Role of Pfmdr1 in in Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine and

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1 AAC Accepts, published online ahead of print on 8 September 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved Role of Pfmdr1 in in Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine and Dihydroartemisinin Running title: Role of Pfmdr1 in antimalarial susceptibility Nathalie Wurtz, 1,2 Bécaye Fall, 3 Aurélie Pascual, 1,2,4 Mansour Fall, 5 Eric Baret, 1,2,3,4 Cheikhou Camara, 6 Aminata Nakoulima, 7 Bakary Diatta, 5 Khadidiatou Ba Fall, 8 Pape Saliou Mbaye, 9 Yaya Diémé, 3 Raymond Bercion, 3,10 Boubacar Wade, 11 Bruno Pradines, 1,2,3,4,12 * Unité de Parasitologie, Département d Infectiologie de Terrain, Institut de Recherche Biomédicale des Armées, Marseille, France 1 ; Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France 2 ; Laboratoire d Etude de la Chimiosensibilité du Paludisme, Fédération des Laboratoires, Hôpital Principal de Dakar, Dakar, Sénégal 3 ; Centre National de Référence du Paludisme, Marseille, France 4 ; Service de réanimation Médicale, Hôpital Principal de Dakar, Dakar, Sénégal 5 ; Service des Urgences, Hôpital Principal de Dakar, Dakar, Sénégal 6 ; Service de Pédiatrie, Hôpital Principal de Dakar, Dakar, Sénégal 7 ; Service de Pathologie Infectieuse, Hôpital Principal de Dakar, Dakar, Sénégal 8 ; Département de Médecine Interne et Spécialités Médicales de Pathologie Tropicale, Hôpital Principal de Dakar, Dakar, Sénégal 9 ; Laboratoire de Biologie, Institut Pasteur de Dakar, Dakar, Sénégal 10 ; Chefferie, Hôpital Principal de Dakar, Dakar, Sénégal 11 ; Unité de Parasitologie et d Entomologie, Département de Microbiologie, Institut de Recherche Biomédicale des Armées, Brétigny sur Orge, France 12

2 26 27 Keywords: malaria, Plasmodium falciparum, in vitro, ex vivo, resistance, antimalarial drug, molecular marker, Pfmdr1, Pfcrt 28

3 Abstract The involvement of Pfmdr1 polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ) and dihydroartemisinin (DHA) in174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (pvalue = ), LMF (p-value = ), DHA (p-value = ) and MQ (p-value = ). The N86Y mutation was not associated with CQ (p-value = 0.214) or QN (p-value = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (p-values = for CQ; for MDAQ; for LMF; for DHA; for QN and for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ p-value = ), LMF (p-value = ) and MQ (p-value = ). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (p-value < 00001), LMF (p-value < 00001), MQ (p-value < 00001) and QN (p-value = ) in wild type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to CQ (p-value = ) in Pfcrt 76T CQ-resistant parasites. 50

4 Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine (CQ) and other anti-malarial drugs. In response to increasing CQ resistance Senegal switched, in 2004, to sulphadoxine-pyrimethamine with amodiaquine as the first-line therapy. In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. The combination sulphadoxine-pyrimethamine and amodiaquine treatment was changed to artemether-lumefantrine and artesunate-amodiaquine. Since 2006, more than 1.5 million ACT-based treatments have been administered in Senegal (1). In 2006, the Senegalese National Malaria Control Programme also recommended testing for all suspected cases of malaria with the P. falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT). Since this time, ACT use has been restricted to confirmed malaria cases to reduce drug pressure. In 2009, 184,170 doses of ACT were dispensed in Senegal (2). The ability to maximize the efficacy and longevity of anti-malarial drugs for malaria control will depend critically on intensive research to identify in vitro markers along with ex vivo and in vivo surveillance programs. Furthermore, it is necessary to identify molecular markers that predict antimalarial resistance or decreased susceptibility so that active surveillance can monitor temporal trends in parasite susceptibility. Although the molecular mechanisms underlying multidrug resistance by P. falciparum remain largely unknown, polymorphisms within the Pfmdr1 (Plasmodium falciparum multidrug resistance 1) gene, which encodes a trans-membrane homolog of the PGH1 protein, have been implicated. Field work has shown that the predictive value of CQ resistance and point mutations in the Pfmdr1 sequence resulting in amino acid changes varies depending on the geographic area (3, 4). Five point mutations have been described: N86Y, Y184F, S1034C, N1042D and D1246Y. Point mutations, most notably 86Y, have been associated with a decrease in CQ susceptibility (5). However, in some epidemiological studies, the number of CQ-susceptible samples is too

