we screened by microscopy 4,980 patients with febrile illness who sought care at a malaria clinic in kachin state , northeastern myanmar , during 20072009 ; a total of 27.9% had malaria infections . p. falciparum , p. vivax , and mixed species infections accounted for 56.7% , 41.1% , and 2.2% of malaria cases , respectively . finger - prick blood samples were obtained from 260 patients with uncomplicated p. falciparum infection who had not used antimalarial drugs during the previous 2 weeks . parasite dna was extracted from filter papers and genotyped at 3 polymorphic genes , which detected 54.6% of samples containing mixed - strain infections ( 5 ) . of the 118 samples with monoclonal infection , 117 samples were successfully genotyped by pcr and sequencing at 5 known and putative drug - resistance genes ( technical appendix ) , some of which have been widely used for resistance surveillance and as predictors of clinical efficacy of antimalarial drugs . sequencing of 2 fragments in the p. falciparum chloroquine resistance transporter ( pfcrt ) gene covering single nucleotide polymorphisms ( snps ) at codons 7276 and 220 , respectively ( 6 ) , showed that the major chloroquine resistance determinant k76 t mutation has reached fixation in the parasite population ( figure 1 ) . all parasites had sequence cviet at positions 7276 , compared with the wild - type sequence svmnk . in addition , the a220s mutation associated with chloroquine resistance was predominant ( 99.1% ) . wild - type and mutated amino acids are shown in white and black , respectively . prevalence ( % ) of each multilocus genotype in each year is indicated in the right columns . sequencing of 2 p. falciparum multidrug resistance 1 ( pfmdr1 ) fragments as described ( 5 ) detected only the n86y , y184f , and n1042d mutations making up 5 haplotypes ( figure 2 ) . the overall haplotype prevalence differed significantly among the years ( p<0.001 ,   =  31.39 , df  =  8).the prevalence of wild - type haplotype was 59.0% . among the 3 mutant codons , only y184f reached a relatively high frequency ( 35.9% ) . analysis of pfmdr1 copy number from monoclonal infections by real - time pcr with 3d7 and dd2 strains as negative and positive controls , respectively ( 6 ) , detected no pfmdr1 amplification . annual prevalence of plasmodium falciparum dihydrofolate reductase haplotypes among clinical samples collected from kachin state , northeast myanmar , 20072009 . the x - axis shows the 5 haplotypes ( the amino acids at positions 86 , 184 , and 1042 with mutated amino acids in boldface ) . the  test was performed to compare prevalence of 2 major haplotypes between years . for each haplotype , ns denotes no significant difference ( p>0.05 ) between years ; asterisk ( * ) denotes significant difference ( p<0.05 ) between years . sequencing of the dihydrofolate reductase ( pfdhfr ) and dihydropoteroate synthase ( pfdhps ) genes ( technical appendix ) detected 4 snps in pfdhfr associated with pyrimethamine resistance ( n51i , c59r , s108n , and i164l ) and 4 snps in pfdhps associated with sulfadoxine resistance ( s436a , a437 g , k540e / n , and a581 g ) ( table 1 ) . the overall haplotype prevalence of the 2 genes differed significantly between the years ( p<0.0001 ,  =  76.49 , df  =  28 ) . of the 5 pfdhfr haplotypes , wild - type ncsi was observed only in 1 sample in 2007 ; the remaining samples contained at least double mutations 59r/108n . two triple - mutation haplotypes ( nrnl and irni , mutations in boldface ) were detected with nrnl being more frequent than irni in each year . overall , quadruple mutations ( irnl ) were found in > 50% of the samples . in addition , frequency of triple and quadruple mutations increased gradually from 2007 to 2009 . we found all 5 haplotypes in 2007 but only triple and quadruple mutations in 2009 . n mutations were highly prevalent : 98.3 and 96.6% , respectively ( table 1 ) . similarly , the wild - type pfdhps haplotype sgka was found in only 2 samples . agea was the most common haplotype in each year and reached an overall frequency of 48.7% . * pfdhfr , plasmodium falciparum dihydrofolate reductase ; pfdhps , p. falciparum dihydropoteroate synthase ;  , no such haplotype detected . test was performed with all data points of the 2 genes , which showed that overall haplotype prevalence differed significantly between the years (  test , p<0.0001 ,   =  76.49 , df  =  28 ) . major dhfr and dhps haplotypes were individually compared . for