in august 2000 , a 38-year - old thai man came to an outpatient department of king chulalongkorn memorial hospital , bangkok , with daily fever , headache , intermittent chill , sweating , and malaise for 4 days .
his home was in a suburb of bangkok , where no malaria transmission has been reported . during the past few months before the present illness
, he spent several few weeks in a hilly forest area in prachuap khiri khan province in southern thailand , 300 km from bangkok near the thai - myanmar border .
he did not know of any underlying illness and had not experienced any previous malaria attacks .
although he stayed in a cottage and slept inside a mosquito net , he remembered being bitten frequently by mosquitoes , especially at dusk and dawn . upon examination ,
his hemoglobin was 14.0 g / dl , hematocrit was 0.4 , and erythrocyte count was 4.2 x 10 cells/l .
levels of other laboratory investigations , including urinalysis , blood sugar , liver function test , blood urea nitrogen , and creatinine , were normal .
examination of giemsa - stained thin blood films showed 10% young trophozoites , 45% growing trophozoites , 40% schizonts , and 5% gametocytes ( n = 300 ) .
the parasite structure was compatible with that of p. malariae . the parasite density inferred from the number of malarial parasites per 500 leukocytes in thick blood smear yielded 1,155/l or equivalent to parasitemia 0.03% .
the patient was treated with 10 mg / kg of oral chloroquine initially , followed by 5 mg / kg , 6 hours later on the day 1 , and 5 mg / kg / day for the next 2 days . on day 2 , with a temperature of 37.5c , he came to the hospital .
meanwhile , we recently evaluated a dna - based diagnostic method by the polymerase chain reaction ( pcr ) targeting the small subunit ribosomal rna ( ssu rrna ) genes of all four species of human malaria as reported ( 8) .
ten isolates each for p. falciparum , p. vivax , and p. malariae and four isolates of p. ovale were used as positive controls .
results showed that all isolates gave concordant positive pcr products with those diagnosed by microscopy except an isolate from this patient ( data not shown ) .
retrospective examination of blood smears has shown several developmental stages of malaria parasites similar to those typically seen in p. malariae . however , some erythrocytes that harbored mature asexual parasites possessed fimbriated margins .
the cytoplasm of some young trophozoites appeared spread out into the network of irregular pseudopodia , and the chromatin was distributed into fragments , conforming to the tenue forms .
pinkish dots varying from fine to large irregular masses called sinton and mulligan 's stippling developed intracorpuscularly with the maturation of some parasites ( figure 1 ) .
giemsa - stained thin blood films depicting a ) ring stage , b ) tenue form of young trophozoite , c ) band - shaped growing trophozoite , d ) growing trophozoite with little or no amoeboid activity , e ) double growing trophozoites , f ) early schizont , g ) late schizont in an erythrocyte with fimbriated margins , and h ) mature macrogametocyte .
discernible sinton and mulligan stippling is in c , d , and f. to elucidate the species of malaria infecting our patient , we determined the ssu rrna gene by using similar methods as described by others ( 9 ) , except that extaq dna polymerase ( takara , japan ) , pgem - t vector ( promega , usa ) , and escherichia coli strain jm109 were used .
results showed that the ssu rrna sequence contained 97.8% to 99.6% homology with those of p. knowlesi transcribed during asexual stages or the type a gene ( genbank accession no .
nucleotide sequence data reported in this study are available in the embl , genbank , and ddjb databases under the accession no .
phylogenetic tree showed that p. knowlesi in this study was closely related to the w1 and nuri strains , although its divergence from malaysian human isolates was not supported by bootstrap analysis ( figure 2 ) .
consistently , the mitochondrial cytochrome b gene of this isolate , determined by the methods similar to previous report except the pcr primers ( mtpk - f:5-aggtattatattctttatacaaatattaac-3  and mtpk - r:5-tcttttataatgaacaagtgtaaataatc-3  ) , displayed perfect sequence identity with that of p. knowlesi strain h from monkey ( af069621 ) ( 4 ) . neighbor - joining tree based on the asexually transcribed ssu rrna sequences displaying the phylogenetic position of isolate a1 in this study in relation to other plasmodium knowlesi isolates ( ay327549-ay327556 from humans , and l07560 , u72542 , and ay327557 from monkeys ) and p. fragile ( m61722 ) .
p. knowlesi is prevalent among crab - eating macaques , macaca fascicularis , in the malaysian peninsula and the philippines ( 2,10 ) .
other known natural hosts include pig - tailed macaques , m. nemestrina , and leaf monkeys , presbytis melalophos ( 2,10 ) .