5 limited to provide a statistically meaningful analysis (4, 6). Using precautions, no relationship or only weak relationships have been established between CQ resistance and mutations in Pfmdr1 in P. falciparum (3). However, the risk of therapeutic failure with CQ is greater for patients harboring the 86Y mutation, with an Odds ratio (OR) of 2.2 (95%CI: ) for a 14-day follow-up and 1.8 (95%CI: ) for a 28-day follow-up (7). In addition, the risk of therapeutic failure with amodiaquine is greater for patients harboring the 86Y mutation, with an OR of 5.4 (95%CI: , meta-analysis of six studies) (7); this mutation increases the risk of failure with amodiaquine plus sulfadoxine-pyrimethamine by 7.9 (8). It has been shown through heterologous expression that Pfmdr1 mutations at codons 1034 and 1042 abolish or reduce the level of resistance to mefloquine (MQ) (9). Moreover, transfection with a wild-type Pfmdr1 allele at codons 1034, 1042 and 1246 confers MQ resistance to susceptible parasites (10). However, mutations at Pfmdr1 codons 1034, 1042 and 1246 in P. falciparum isolates are not sufficient to explain the variations in MQ susceptibility (11). Analyses of P. falciparum isolates show an association between mutation at codon 86 and an increase in susceptibility to MQ, halofantrine or artemisinin derivatives (12-14). The selection of Pfmdr1 polymorphisms by the combination of artemether and lumefantrine has recently been observed (15). Mutation in Pfmdr1 has also been associated with decreased susceptibility to artemether and lumefantrine (LMF) drugs separately (16-18). However, mutations at Pfmdr1 gene seem to be not sufficient to explain in vitro resistance to antimalarial drugs and additional gene mutations are necessary. The Pfcrt gene was first identified in 2000 (19). To date, at least 20 point mutations have been described (19-21), but only one is the reference mutation (K76T), which is a marker of the CQ-resistant phenotype. This mutation is often associated with other mutations in the Pfcrt gene, whose role is not yet defined. The odds ratio (OR) for CQ failure associated with

6 the K76T mutation was 2.1 (95% confidence interval: , meta-analysis of 13 studies) for a 14-day follow-up and 7.2 (95%CI: , meta-analysis of 12 studies) for a 28-day follow-up (7). However, the existence of CQ-susceptible strains associated with the K76T mutation suggests that other genes could be involved in the resistance to CQ. The 76T mutation is necessary, but not sufficient, for influencing CQ susceptibility (22). The aim of this study was first to evaluate the association between polymorphisms in Pfmdr1 and in vitro responses to CQ, MQ, LMF, quinine (QN), monodesethylamodiaquine (the metabolite of amodiaquine) (MDAQ) and dihydroartemisinin (DHA) in Senegal. Then, the association between Pfcrt K76T and Pfmdr1 mutations and in vitro susceptibility to the six drugs was investigated. MATERIALS AND METHODS Patients and sample collection. P. falciparum isolates from patients with malaria who live in Dakar (>80%) and the surrounding area and did not travel during the previous month were obtained during the rainy seasons of October 2009 to January 2010 (172 patients, 42% female) and August 2010 to January 2011 (129 patients, 38% female). The patients with malaria were recruited at the Hôpital Principal de Dakar, a military hospital, within the context of an evaluation of ex vivo malaria susceptibility to anti-malarial drugs in Dakar (23, 24). Venous blood samples were collected in Vacutainer ACD tubes (Becton Dickinson, Rutherford, NJ, USA) prior to patient treatment. Of the 301 patients, 54% were recruited from the emergency department during each of the 2 seasons; other patients were recruited from the intensive care unit (18% during October 2009 January 2010 and 20% during August 2010 January 2011), pediatric department (9% and 5%) and other units (19% and 21%). No significant differences were found between the 2 seasons for parasitemia (p-value = 0.160),

