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150 third trimester of pregnancy, were administrated under supervision during 528 antenatal care visit in the controlled IPT group. In the non controlled IPT group, drug was given free to women and it was recommended to take it chloroquine-resistAnt Plasmodium falciParum at home. Women were individually randomized to receive non controlled hAplotypes from the BrAZiliAn AmAZon IPTp (n =130) or controlled IPT (n= 125) with SP. Women were followed up during pregnancy and at delivery. A thin and thick smear was done at the giselle m. Viana, Marinete M. Póvoa ANC every month and if patient presented signs/ symptoms suggestive of Instituto Evandro Chagas, Ananindeua, Brazil malaria at any time. Low birth weight, defined as below 2,500 g, maternal Most Brazilian malaria cases occur in the Amazon region and anaemia, placental malaria infection detected by thick blood smear, Plasmodium falciparum accounts for approximately 15% of those with Rapid diagnostic Test and Biopsy were assessed. Also antenatal parasite a focal distribution. P. falciparum isolates started to show resistance to prevalence and delivery outcomes in all parity groups were investigated. chloroquine, a mainstay of Brazilian antimalarial policy, in 1960. CQ The prevalence of anaemia (as defined by a haemoglobin concentration resistance has been linked to mutations in the P. falciparum CQ resistance of 11.0 g/dL) at delivery were comparable between the controlled and transporter (pfcrt) with the K76T as the critical event while additional non controlled groups. Effects on of low birth weight (LBW, < 2.5 kg) mutations (72-76 residue) enhance the resistance. Our objective was to were non-statistically significant. The prevalence of LBW was 1.5%( IPTp characterize the geographic and temporal extent of CQ-resistant pfcrt non controlled group) and 1.6% (IPTp controlled group). Unfavourable alleles in the Brazilian Amazon. For this purpose, we examined 177 P. pregnancy outcomes, including one abortion was observed on IPTp non falciparum blood isolates from Para, Amapa, and Rondonia states from controlled group. Placental P. falciparum infection was observed in 1.6% the 1980s to 2000s. We used direct sequencing to determine the presence (Rapid Diagnostic Test) and 3.1% respectively on IPTp controlled groug of mutations in pfcrt and examined four microsatellite loci that cover 11 and IPTp non controlled group. In conclusion, Intermittent preventive kilobases (kb) around pfcrt. No ancestral-type parasites (CVMNK) were treatment of malaria in pregnancy (IPTp) with sulphadoxine-pyrimethamine found and all isolates carried the sVMNt genotype, which has been (SP) reduces the incidence of low birth-weight, pre-term delivery, reported to be a highly resistant CQ genotype in South America. However, intrauterine growth-retardation and maternal anaemia. there are two SVMNT alleles, one where S is coded for by TCT and the other where it is coded for by AGT. STCTVMNT allele was more frequently 527 found. Most alleles share a common haplotype which suggests a common founder allele for these genotypes. In addition, this haplotype is similar inVestigAtion of genetic mutAtions in to that found in other South American countries. Overall this finding Plasmodium vivax dihydrofolAte reducAtAse And suggests strong CQ selective pressure has fixed SVMNT alleles in these susceptiBility to conVentionAl And new AntifolAte Brazilian states. AntimAlAriAls using A Plasmodium falciParum expression system 529 Alyson m. Auliff1, Michael T. O’Neil2, Don L. Gardiner3, John H. 4 1 A proBABle cAse of chloroquine-resistAnt Adams , Qin Cheng Plasmodium vivax in the peruViAn AmAZon 1Department of Drug Resistance and Diagnostics, Australian Army Malaria 1 1 1 Institute, Enoggera, Australia, 2Division of Experimental Therapeutics, Salomon Durand , meddly santolalla , Carola Salas , Valeria 1 2 2 Walter Reed Army Institute of Research, Silver Spring, MD, United States, Soberon , Carlos Alvares Antonio , Carmen Montalvan , Mariella 2 3 3 3Queensland Institute of Medical Research, Brisbane, Australia, 4Global Galves Montoya , Andrea McCollum , Michael D. Green , Carmen 1 3 1 Health Infectious Disease Research Program, University of South Florida, Lucas , Venkatachalam Udhayakumar , Paul C. Graf , David J. 4 Tampa, FL, United States Bacon 1 2 With the high prevalence of Plasmodium vivax resistance to antifolates Naval Medical Research Center Detachment, Lima, Peru, Regional Health 3 throughout Australasia, it is critical to understand the determinants for Directorate, Loreto, Peru, Centers for Disease Control and Prevention, 4 resistance and for development of new treatments. Like P. falciparum, Atlanta, GA, United States, Navy Environmental and Preventative resistance to antifolates such as pyrimethamine