We describe here the results of an analysis of Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene copy number from 440 field isolates from Papua New Guinea. No multiple copies of the gene were found, which corresponds to the lack of usage of mefloquine. These data extend regional knowledge about the distribution of multidrug-resistant P. falciparu

Beck, Hans-Peter

Borrmann, Steffen

Genton, Blaise

Hodel, Eva Maria

Marfurt, Jutta

Müller, Dania

Müller, Ivo

Reeder, John C.

Rippert, Anja

Siba, Peter

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Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 1151—1153avai lab le at www.sc iencedi rec t .comjourna l homepage: www.e lsev ierhea l th .com/ journa ls / t rs tSHORT COMMUNICATIONLack of multiple copies of pfmdr1 genein Papua New GuineaEva Maria Hodela, Jutta Marfurta,1, Dania Mu¨llera, Anja Rippertb,Steffen Borrmannb, Ivo Mu¨llerc, John C. Reederc,2, Peter Sibac,Blaise Gentona, Hans-Peter Becka,∗a Swiss Tropical Institute, Socinstrasse 57, P.O. Box 4002 Basle, Switzerlandb Institute of Hygiene, University of Heidelberg School of Medicine, Im Neuenheimer Feld 324, 69120 Heidelberg, Germanyc Papua New Guinea Institute of Medical Research, P.O. Box 60, Goroka, EHP 411, Papua New GuineaReceived 4 April 2008; received in revised form 22 May 2008; accepted 22 May 2008Available online 2 July 2008KEYWORDSMalaria;Plasmodiumfalciparum;Summary We describe here the results of an analysis of Plasmodium falciparum multidrugresistance protein 1 (pfmdr1) gene copy number from 440 ﬁeld isolates from Papua New Guinea.No multiple copies of the gene were found, which corresponds to the lack of usage of meﬂo-quine. These data extend regional knowledge about the distribution of multidrug-resistantP. falciparum.Drug resistance;Gene ampliﬁcation; © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rightsptrpfmdr1;Papua New Guineareserved.1. IntroductionRecent in vitro and in vivo studies showed an associa-tion between artesunate—meﬂoquine therapy failure andgenetic changes in Plasmodium falciparum multidrug resis-tance protein 1 (pfmdr1) gene (Price et al., 2004; Sidhuet al., 2006). These ﬁndings underline the contribution of∗ Corresponding author. Tel.: +41 61 284 81 16;fax: +41 61 271 86 54.E-mail address: hans-peter.beck@unibas.ch (H.-P. Beck).1 Current address: Menzies School of Health Research, P.O. Box41096, Casuarina, Northern Territory 0811, Australia.2 Current address: Burnet Institute for Medical Research and Pub-lic Health, G.P.O. Box 2284, Melbourne, Victoria 3001, Australia.aodpapfgAc(h0035-9203/$ — see front matter © 2008 Royal Society of Tropical Medicindoi:10.1016/j.trstmh.2008.05.017fmdr1 copy number to the susceptibility of P. falciparumo antimalarial drugs. Most malaria-endemic countries haveecently adopted artemisinin combination therapies (ACTs)s ﬁrst-line therapy for P. falciparum infections becausef increasing resistance to all currently used antimalarialrugs.As the world’s most multidrug-resistant P. falciparumarasites are found along the borders of Thailand and arelso emerging elsewhere in Southeast Asia, many studies onfmdr1 gene copy number have been performed on samplesrom Southeast Asia. Studies in Africa indicate that pfmdr1ene copy number is rare in the ﬁeld outside Southeastsia (Ursing et al., 2006). However, no research has beenonducted on pfmdr1 copy number in Papua New GuineaPNG), where drug pressure of meﬂoquine on P. falciparumas been minimal so far. Until recently, standard ﬁrst-linee and Hygiene. Published by Elsevier Ltd. All