7 sex ratio (p-value = 0.446), area of residence (p-value = 0.651) or hospital admission status (p-value = 0.567). Information on antimalarial treatment prior to admission was not available. Informed verbal consent from the patients and/or their parents/guardians was obtained before blood collection; the study was approved by the ethical committee of the Hôpital Principal de Dakar. An assessment of P. falciparum susceptibility to anti-malarial drugs was performed using the same venous blood sample used for this diagnostic analysis. Thin blood smears were stained using a RAL kit (Réactifs RAL, Paris, France) and examined to determine the P. falciparum density and confirm monoinfection. Parasitized erythrocytes were washed three times with RPMI 1640 medium (Invitrogen, Paisley, UK) buffered with 25 mm HEPES and 25 mm NaHCO 3. If parasitaemia exceeded 0.5%, the infected erythrocytes were diluted to 0.5% with uninfected erythrocytes (human blood type A+) and re-suspended in RPMI 1640 medium supplemented with 10% human serum (Abcys S.A. Paris, France), for a final hematocrit of 1.5%. Drugs. CQ, QN and DHA were purchased from Sigma (St. Louis, MO, USA). MDAQ was obtained from the World Health Organization (Geneva, Switzerland), MQ was purchased from Roche (Paris, France), and LMF was purchased from Novartis Pharma (Basel, Switzerland). QN, MDAQ, MQ and DHA were first dissolved in methanol and then diluted in water to final concentrations ranging from 5 nm to 3200 nm for QN, 1.56 nm to 1000 nm for MDAQ, 3.2 nm to 400 nm for MQ and 0.1 nm to 100 nm for DHA. CQ was re-suspended and diluted in water to final concentrations ranging from 5 nm to 3200 nm. LMF was resuspended and diluted in ethanol to obtain final concentrations ranging from 0.5 nm to 310 nm. Batches of plates were tested and validated using the chloroquine-susceptible strain 3D7 (isolated in West Africa; obtained from MR4, VA, USA) and the chloroquine-resistant W2 (isolated in Indochina; obtained from MR4, VA, USA) in three to six independent

8 experiments using the conditions described below. The two strains were synchronized twice with sorbitol before use (25), and clonality was verified every 15 days through PCR genotyping of the polymorphic genetic markers msp1 and msp2 and microsatellite loci (26, 27). Additionally, clonality was verified each year by an independent laboratory from the Worldwide Anti-malarial Resistance Network (WWARN). Ex vivo assay. For in vitro isotopic microtests, 200 µl of parasitized red blood cells (final parasitemia, 0.5%; final hematocrit, 1.5%) was aliquoted into 96-well plates pre-dosed with anti-malarial drugs. The plates were incubated in a sealed bag for 42 h at 37 C with atmospheric generators for capnophilic bacteria (Genbag CO2 ) at 5% CO 2 and 15% O 2 (BioMérieux; Marcy l Etoile, France) (28). After thawing the plates, the hemolysed cultures were homogenized by vortexing the plates. Both the success of the drug susceptibility assay and the appropriate volume of hemolysed culture to use for each assay were determined for each clinical isolate during a preliminary pldh ELISA. Both the pre-test and subsequent experimental ELISAs were performed using a commercial kit (ELISA-Malaria antigen test, ref , DiaMed AG, Cressier s/morat, Switzerland), as previously described (29). The optical density (OD) of each sample was measured with a spectrophotometer (Multiskan EX, Thermo Scientific, Vantaa, Finland). The concentration at which the drugs were able to inhibit 50% of parasite growth (IC 50 ) was calculated using the inhibitory sigmoid Emax model with an estimation of the IC 50 through non-linear regression using a standard function of the R software (ICEstimator version 1.2) (30). The IC 50 values were validated only if the OD ratio (OD at concentration 0 / OD at concentration max) was higher than 1.8 and the confidence interval ratio (upper 95% confidence interval of the IC 50 estimation/lower 95% confidence interval of the IC 50 estimation) was lower than 2.0 (30).

