a 12-year - old male presented on 13 august 2010 with a nine day history of fever . he had recently spent 6 weeks in india , where he did not take any malaria chemoprophylaxis or use a mosquito net . he developed a 105.7  f fever on 5 august on the flight home , which recurred every morning between 6:00 and 11:00 a.m. , ranged from 104  f to 105  f , and was associated with headaches . he had treated his fevers only with tylenol . additionally , he presented with a five day cough , and one episode of diarrhea and three episodes of emesis in the two days prior to presentation . the day after his flight home , the patient saw his primary care physician ; a complete blood count and liver panel were ordered ( table 1 ) . a malaria smear was also ordered and was negative for plasmodium and babesia . a second complete blood count and a liver panel urinalysis revealed small bilirubin , trace ketones and protein 30 , but was otherwise negative . the patient was negative by monospot test , as well as for influenza a and b , and had had a negative purified protein derivative test 3 years prior . negative results were also obtained for antibodies ( igm and igg ) to chickungunya and dengue fever and for ehrlichia pcr . blood films ( figure 1 ) demonstrated the diagnostic features of plasmodium vivax infection : all parasite forms were present ( trophozoite , schizont , and gametocyte ) ; not all red blood cells were infected , unlike p. falciparum infections ; and the schizonts were larger than uninfected red blood cells ( figure 1 ) , and merozoites and schuffner 's dots were present , both characteristic of p. vivax . several months subsequent to treatment , remnant whole - blood and plasma specimens collected the day of presentation were de - identified and sent to ibis biosciences for analysis with the vector - borne bpn assay using the plex - id ( abbott molecular , des plaines , il , usa ) . this technology employs broad - range pcr and electrospray ionization mass spectrometry ( pcr / esi - ms ) and has been used to detect a range of vector - borne organisms . specifically , pcr / esi - ms involves generation of short pcr amplicons , followed by electrospray mass spectrometry to determine the mass of the amplicon . based on the amplicon mass , the numbers of each of the four nucleotides in the amplicon , the basecount , can be calculated and compared to a database of basecounts to identify the microorganism . this assay , which is not available for diagnostic use , is designed to detect species of anaplasma , babesia , bartonella , borrelia , ehrlichia , francisella and rickettsia . modifications consisted of the following : homogenized lysates were incubated in lysis buffer for 1  h at 30  c prior to loading on the kingfisher flex ; isopropanol was used instead of ethanol in wash 1 buffer ; specimens were washed twice with wash i buffer and twice with wash ii buffer ; and the temperature of the initial binding step on the kingfisher was changed to 30  c for 22.5  min . ten microliters of the resulting 200  l extracts was used in each well of the vector - borne bpn assay plate . negative controls included water extractions ; all controls were negative . a novel base count , a28 g24 c16 t18 , was detected in both specimen extracts ( figure 2 ) with primer pair inv4855 , designed to target the babesia -tubulin gene . to identify the detection , initially thought to be a species of babesia , the specimen extracts were amplified with 4 sets of primers targeting the 18s rdna , its1 , its2 and hsp70 genes of babesia spp . using the platinum taq high fidelity kit ( invitrogen , carlsbad , ca , usa ) . the reactions were performed on an mj thermocycler with the following pcr cycle : 95  c for 2  min , followed by eight cycles of 95  c for 15  s , 50  c for 45  s and 68  c for 90  s with the 50  c annealing temperature increasing 0.6  c each cycle . the pcr was then continued for 37 additional cycles of 95  c for 15  s , 60  c for 15  s and 68  c for 60  s. the cycle ended with a final extension of 4  min at 72  c and a 4  c hold . pcr products were cloned with the zeroblunt topo pcr kit ( invitrogen ) according to the manufacturer 's instructions , and then sequenced with sp6 and t7 promoter primers by seqwright ( houston , tx , usa ) . six of the its2 clones tested had sequences bearing 97.3% ( 397/408 ) identity with plasmodium vivax ( genbank accession number af316892.1 ) . based upon the initial clinical findings in august 2010 , the patient was diagnosed with vivax malaria and treated with malarone ( atovaquone - proguanil 250/100  mg ) for 3 days , and then received 14 days of primaquine ( 15  mg base per day ) . malaria is an important cause of illness in travelers returning to the usa from endemic countries . p. vivax is the second most common etiological agent of malaria after p. falciparium and , although vivax malaria is not as severe as that caused by p. falciparum , p. vivax is more geographically widespread , particularly in temperate climates . 2010 saw a significant upswing in the number of imported malaria cases in the usa , with the highest reporting levels seen since 1980 . of the 1479 cases of imported malaria from all infecting species , 216 ( 15% ) were acquired in india , a 57% increase in the number of imported cases acquired in india over 2009 . among the 1366 cases of imported malaria in the usa for which the etiological agent and the region of acquisition were known , p. vivax infection also displays a seasonal transmission cycle ; in 2010 , the incidence of imported p. vivax cases peaked in august , with a secondary peak in november . this case marks the first report of malaria detection using the pcr / esi - ms technique with the plex - id . this technique has previously been used to rapidly detect and identify nucleic acids from pathogens in specimens collected from patients diagnosed with various infectious diseases . the particular assay used in this report , the vector - borne bpn assay , was designed to detect and identify common vector - borne pathogens from the usa and europe , including babesia . while not specifically designed to target plasmodium , the broad - range primer , inv4855 , designed to target the related babesia -tubulin gene , amplified p. vivax and produced a unique basecount signature allowing for the identification of the organism . bioinformatic analysis has since determined that primer pair inv4855 can also detect and differentiate p. falciparum and p. knowlesi . that there is conservation in -tubulin gene between babesia and plasmodium species there are several advantages of using pcr / esi - ms over traditional means of malaria detection . the technique offers a rapid molecular method for the identification of pathogen dna from human specimens , which does not require the screening of multiple blood films to identify infected red blood cells and provides a molecular basis for species differentiation rather than one based on visual inspection of parasite forms . additionally , the vector - borne bpn assay was able to detect p. vivax dna in a plasma specimen , indicating that sample types other than whole blood can be used to detect active malaria infection . finally , the use of the vector - borne bpn assay in particular offers the advantage of being able to screen for multiple vector - borne pathogens simultaneously , which proved useful in this case for differentiating an imported p. vivax infection from infection with domestic vector - borne pathogens associated with high fevers , such as babesia microti and ehrlichia chaffeensis .
plasmodium vivax is a common cause of imported malaria in the usa , second only to p. falciparum . 
 we present a case of p. vivax malaria in a child returning from india . 
 p. vivax was initially diagnosed by standard methodology and detected retrospectively by use of broad - range pcr and electrospray ionization mass spectrometry using a panel of primers designed to detect vector - borne pathogens . 
 this is the first reported case of p. vivax detection using pcr and electrospray ionization mass spectrometry .