameters are associated with malarial infection in the East Sepik province of Papua New Guinea (PNG), a remote area where malaria is a major cause of morbidity and mortality. METHODS: A global positioning system (GPS) unit was used at each village to collect elevation, latitude and longitude data. Concurrently, a sketch map of each village was generated and the villages were sub-divided into regions of roughly equal populations. Blood samples were taken from subjects in each region using filter paper collection. The samples were later processed using nested PCR for qualitative determination of malarial infection. The area was mapped using the GPS-information and overlaid with prevalence data. Data tables were examined using traditional chi square statistical techniques. A logistic regression analysis was then used to determine the significance of geographic risk factors including, elevation, distance from administrative centre and village of residence. RESULTS: Three hundred and thirty-two samples were included (24% of the total estimated population). Ninety-six were positive, yielding a prevalence of 29%. Chi square testing within each village found a non-random distribution of cases across sub-regions (p < 0.05). Multivariate logistic regression techniques suggested malarial infection changed with elevation (OR = 0.64 per 10 m, p < 0.05) and distance from administrative centre (OR = 1.3 per 100 m, p < 0.05). CONCLUSION: These results suggest that malarial infection is significantly and independently associated with lower elevation and greater distance from administrative centre in a rural area in PNG. This type of analysis can provide information that may be used to target specific areas in developing countries for malaria prevention and treatment.

Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening.

BACKGROUND: Until recently, Plasmodium knowlesi malaria in humans was misdiagnosed as Plasmodium malariae malaria. The objectives of the present study were to determine the geographic distribution of P. knowlesi malaria in the human population in Malaysia and to investigate 4 suspected fatal cases. METHODS: Sensitive and specific nested polymerase chain reaction was used to identify all Plasmodium species present in (1) blood samples obtained from 960 patients with malaria who were hospitalized in Sarawak, Malaysian Borneo, during 2001-2006; (2) 54 P. malariae archival blood films from 15 districts in Sabah, Malaysian Borneo (during 2003-2005), and 4 districts in Pahang, Peninsular Malaysia (during 2004-2005); and (3) 4 patients whose suspected cause of death was P. knowlesi malaria. For the 4 latter cases, available clinical and laboratory data were reviewed. RESULTS: P. knowlesi DNA was detected in 266 (27.7%) of 960 of the samples from Sarawak hospitals, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. Only P. knowlesi DNA was detected in archival blood films from the 4 patients who died. All were hyperparasitemic and developed marked hepatorenal dysfunction. CONCLUSIONS: 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.

Knowlesi malaria: newly emergent and of public health importance?

Several questions on public health impact have arisen from the discovery of a large focus of the simian malaria parasite, Plasmodium knowlesi, in the human population. P. knowlesi malaria is not newly emergent and was overlooked until molecular tools to distinguish between P. knowlesi and the morphologically similar Plasmodium malariae became available. Knowlesi malaria is a zoonosis that is widely distributed in Southeast Asia and can be fatal. Information on knowlesi malaria should be included in medical and public health guidelines to encourage the accurate diagnosis and treatment of patients, and monitor the incidence and distribution of cases. A complete emergence of P. knowlesi into the human population could be overwhelming and, although challenging, the prevention of this situation deserves serious consideration.

Comparison of artemether-lumefantrine with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in eastern Nepal.

Because available data suggest that resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is increasing in Nepal, an open-label, parallel-group efficacy/safety study was conducted in 99 Nepalese patients with uncomplicated falciparum malaria randomized 2:1 to artemetherlumefantrine (AL) or SP. Efficacy was assessed from clinical and microscopic evidence of treatment failure. Four SP-treated patients (12.1%; 95% CI, 4.0-29.1%) redeveloped parasitemia during the 28-day follow-up versus 0% (95% CI, 0-6.9%) in the AL group (P = 0.011), a difference that was confirmed by polymerase chain reaction (PCR) analysis of parasite DNA. PCR detected an additional six patients (two SP and four AL) with sub-microscopic gametocytemia or breakthrough parasitemia between Days 14 and 28, suggesting that AL efficacy was lower than estimated by microscopy. Dhfr and dhps mutations were not associated with outcome. AL is more effective than SP for uncomplicated malaria in Nepal, but regular monitoring of its efficacy should be carried out if this combination therapy is introduced.

A large focus of naturally acquired Plasmodium knowlesi infections in human beings.

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. METHODS: We took blood samples from 208 people with malaria in the Kapit division between March, 2000, and November, 2002. The small subunit ribosomal RNA and the circumsporozoite protein genes were sequenced for eight isolates that had been microscopically identified as P malariae. All blood samples were characterised with a genus-specific and species-specific nested PCR assay together with newly designed P knowlesi-specific primers. FINDINGS: All DNA sequences were phylogenetically indistinguishable from those of P knowlesi, a malaria parasite of long-tailed macaque monkeys, but were significantly different from other malaria parasite species. By PCR assay, 120 (58%) of 208 people with malaria tested positive for P knowlesi, whereas none was positive for P malariae. P knowlesi parasites in human erythrocytes were difficult to distinguish from P malariae by microscopy. Most of the P knowlesi infections were in adults and we did not note any clustering of cases within communities. P knowlesi infections were successfully treated with chloroquine and primaquine. INTERPRETATION: Naturally acquired P knowlesi infections, misdiagnosed by microscopy mainly as P malariae, accounted for over half of all malaria cases in our study. Morphological similarities between P knowlesi and P malariae necessitate the use of molecular methods for correct identification. Further work is needed to determine whether human P knowlesi infections in the Kapit division are acquired from macaque monkeys or whether a host switch to human beings has occurred.