9 Nucleic acid extraction. The total genomic DNA of each strain was isolated using the QIAampW DNA Mini kit according to the manufacturer s recommendations (Qiagen, Germany). Pfmdr1 single-nucleotide polymorphisms. Two primer pairs were used to amplify Pfmdr1 fragments carrying the five key codons (31). A 590-base pair fragment was amplified with the primer pair sense 50-AGA GAA AAA AGA TGG TAA CCT CAG-30 and antisense 50-ACC ACA AAC ATA AAT TAA CGG-30 to determine the sequences of codons 86 and 184 (MDR1-1), and a second fragment (968 base pairs) was amplified with the primer pair sense 50-CAG GAA GCA TTTTAT AAT ATG CAT-30 and antisense 50-CGT TTAACA TCT TCC AAT GTT GCA-30 to determine the sequences of codons 1034, 1042 and 1246 (MDR1-2) (31). The reaction mixture consisted of approximately 2.5 μl of genomic DNA, 0.5 μm of forward and reverse primers, 2.5 μl of 10X reaction buffer (Eurogentec), 2.5mM MgCl 2, 200 μm deoxynucleoside triphosphate mixture (dgtp, datp, dttp and dctp) (Euromedex, Souffelweyersheim, France) and 1 U of Red Gold StarW DNA polymerase (Eurogentec) in a final volume of 25 μl. The thermal cycler (T3 Biometra) was programmed for MDR1-1 as follows: an initial step at 94 C for 5 min; 40cycles of 94 C for 30 sec, 52 C for 30 sec and 72 C for1 min and a final 10-min extension step at 72 C. ForMDR1-2, the parameters were as follows: an initial step at 94 C for 5 min; 40 cycles of 94 C for 30 sec, 56 C for1 min and 72 C for 1 min 30 sec and a final 10-minextension step at 72 C. The PCR products were separated using a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Amplicons were purified using the QIAquick96 PCR BioRobot Kit and an automated protocol on the BioRobot 8000 workstation (Qiagen, Courtaboeuf, France). The purified fragments were sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using the primers described above. The sequencing reaction products were purified using the Big Dye XTerminatorW Purification Kit (Applied Biosystems) in

10 accordance with the manufacturer s instructions. The purified products were sequenced using an ABI Prism 3100 analyzer (AppliedBiosystems), and the sequences were analyzed using VectorNTI advance (TM) software (version 11, Invitrogen, Cergy Pontoise, France). Pfcrt single-nucleotide polymorphisms (SNPs). A 546-nucleotide fragment of the Pfcrt gene (containing codon 76) was amplified by PCR using CRTP1-sense 5 -CCG TTA ATA ATA AAT ACA CGC AG-3 and CRTP1-antisense 5 -CGG ATG TTA CAA AAC TAT AGT TAC C-3 primers (32). The reaction mixture for PCR amplifications included 2.5 µl of genomic DNA, 2.5 µl of 10X reaction buffer (Eurogentec), 0.5 µm of each primer, 200 µm of a deoxynucleoside triphosphate mixture (dgtp, datp, dttp and dctp) (Euromedex, Souffelweyersheim, France), 2.5 mm MgCl 2 and 1 unit of RedGoldStar DNA polymerase (Eurogentec) in a final volume of 25 µl. The thermal cycler (T3 Biometra, Archamps, France) was programmed as follows: an initial 94 C incubation for 5 min; 40 cycles of 94 C for 20 sec, 56 C for 20 sec and 60 C for 40 sec; and a final 5-min extension step at 60 C. The PCR products were loaded on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The PCR products were diluted 1:100 in distilled water, and 2.5 µl of the final dilution was used for the second PCR. This PCR amplified a 275 bp segment around the mutation using a common inner primer CRTP3-sense 5 -TGA CGA GCG TTA TAG AG-3 coupled with either CRTP4m-antisense 5 -GTT CTT TTA GCA AAA ATT G-3 (detects the 76T codon), or CRTP4w-antisense 5 -GTT CTT TTA GCA AAA ATT T-3 (detects the 76K codon) (20). The reaction mixture for the PCR amplifications included 2.5 µl of diluted PCR product, 2.5 µl of 10X reaction buffer (Eurogentec), 0.5 µm of each primer, 200 µm deoxynucleoside 221 triphosphate mixture, 1.5 mm MgCl 2 and 0.75 U of RedGoldStar DNA polymerase (Eurogentec) in a final volume of 25 µl. The PCR conditions were as follows: initiation at 94 C for 5 min; 15 cycles at 94 C for 20 sec, 48.5 C for 20 sec and 64 C for 40 sec; and a final 5 min extension step at 64 C. Purified genomic DNA from P. falciparum clones 3D7