and cycolguanil in P. Medicine Unit-2, Norfolk, VA, United States vivax, are caused by point mutations within the parasites dihydrofolate Chloroquine is nearly universally used as the first line therapy for reducatase (DHFR)-thymidylate synthase genes. However several unique Plasmodium vivax malaria due to its high efficacy and low cost. However, mutations have been reported in P. vivax DHFR and their roles in resistance isolated cases of CQ resistance (CQR) have been reported in South to classic and novel antifolates are not entirely clear. We have assessed America including two cases in Peru. To date very little has been published the in vitro expression of the P. vivax wild- type and various mutant on the identification of specific molecular markers in the genome of dhfr alleles using both episomal and piggyBac transposon integrated P. P. vivax that confer CQR. We conducted a 2 years study to evaluate 3 falciparum expression systems and compared the effect of these alleles regimens of primaquine (PQ) in the Amazon Basin of Peru. We enrolled to susceptibility to antifolates. We show that the P. falciparum parasites 540 patients between the ages of 1 and 77 years. All patients had fever transfected with wild-type pvdhfr, in both expression systems, is as or history of fever, and their geometric mean parasite density was 4 271 susceptible to classic and novel antifolates as the P. falciparum with wild- parasites/μL. All subjects received directly observed therapy with 25 mg/ type pfdhfr, while P. falciparum parasites transfected with episomal mutant kg of CQ over three days and 0.5 mg/kg PQ daily for 5 or 7 days or 0.25 pvdhfr are resistant to classic antifolates as mutant P. falciparum and are mg/kg PQ daily for 14 days. Four out of 540 patients had a recurrence of notably more resistant to a novel antifolate drug WR99210. Our results P. vivax parasitemia within 35 days of treatment, indicative of CQ failure. show that episomal expression of pvdhfr alleles in P. falciparum, a closely The treatment failures were detected on days 28 (twice), 30 and 32, in related biological system, help identify the role and importance of specific patients between 4 and 11 years old, with an average geometric mean mutations against current and new antifolate treatments and provide a parasitemia of 382 parasite/μL on D-F. Only one of the four had a total CQ system for the quick assessment of the potency of new antifolate drugs level of 95 ng/mL whole blood at the time of reappearance of parasitemia; against P. vivax with different dfhr alleles. Whereas, integrated expression the other three had sub-efficacious levels of total CQ in their blood on D-F. systems would help to provide a more stable and reproducible assessment. Pvmdr1 gene sequencing and neutral microsatellite markers analysis were performed to distinguish between recrudescence and reinfection. Two out of four had the same genotype at all loci between D-0 and D-F, including the patient with the blood CQ level of 95 ng/mL. No obvious mutation linked to CQR was seen by direct sequencing of the entire ORFs of Pvmdr1 and Pvcrt. www.astmh.org 151 530 532 “yeAst optimiZed” Plamodium falciParum CRT long term persistence of Plasmodium falciParum (pfCRT) And mdr1 proteins (pfCRT): purificAtion, clones And potentiAl BiAs in meAsurement of drug reconstitution And putAtiVe drug Binding efficAcy in low trAnsmission AreAs perri g. pleeter, Linda Amoah, Jacqueline Lekostaj, Paul Roepe claribel murillo1, Diego F. Echeverry1, Lyda Osorio1, Sanjay 2 2 2 Georgetown University, Washington, DC, United States Menon , Shalini Nair , Tim Anderson 1 Nearly two decades ago, a genetic cross of chloroquine-resistant (CQR) Centro Internacional de Entranamiento e Investigaciones Médicas 2 and chloroquine-sensitive (CQS) malaria parasites yielded progeny whose (CIDEIM), Cali, Colombia, Southwest Foundation for Biomedical Research, chloroquine-resistance phenotype was dependent on a single locus. It is San Antonio, TX, United States now known that mutations in a single gene within this locus (pfcrt) are Examination of parasite genotypes before and after treatment is frequently primarily responsible for the CQR phenotype. However, mutation and / or used to correct estimates of antimalarial drug efficacy. However, when increased expression of P. falciparum multidrug resistance (PfMDR) protein identical clones are common within parasite populations this may result may subtly alter the degree of resistance to some quinolines, and perhaps in overestimation of failure rates because patients may be reinfected other compounds. Multiple isoforms of PfCRT and PfMDR1 protein have with identical clones. We investigated the population structure of malaria been successfully overexpressed in Pichia Pastoris using
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