( 11 ) had shown that p. knowlesi isolated from monkey could be infectious to humans , the first naturally acquired human infection with p. knowlesi was not reported until 1965 ( 6 ) ; the patient was infected in a malaysian forest . in
1971 the second case , albeit presumptive , occurred in a man who also acquired the infection in a forest in malaysia ( 12 ) .
recently , a large cluster of human infections caused by p. knowlesi has been identified from malaysian borneo ( 9 ) .
our report has expanded the geographic range for natural transmission of p. knowlesi to a forest in thailand near southern myanmar border , where wild populations of crab - eating macaques , despite being considered endangered , are still substantial .
the prevalence of naturally acquired primate malaria in humans can be underestimated from examination of blood films .
the reported abundance of ring stages of p. knowlesi found in the first naturally acquired human case led to the initial diagnosis of p. falciparum , while the mature parasites could masquerade as those of p. malariae , as we encountered in this patient ( 6 ) .
although structural descriptions of young trophozoites of p. knowlesi have been delineated , we were unable to find the ring form with double chromatin dots ( 9 ) .
conversely , a few young trophozoites resembled the tenue forms , proposed by stephens in 1914 ( 13 ) to be a distinct species .
however , the tenue form has recently been recognized to be a p. malariae variant found in myanmar ( 8) . the presence of the tenue form in the blood smears of our patient , despite the low number , rather suggests a shared structural feature among species of malaria .
the possibility of coinfection between p. knowlesi with one or more of the four human malaria species was not supported by our pcr detection .
the structure of p. knowlesi is highly dependent on the host erythrocytes , i.e. , resembling p. vivax in m. fascicularis , p. falciparum in rhesus monkeys , and p. malariae in humans ( 2,9,11,12 ) .
although stippling was not seen among p. knowlesi  infected blood smears of sarawak 's patients , the presence of sinton - mulligan stippling in infected erythrocytes in this study is in accord with the report by fong et al . , in which erythrocytic stippling served as one of the diagnostic feature ( 9,12 ) .
such discrepancy could partly arise from differences in the condition for giemsa staining , infecting parasite strains , or both .
the complete asexual erythrocytic cycle of p. knowlesi in human and its natural macaque host requires 24 hours , coinciding with a quotidian fever pattern .
however , fever pattern per se may not be a precise indicator for differentiating malaria caused by p. knowlesi and p. malariae .
although the merogony cycle of p. malariae has been generally known to be 72 hours , fever patterns might not be strictly quartan ( 14 ) .
meanwhile , the preexisting immunity to p. vivax has reportedly conferred partial resistance to induced infection during malariotherapy ( 2 ) .
whether naturally acquired immunity against p. vivax can reduce symptoms in p. knowlesi infection requires further investigation . to date , little is known about the extent of variation in the p. knowlesi population .
analysis of the ssu rrna gene from the isolate in this study has shown minor difference from those of p. knowlesi from monkeys and patients in malaysian borneo ( 3,9 ) .
evidence from malariotherapy showed that p. knowlesi could lose or increase its virulence on blood passage in humans , which suggests that strain difference could occur in wild populations and might effect humans differently ( 2 ) . in conclusion
, p. knowlesi could contribute to the reemergence of simian malaria in thailand and southeast asia , where its vectors , anopheles leucosphyrus group , are abundant ( 15 ) .
 we describe a case of naturally acquired infection with plasmodium knowlesi in thailand . 
 diagnosis was confirmed by the small subunit ribosomal rna and the mitochondrial cytochrome b sequences . 
 the occurrence of simian malaria in human has signified the roles of wild primate populations in disease transmission in some malaria - endemic areas . 
The Study
Conclusions