11 (CQ susceptible) and W2 (CQ resistant) were used as positive controls, and water and human DNA were used as negative controls. The PCR products from the amplification reactions were evaluated by electrophoresis on 2% agarose gels Statistical analysis. The data were analyzed using R software (version ). Differences between the IC 50 values of the isolates and the Pfmdr1 and/or Pfcrt polymorphisms were compared using the Wilcoxon rank sum test, the Kruskall Wallis test and the Welch two-sample t-test. RESULTS Of the 301 isolates tested for Pfmdr1 polymorphisms, 174 were successfully evaluated by ex vivo testing. The average parameter estimates for the 6 antimalarial drugs used against the P. falciparum isolates are given in Table 1. The Pfmdr1 86Y mutation was identified in 14.9% of the samples (26 out of 174 isolates), and the 184F mutation was identified in 71.8% of the isolates (125 out of 174). No 1034C, 1042N or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (Wilcoxon rank sum test, p-value = ), LMF (p-value = ), DHA (p-value = ) and MQ (p-value = ). For the 86Y mutation isolates, the IC 50 values were significantly reduced for MDAQ (14.4 nm versus 30.5 nm, p-value = ), LMF (9.2 nm versus 25.0 nm, p-value = ), MQ (18.6 nm versus 42.0 nm, p-value < ) and DHA (2.1 nm versus 3.0, p-value = 0.049) (Table 2). The N86Y mutation was not associated with CQ (p-value = 0.214) or QN (p-value = 0.287) responses. The Pfmdr1 184F mutation was not associated with variable susceptibility responses to the 6 antimalarial drugs (Wilcoxon rank sum test, p-values = for CQ; for MDAQ; for LMF; for DHA; for QN and for MQ). There was no significant

12 difference in IC 50 between the isolates with Y184 or 184F (Welch two sample t-test, p-values from to 0.776, Table 2). The IC 50 values distribution for the four haplotypes (N86-Y184, N86-F184, 86Y-Y184 and 86Y-184F) was presented in Fig. 1. The 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (mean IC 50 = 3.8 nm, p-value = , Kruskall Wallis test), LMF (mean IC 50 = 5.7 nm, p-value = ) and MQ (mean IC 50 = 9.0 nm, p- value = ) (Table 2). Only CQ responses were significantly associated with Pfcrt 76T. The CQ IC 50 values were significantly increased in the group of isolates with the 76T mutation (53.4 nm versus nm, p-value = , Welch two sample t-test) (Table 3). The IC 50 values for the Pfcrt K76 Pfmdr1 86Y haplotype combination were significantly lower for MDAQ (p-value = , Kruskall Wallis test), LMF (p-value = ), MQ (p-value = ) and QN (p-value = ) (Table 3). The CQ IC 50 values were significantly higher for the Pfcrt 76T Pfmdr1 N86 haplotype combination (p-value = ) (Table 3). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (p-value < 00001), LMF (p-value < 00001), MQ (p-value < 00001) and QN (p-value = ) in wild type Pfcrt K76 parasites (Table 3). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to CQ (p-value = ) in Pfcrt 76T CQresistant parasites. The association between Pfmdr1 Y184F and Pfcrt K76T didn t significantly influence the IC 50 for CQ, MDAQ, LMF, QN, MQ or DHA (Table 4). DISCUSSION