rights reserved.1tadnwm2PpdSapl(cWtacp2wtCpsvaf17Hsrsuoespu3OﬁtosPﬁT1i4TmctPasnpfopqippnsnspPhpAIEHtrtAfFPCEGRC152reatment in PNG for malaria consisted of chloroquine ormodiaquine plus sulfadoxine—pyrimethamine. In order toescribe the baseline situation, we determined pfmdr1 copyumbers in 440 ﬁeld isolates from PNG. With this analysise extend the knowledge about the global distribution ofultidrug-resistant P. falciparum.. Materials and methodsre-treatment ﬁngerprick samples were collected from 440atients within several in vivo drug efﬁcacy studies con-ucted between 2003 and 2005 in three health centres inimbu, East Sepik and Madang Province in PNG (Marfurt etl., 2007).The pfmdr1 gene copy number was assessed as describedreviously (Price et al., 2004). The assays were run in trip-icate on an Applied Biosystems 7500 real-time PCR systemApplied Biosystems, Rotkreuz, Switzerland) and every runontained two calibrator DNA samples from clones 3D7 and2-mef, having pfmdr1 copy numbers of 1 and 3, respec-ively. The Ct threshold was set manually at 0.05 for pfmdr1nd at 0.03 for -tubulin, respectively, and the baseline wasalculated automatically. Results were analysed by the com-arative Ct method described previously (Price et al.,004). Assays were repeated if one of the following resultsas obtained: no Ct value for more than one sample in theriplicate; calibrator copy number /= 3; Ct spread >1.5;t values >35.Themultiplicity of infection was determined as describedreviously by fragment-sizing of P. falciparum merozoiteurface protein 2 (pfmsp2) using GeneMapper Software,3.7 (Applied Biosystems) (Falk et al., 2006). The primarynd nested PCR protocol was changed to 2min at 94 ◦Collowed by 25 cycles (30 s at 94 ◦C, 45 s at 50 ◦C andmin 30 s at 70 ◦C) with a ﬁnal elongation for 10min at0 ◦C each. Of the diluted nested PCR product (1:10 in2O), 2.5l were combined with 10l diluted ROX-500 sizetandard (1:40 in H2O; Applied Biosystems). Assays wereepeated if one of the following results was obtained: noizing data; pfmsp2 PCR artefacts; no pfmsp2 PCR prod-ct.Samples were excluded from the ﬁnal analysis if oner more of the following results were obtained after rep-tition of the assay: no Ct value for more than oneample in triplicate; Ct spread >1.5; Ct values >35;fmsp2 PCR artefacts; no sizing data; no pfmsp2 PCR prod-ct.. Resultsf the 440 samples analysed, 35 were excluded from thenal analysis (15 no Ct value for more than one sample inriplicate; one Ct value >35; three no Ct value for more thanne sample in triplicate and no pfmsp2 PCR product; six noizing data; three pfmsp2 PCR artefacts; seven no pfmsp2CR product). The remaining 405 samples included in thenal analysis showed only one copy of the pfmdr1 gene.he mean multiplicity of infection found in the patients was.56± 0.76 (mean± SD), with 240 patients showing singlenfections.FE.M. Hodel et al.. Discussionhe number of samples excluded from the ﬁnal analysisight be explained by the fact that ﬁeld samples sometimesontain very low levels of parasite DNA, thus leading to inde-erminable Ct values in the real-time PCR or missing pfmsp2CR products.Meﬂoquine use has not been signiﬁcant yet in PNG,nd the occurrence of only one gene copy of pfmdr1trengthens the assumption that meﬂoquine selects for copyumber increase. Studies that have tested different P. falci-arum isolates from PNG for meﬂoquine resistance showedull in vitro susceptibility (Hombhanje, 1998). By contrast,ther authors previously have shown a high frequency offmdr1 sequence polymorphisms, conﬁrming that chloro-uine selects for