a 38-year - old man came to the netherlands in january 2009 to work as a rigger in the harbor of rotterdam . since october 2008
, he had lived in kapit , sarawak , in borneo and hunted wild animals in the surrounding jungles .
one week after arriving in the netherlands , he came to our hospital with a 5-day history of fever , myalgia , headache , and low back pain .
his medical history was uneventful , and he had not experienced any previous malaria attacks .
laboratory investigations showed a moderate anemia ( hemoglobin 7.8 mmol / l [ reference range 8.511.0 mmol / l ] ) , a normal leukocyte count ( 5.8  10/l [ reference range 4.310  10/l ] ) , thrombocytopenia ( platelet count 22  10/l [ reference range 150400  10/l ] ) , an increased level of c - reactive protein ( 158 mg / l [ reference range < 10 mg / l ] ) , and liver function abnormalities ( serum alanine aminotransferase 199 u / l [ reference range < 41
u / l ] ; aspartate aminotransferase 128 u / l [ reference range < 37 u / l ] ; lactate dehydrogenase [ ldh ] 1,059
u / l [ reference range < 450 u / l ] ; gamma - glutamyltransferase 183 u / l [ reference range < 50 u / l ] ; alkaline phosphatase 285 u / l [ reference range < 120
u / l ] ; and total bilirubin 99 mol / l [ reference range < 17 mol / l ] ) .
plasma lactate level was within normal limits . in a rapid diagnostic test for malaria ( binaxnow malaria test ; binax ,
scarborough , me , usa ) , his blood sample was negative for p. falciparum histidine - rich protein 2 but showed a positive reaction with pan - malarial aldolase antigen , which suggested a non  p .
results of quantitative buffy coat analysis were positive for malaria trophozoites , schizonts , and gametocytes .
a thin blood film showed parasite density of 2% infected erythrocytes ( 84,000 trophozoites/l ) , schizonts , and gametocytes with an inconclusive morphologic appearance ( figure 1 ) .
a p. knowlesi infection was suspected because of his recent stay in kapit , malaysian borneo .
the patient was treated orally with chloroquine , 10 mg / kg , followed by 5 mg / kg after 6 , 24 , and 48 hours , which resulted in a rapid relief of symptoms and fever .
results of quantitative buffy coat analysis , pan - malarial aldolase antigen reactivity , and thick and thin blood smears were negative within 40 hours after administration of chloroquine .
infected erythrocytes were not enlarged , lacked schuffner stippling , and contained much pigment . shown are examples of trophozoites ( a  f ) , a schizont ( g ) , and a gametocyte ( h ) .
subsequently , pcr analysis of blood samples taken at admission was performed to determine the plasmodium species .
human plasmodium species were excluded by using a conventional nested pcr and real - time pcr ( 8,9 ) .
in addition , pcr analysis was performed on a blood sample by using diagnostic primers for plasmodium small subunit ( ssu ) rrna as described ( 3 ) , including genus - specific and species - specific primers .
( 3 ) , nested pcr was not necessary because of high parasitemia and availability of fresh material . instead
, pcrs were performed directly on 2 l of blood in 25-l volumes by using the phusion blood pcr kit ( finnzymes , espoo , finland ) .
specific primers generated pcr products , providing evidence that the patient had p. knowlesi malaria . to confirm the pcr result , we sequenced the cloned amplification product generated with primers rplu1 and rplu5 .
sequences were compared with known plasmodium a - type ssu rrna sequences by using the neighbor - joining method ( figure 2 ) .
fj804768 ) clustered strongly with p. knowlesi a - type ssu rna sequences , confirming that the patient was infected with the p. knowlesi parasite .
phylogenetic tree constructed according to the neighbor - joining method based on a - type small subunit rna sequences of several plasmodium species (
the sequence of the clinical isolate pkhhr - bprc1 ( in boldface ) ( genbank accession no .
fj804768 ) clusters with all other p. knowlesi strains ( indicated by pk isolate numbers ) .
pfrag , p. fragile ; pinui , p. inui ; pcyn , p. cynomolgi ; pfalc , p. falciparum ; pmal , p. malariae .
obtaining a correct diagnosis of malaria may be troublesome in centers where laboratory staff are less skilled in the proper identification and quantification of causative plasmodium species , as may occur in countries in which malaria is not endemic .
in contrast to our case , bronner et al . reported that the binaxnow malaria test did not detect a p. knowlesi infection in a traveler from sweden who had a p. knowlesi infection acquired in malaysian borneo ( 2 ) .
low parasitemia ( 0.1% ) in this patient may have caused the lack of reactivity with the pan - malarial antigen aldolase ( 2 ) .
we evaluated the binaxnow malaria and the optimal rapid malaria ( diamed , cressier , switzerland ) tests for detection of p. knowlesi in human blood by analysis of consecutive blood samples taken after admission .
these samples were stored at 20c for 2 weeks until tests were performed ( table ) .
the blood sample taken on admission ( 2% infected erythrocytes ) did not react with p. falciparum 
specific antibody against histidine - rich protein 2 , but reacted with the pan - malarial antigen aldolase in the binaxnow malaria test .
specific ldh and pan - malarial ldh in the optimal rapid malaria test , confirming the cross - reactivity of p. knowlesi ldh with monoclonal antibody 17e4 against p. falciparum ldh , as shown by mccutchan et al .
this antibody is also used in the optimal rapid malaria test ( diamed , pers .
therefore , a positive test result for the p. falciparum ldh in the optimal rapid malaria test is not specific for p. falciparum because it can also be caused by a p. knowlesi infection .
the positive result for ldh and aldolase in either test became negative after treatment ( table ) , which indicates rapid clearance of parasites after treatment .
results of our comparative study suggest that the optimal rapid malaria test may be able to detect lower levels of p. knowlesi parasitemia than the binaxnow malaria test .
* hrp-2 , histidine - rich protein 2 ; ldh , lactate dehydrogenase ; nd , not detectable .
all tests were performed on blood samples collected in edta and frozen at 20c for 2 weeks . 
binax , inc . , scarborough , me , usa .  diamed , cressler , switzerland . 
our results indicate that commercially available rapid diagnostic antigen tests for human plasmodium species can detect p. knowlesi infections in humans , although infections with a low parasitemia will not be detected .
a negative test result does not exclude a p. knowlesi infection , as it does not exclude infections by other human plasmodium species ( 11 ) .
 we describe a pcr - confirmed case of plasmodium knowlesi infection with a high parasitemia level and clinical signs of severe malaria in a migrant worker from malaysian borneo in the netherlands . 
 investigations showed that commercially available rapid antigen tests for detection of human plasmodium infections can detect p. knowlesi infections in humans . 
The Case
Conclusions