13 Maximizing the efficacy and longevity of antimalarial drugs remains important and will critically depend on the pursuit of intensive research toward the identification of in vitro markers of resistance and the implementation of ex vivo and in vivo surveillance programs, such as those championed by the WorldWide Antimalarial Resistance Network (33, 34). Specifically, there is a need to identify molecular markers that effectively predict antimalarial drug resistance and enable the active surveillance of temporal trends in parasite susceptibility (35). The role of polymorphism in Pfmdr1 is still debated. However, single-nucleotide polymorphisms in Pfmdr1 have been shown to be associated with differential in vivo efficacy and in vitro P. falciparum to ACT antimalarial partner drugs, including amodiaquine (36, 37), mefloquine (10, 38), lumefantrine (39, 40) and artemisinin derivatives (13, 14, 38). Pfmdr1 mutations at codons 86, 1034, 1042 and 1246 have been associated with in vitro resistance to MQ and artesunate in genetically modified parasite lines (10, 38) and clinical isolates (18, 41). It has been shown through heterologous expression that Pfmdr1 mutations at codons 1034 and 1042 abolish or reduce the level of resistance to MQ (9). Moreover, transfection with a wild-type Pfmdr1 allele at codons 1034, 1042 and 1246 confers MQ resistance to susceptible parasites (10). However, mutations at codons 1034, 1042 and 1246 in P. falciparum Pfmdr1 isolates are not sufficient to explain variations in MQ susceptibility (11). Analyses of P. falciparum isolates revealed an association between mutation at codon 86 and an increase in susceptibility to MQ (12-14). However, as no mutation at codons 1034, 1042 and 1246 was found in the 174 P. falciparum isolates from Dakar, these polymorphisms cannot explain the variation in in vitro MQ susceptibility in Senegal. Nonetheless, we show that the 86Y mutation was significantly associated with increased susceptibility to MQ (pvalue = ). These results are in accordance with previous studies in Asia (12, 42) and

14 in Benin (43) and are strengthened by field studies demonstrating MQ selection of the N86 allele in recurrent infections after treatment with artesunate plus MQ (44). We show that the N86 allele can predict in vitro decreased susceptibility to LMF, whereas the 86Y mutation was significantly associated with increased susceptibility to LMF (p-value = ). These results are in accordance with previous in vitro studies in Asia (45), in Kenya (17) and in Benin (43). Field studies in East Africa have also shown selection of the 86N allele in recurrent infections after treatment with artemether plus LMF (39, 40, 46, 47), which suggests that 86N could be a potential marker of LMF resistance in vivo. We show that the Pfmdr1 N86 allele can predict in vitro decreased susceptibility to MDAQ, the metabolite of amodiaquine; in contrast, the 86Y mutation was significantly associated with increased susceptibility to MDAQ (p-value = ). However, these data are in contrast to previous in vitro works. There was no difference in MDAQ IC 50 between the two haplotypes N86 and 86Y in isolates from Benin (43). In other works, mutant Pfmdr1 86Y allele showed increased MDAQ IC 50 in isolates from Nigeria (48). The Pfmdr1 86Y mutation has been shown to be associated with treatment failure after monotherapy with amodiaquine (36, 49) or after combination therapy with artesunate-amodiaquine (50). In a meta-analysis, the Pfmdr1 86Y mutation was found to be associated with amodiaquine failure, with an odds ratio of 5.4 (7). The pfmdr1 1246Y mutation has also been found to be associated with in vitro resistance to amodiaquine (51) and with recrudescent infection after treatment with amodiaquine or amodiaquine-artesunate (50, 49). Here, we show that the N86 allele can predict in vitro decreased susceptibility to DHA and that the 86Y mutation was significantly associated with increased susceptibility to DHA (pvalue = ). These results are in accordance with previous in vitro studies on parasite laboratory strains (13, 14, 38) or in clinical isolates (44). The pfmdr1 86Y mutation has been shown to be associated with in vivo resistance to ACT after combination therapy with