polymorphisms (Nagesha et al., 2001).The multiplicity of infections found is similar to resultsn other settings in PNG (Cortes et al., 2004). However, it isossible that a small percentage of the parasites in multi-le infections carry more than one copy of pfmdr1 and willot appear as multiple copies in the analysis due to lack oftatistical power. In addition, the fact that within the largeumber of single infections there was no gene ampliﬁcationupports the idea that pfmdr1 is still not ampliﬁed in PNG.In conclusion, we show that drug-resistance-associatedfmdr1 gene ampliﬁcations have not yet been selected inNG. Therefore meﬂoquine and artesunate might still beighly effective ﬁrst-line treatment options against P. falci-arum infections.uthors’ contributions: HPB conceived the study; JM, SB,M, JCR, PS and BG designed the study; JM collected samples;MH, DM and AR carried out the laboratory work; EMH andPB analysed the data; SB, IM, JCR, PS and BG interpretedhe data; EMH and HPB wrote the manuscript. All authorsead and approved the ﬁnal manuscript. HPB is guarantor ofhe paper.cknowledgements: The authors thank the study subjectsor their participation in the in vivo studies.unding: Provided by the Swiss National Science Foundationroject 320000-112479/1.onﬂicts of interest: None declared.thical approval: The study was approved by the Papua Newuinea Medical Research Advisory Committee.eferencesortes, A., Mellombo, M., Benet, A., Lorry, K., Rare, L., Reeder,J.C., 2004. Plasmodium falciparum: distribution of msp2 geno-types among symptomatic and asymptomatic individuals fromthe Wosera region of Papua New Guinea. Exp. Parasitol. 106,22—29.alk, N., Maire, N., Sama, W., Owusu-Agyei, S., Smith, T.,Beck, H.P., Felger, I., 2006. Comparison of PCR-RFLP andGenescan-based genotyping for analyzing infection dynamics ofPlasmodium falciparum. Am. J. Trop. Med. Hyg. 74, 944—950.aPSLack of multiple copies of pfmdr1 gene in Papua New GuineHombhanje, F.W., 1998. In vitro susceptibility of Plasmodium fal-ciparum to four antimalarial drugs in the Central Province ofPapua New Guinea. PNG Med. J. 41, 51—58.Marfurt, J., Mueller, I., Sie, A., Maku, P., Goroti, M., Reeder, J.C.,Beck, H.P., Genton, B., 2007. Low efﬁcacy of amodiaquine orchloroquine plus sulfadoxine—pyrimethamine against Plasmod-ium falciparum and P. vivax malaria in Papua New Guinea. Am.J. Trop. Med. Hyg. 77, 947—954.Nagesha, H.S., Din, S., Casey, G.J., Susanti, A.I., Fryauff, D.J.,Reeder, J.C., Cowman, A.F., 2001. Mutations in the pfmdr1, dhfrand dhps genes of Plasmodium falciparum are associated within-vivo drug resistance in West Papua, Indonesia. Trans. R. Soc.Trop. Med. Hyg. 95, 43—49.U1153rice, R.N., Uhlemann, A.C., Brockman, A., McGready, R., Ashley,E., Phaipun, L., Patel, R., Laing, K., Looareesuwan, S., White,N.J., Nosten, F., Krishna, S., 2004. Meﬂoquine resistance in Plas-modium falciparum and increased pfmdr1 gene copy number.Lancet 364, 438—447.idhu, A.B., Uhlemann, A.C., Valderramos, S.G., Valderramos,J.C., Krishna, S., Fidock, D.A., 2006. Decreasing pfmdr1 copynumber in Plasmodium falciparum malaria heightens suscepti-bility to meﬂoquine, lumefantrine, halofantrine, quinine, andartemisinin. J. Infect. Dis. 194, 528—535.rsing, J., Kofoed, P.E., Rombo, L., Gil, J.P., 2006. No pfmdr1 ampli-ﬁcations in samples from Guinea-Bissau and Liberia collectedbetween 1981 and 2004. J. Infect. Dis. 194, 716—718.

Lack of multiple copies of pfmdr1 gene in Papua New Guinea

Lack of multiple copies of pfmdr1 gene in Papua New Guinea

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