each haplotype ( in the same row ) , the labeling by different letters denotes significant difference between the years (  test , p<0.05 ) . molecular analysis of drug - resistance markers in monoclonal infections enabled us to obtain multilocus genotypes of the parasites . genotyping each of the 117 parasite isolates at 16 drug resistance  related codons in the pfcrt , pfmdr1 , pfdhfr , and pfdhps genes showed 41 haplotypes ( figure 1 ) . among these haplotypes , parasites containing > 10 mutated codons accounted for 93.2% of the samples . polymorphisms in the minisatellite ms4760 of p. falciparum na / h exchanger ( pfnhe1 ) are associated with quinine sensitivity ( 5,710 ) . sequencing of the pfnhe1 fragment containing the ms4760 minisatellite from 79 monoclonal infections showed 10 haplotypes , with haplotype 7 the most predominant ( 54.4% ) ( table 2 ) . more than 64% of samples tested contained > 3 copies of the dnnnd repeat ( rep1 ) ; 76% contained 1 copy of the nhndnhnnddd repeat ( rep2 ) . accordingly , > 60% of parasite isolates had a rep1:rep2 ratio of > 3:1 . * in myanmar , high - level resistance to chloroquine and pyrimethamine  sulfadoxine was reported more than a decade ago ( 1113 ) . our molecular survey showed that the major chloroquine resistance allele cviet has reached fixation , and triple and quadruple mutations in pfdhfr and pfdhps were highly prevalent in this region . these findings strongly suggest that a large proportion of parasites might show clinical resistance to chloroquine and antifolate drugs . although chloroquine has been withdrawn from treating p. falciparum malaria for decades in some regions , the pfcrt resistance alleles showed no sign of abating ( 6 ) . furthermore , despite adoption of artemisinin combination therapy in 2002 , the frequency of highly mutated pfdhfr and pfdhps haplotypes appeared to have increased during this study , which suggested that artemisinin combination therapy might not have retarded the spread of antifolate - resistant parasites . this situation differs from that in the western myanmar border area but is similar to that in thailand and cambodia , where highly mutated pfdhfr and pfdhps genotypes also were common ( 14,15 ) . mutations in pfmdr1 are associated with resistance to several antimalarial drugs including chloroquine , mefloquine , and quinine and increased pfmdr1 copy number is responsible for mefloquine resistance . we found that 60% of the parasites contained the wild - type pfmdr1 allele , similar to some parasites from the western myanmar border area ( 15 ) . no pfmdr1 amplification was detected , suggesting that parasites from this region might be mefloquine sensitive , consistent with the fact that mefloquine has not been deployed here . in contrast , in vitro mefloquine resistance was observed in southeast myanmar bordering thailand ( 13 ) , possibly because of the extensive use of mefloquine in thailand for the past 2 decades . although the validity of pfnhe1 minisatellite polymorphism for predicting quinine resistance remains uncertain and may depend on the parasites origins ( 79 ) , we detected significant association of decreased quinine susceptibility with increased dnnnd repeat copies ( 5 ) . we have provided further evidence on the high prevalence of parasites with increased dnnnd repeats in pfnhe1 , which suggests that some parasite strains might show reduced sensitivity to quinine . overall , our molecular survey of antimalarial drug resistance in p. falciparum showed high frequency of multidrug - resistant haplotypes in northeastern myanmar . moreover , parasites in this region had unique multilocus genotypes that differed markedly from those in other areas of the gms . these findings suggest that coordinated efforts are necessary to thwart the spread of resistant strains across larger geographic regions . our molecular study showed only the genotypes of the drug resistance genes ; further in vitro and in vivo studies are required to corroborate these findings . table showing primers used to amplify drug resistance - related genes in plasmodium falciparum isolates .
we performed a molecular epidemiologic survey of mutations associated with drug - resistance genes in plasmodium falciparum in northeastern myanmar . in this region , 
 3 highly mutated drug - resistance haplotypes and 1 associated with decreased quinine susceptibility were prevalent , which suggests that parasites may be resistant to multiple commonly used antimalarial drugs .