15 artesunate-amodiaquine [50] or artemether plus LMF (39, 40, 46, 47), which suggests that 86N could be a potential marker of artemisinin derivative resistance in vivo. However, it has been recently shown that this type of in vitro test is not adapted to detect resistance to artemisinin derivatives. A non-standard phenotypic test is currently used and recommended for evaluating ex vivo or in vitro susceptibility to artemisinin derivatives correlating to the clearance delay of parasites (52, 53). In addition, mutations in a new gene, K13-propeller, are associated with in vitro parasite survival rates, as determined by this new assay and in vivo parasite clearance rates (54). The N86Y mutation was not associated with QN (p-value = 0.287). The involvement of the N86Y in QN resistance is still debated: in some studies, the mutation 86Y is associated with increased susceptibility (42, 55) though not in other studies (56). The 1042D mutation has been reported to induce QN resistance (38). The N86Y mutation was not associated with CQ responses (p-value = 0.214) in Senegalese P. falciparum isolates. These results are in accordance with previous in Kenya (17) or in Nigeria (57). However, the 86Y mutation has been associated with a decrease in CQ susceptibility in some works (5). However, in some of these epidemiological studies, the number of CQ-susceptible samples is too limited to provide a statistically meaningful analysis (4, 6). Using precautions, no relationship or only weak relationships are established between CQ resistance and mutations in Pfmdr1 in P. falciparum (3). The 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs tested (p-values = for CQ; for MDAQ; for LMF; for DHA; for QN and for MQ). Thus, the significance of the 184F mutation remains less well understood. Indeed, no clear association between the 184F mutation and MQ failure has been established. A study showed that Asian isolates with a single 184F mutation exhibited increased resistance to MQ and AS (41). A study from Cambodia

16 demonstrated that isolates with a single 184F mutation had significant increased IC 50 values for MQ (58). Similar results were obtained in studies from Tanzania, Uganda and Nigeria, showing the selection of 86N, 184F and 1246D Pfmdr1 alleles in recurrent infections after treatment with artemether plus LMF (39, 47, 59, 60). In addition, the 184F mutation was shown to be associated with in vitro resistance to QN (55). In our study, the 184F mutation was not associated with QN susceptibility responses (p-value = 0.084), though the p-value was not very distant from the limit of significance. The mean IC 50 was nm for the Y184 allele and nm for the 184F allele (p-value = 0.057). Evaluation of the Pfmdr1 86 plus 184 haplotype showed a significant increased in vitro susceptibility to MDAQ, LMF and MQ in parasites with 86Y plus Y184. A similar association was seen for LMF and MQ in previous studies in Benin (43). Similarly, we showed a decreased susceptibility to LMF and MQ in parasites with N86 plus 184F and N86 plus Y184. The parasites with Pfmdr1 86Y plus Y184 and 86Y plus 184F showed a nonsignificant increase of CQ susceptibility (p-value = ). However, univariate analyse showed that there was a significant association between CQ treatment failure and the presence of Pfmdr1 86Y plus Y184 in Nigeria (57). The IC 50 values for the Pfcrt K76 Pfmdr1 86Y haplotype combination were significantly lower for MDAQ (p-value = ), LMF (p-value = ), MQ (p-value = ) and QN (p-value = ). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (p-value < 00001), LMF (p-value < 00001), MQ (p-value < 00001) and QN (p-value = ) in wild type Pfcrt K76 parasites from Senegal. The LMF IC 50 was significantly lower for Pfmdr1 86Y - Pfcrt K76 (mean IC 50 = 6.0) and Pfmdr1 86Y - Pfcrt 76T (mean IC 50 = 14.8). These results are in concordance with previous data from Kenya (17). Artemether plus LMF is largely used in Senegal and the Pfmdr1 N86 is certainly selected in recurrent infection after treatment with artemether plus LMF like in East Africa

17 (34, 35, 46, 47). For MQ, the 86Y-K76 haplotype showed lower IC 50 (mean IC 50 = 12.3 nm) in concordance with previous work in Western-Kenya (61). For MDAQ, the 86Y-K76 haplotype showed lower IC 50 (mean IC 50 = 7.1 nm). In Western-Kenya, the 86Y-K76 haplotype showed the highest IC 50 (61). In Southern Soudan, more than 80% of the isolates MDAQ-susceptible carried the wild type Pfcrt K76 - Pfmdr1 N86 genotype (62). The 86Y- 76T haplotype was found to be associated with recrudescence infection after treatment with amodiaquine (49, 63). The CQ IC 50 values were significantly higher for the Pfcrt 76T - Pfmdr1 N86 haplotype combination (p-value = ). The N86-K76 and 86Y-K76 haplotypes showed the lower CQ IC 50 in concordance with previous work in Kenya (17, 61). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to CQ (p-value = ) in Pfcrt 76T CQ-resistant parasites from Senegal while the 86Y-76T haplotype was shown to be associated with in vitro CQ resistance in isolates from Nigeria (57). The Y184-76T and 184F-76T haplotypes showed the highest CQ IC 50 but without any significant difference between the two haplotypes in Dakar, in concordance with previous data from Papua New Guinea (64). The association between Pfmdr1 Y184F and Pfcrt K76T didn t significantly influence the IC 50 for CQ, MDAQ, LMF, QN, MQ or DHA. Another molecular marker of antimalarial drug susceptibility is the amplification of Pfmdr1; an increase in the copy number of Pfmdr1 is associated with clinical failure and with in vitro resistance to aryl-amino-alcohols, particularly MQ, QN and LMF (65, 66). Pfmdr1 amplification has also been demonstrated to decrease the susceptibility to artemisinin derivatives in the field as well as in vitro (66, 67). However, the role of the amplification of Pfmdr1 in P. falciparum resistance to antimalarial drugs in Africa is debated. We previously analyzed the number of Pfmdr1 copies in these 174 P. falciparum samples, and a significant association was only found for dihydroartemisinin (p = 0.003) (68). The odds ratio for

18 reduced in vitro susceptibility to dihydroartemisinin associated with 2 Pfmdr1 copies was 1.4. However, the number of isolates with duplicated Pfmdr1 copies was small (9 out 174 samples) In summary, we demonstrated that the 86Y mutation is significantly associated with increased susceptibility to MDAQ, LMF, MQ and DHA in P. falciparum parasites from Dakar and the Pfmdr1 86Y-Y184 haplotype significantly increases the in vitro susceptibility to MDAQ, LMF and MQ in parasites. The additional Pfmdr1 86Y mutation increases significantly the in vitro susceptibility to MDAQ, LMF, MQ and QN in wild type Pfcrt K76 parasites and to CQ in Pfcrt 76T CQ-resistant parasites from Senegal. ACKNOWLEDGEMENTS The authors thank Ndeye Fatou Diop and Maurice Gomis for technical support and the staff of the clinical units. This study was supported by the Schéma directeur Paludisme, Etat Major des Armées Françaises (grant LR 607) and by the Ministère des Affaires Etrangères. REFERENCES 1. Ndiaye JLA, Faye B, Gueye A, Tine R, Ndiaye D, Tchania C, Ndiaye I, Barry A, Cisse B, Lameyre V, Gaye O Repeated treatment of recurrent uncomplicated Plasmodium falciparum malaria in Senegal with fixed-dose artesunate plus amodiaquine versus fixed-dose artemether plus lumefantrine: a randomized, open-label trial. Malar. J. 10: Thiam S, Thior M, Faye B, Diouf ML, Diouf MB, Diallo I, Fall FB, Ndiaye JL, Albertini A, Lee E, Jorgensen P, Gaye O, Bell D Major reduction in anti-malarial

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