pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.     

Advances in understanding the genetic basis of antimalarial drug resistance

The acquisition of drug resistance by Plasmodium falciparum has severely curtailed global efforts to control malaria. Our ability to define resistance has been greatly enhanced by recent advances in Plasmodium genetics and genomics. Sequencing and microarray studies have identified thousands of polymorphisms in the P. falciparum genome, and linkage disequilibrium analyses have exploited these to rapidly identify known and novel loci that influence parasite susceptibility to antimalarials such as chloroquine, quinine, and sulfadoxine-pyrimethamine. Genetic approaches have also been designed to predict determinants of in vivo resistance to more recent first-line antimalarials such as the artemisinins. Transfection methodologies have defined the role of determinants including pfcrt, pfmdr1, and dhfr. This knowledge can be leveraged to develop more efficient methods of surveillance and treatment.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.     

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies.     

Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum

Resistance to several anti-malarial drugs has been associated with polymorphisms within the P-glycoprotein homologue (Pgh-1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh-1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasite's digestive vacuole. How polymorphisms within this transporter mediate alter anti-malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh-1 transports vinblastine, an established substrate of mammalian MDR1, and the anti-malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh-1 alter the substrate specificity for the anti-malarial drugs. Wild-type Pgh-1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh-1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh-1. Our data are discussed in terms of the model that Pgh-1-mediates, in a variant-specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question.     

Genetic linkage analyses redefine the roles of PfCRT and PfMDR1 in drug accumulation and susceptibility in Plasmodium falciparum

Resistance to quinoline antimalarial drugs has emerged in different parts of the world and involves sets of discrete mutational changes in pfcrt and pfmdr1 in the human malaria parasite Plasmodium falciparum. To better understand how the different polymorphic haplotypes of pfmdr1 and pfcrt contribute to drug resistance, we have conducted a linkage analysis in the F1 progeny of a genetic cross where we assess both the susceptibility and the amount of accumulation of chloroquine, amodiaquine, quinine and quinidine. Our data show that the different pfcrt and pfmdr1 haplotypes confer drug-specific responses which, depending on the drug, may affect drug accumulation or susceptibility or both. These findings suggest that PfCRT and PfMDR1 are carriers of antimalarial drugs, but that the interaction with a drug interferes with the carriers' natural transport function such that they are now themselves targets of these drugs. How well a mutant PfCRT and PfMDR1 type copes with its competing transport functions is determined by its specific sets of amino acid substitutions.     

The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

ABSTRACT: BACKGROUND: Malaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance. METHODS: A total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods. RESULTS: Mutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported. CONCLUSION: The high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia.     

CRISPR‐Cas9‐modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine‐containing compounds but potentiates artemisinin‐based combination therapy partner drugs

Emerging resistance to first‐line antimalarial combination therapies threatens malaria treatment and the global elimination campaign. Improved therapeutic strategies are required to protect existing drugs and enhance treatment efficacy. We report that the piperazine‐containing compound ACT‐451840 exhibits single‐digit nanomolar inhibition of the Plasmodium falciparum asexual blood stages and transmissible gametocyte forms. Genome sequence analyses of in vitro‐derived ACT‐451840‐resistant parasites revealed single nucleotide polymorphisms in pfmdr1, which encodes a digestive vacuole membrane‐bound ATP‐binding cassette transporter known to alter P. falciparum susceptibility to multiple first‐line antimalarials. CRISPR‐Cas9 based gene editing confirmed that PfMDR1 point mutations mediated ACT‐451840 resistance. Resistant parasites demonstrated increased susceptibility to the clinical drugs lumefantrine, mefloquine, quinine and amodiaquine. Stage V gametocytes harboring Cas9‐introduced pfmdr1 mutations also acquired ACT‐451840 resistance. These findings reveal that PfMDR1 mutations can impart resistance to compounds active against asexual blood stages and mature gametocytes. Exploiting PfMDR1 resistance mechanisms provides new opportunities for developing disease‐relieving and transmission‐blocking antimalarials.     

Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.     

Molecular epidemiology of drug resistance markers of Plasmodium falciparum in Yunnan Province, China

ABSTRACT: BACKGROUND: The mutations in Plasmodium falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes were associated with anti-malaria drug resistance. The aim of this study was to investigate the prevalence of polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps and pfatp6 in Yunnan Province. Finger-prick blood samples were collected from malaria-positive patients from Yunnan Province in 2009-2010. Single-nucleotide polymorphisms (SNPs) in the resistance-related genes were analysed by various PCR-based methods. RESULTS: A total of 108 blood samples were collected. Although chloroquine has not been used to treat falciparum malaria for nearly 30 years, 95.3% of the parasites still carried the pfcrt K76T mutation, whereas the majority of isolates displayed the wild-type pfmdr1 N86 and D1246 sequences. The molecular level of sulphadoxine-pyrimethamine resistance in P. falciparum was high. The most prevalent mutation was pfdhfr C59R (95.9%), whereas the frequencies of the quadruple, triple and double mutants were 22.7% (N51I/C59R/S108N/I164L), 51.5% (N51I/C59R/S108N, N51I/C59R/I164L and C59R/S108N/ I164L) and 21.6% (N51I/ C59R, C59R/S108N and C59R/I164L), respectively. A437G (n=77) and K540E (n=71) were the most prevalent mutations in pfdhps, and 52.7% of the samples were double mutants, among which A437G/K540E was the most common double mutation (37/49). Quadruple mutants were found in 28.0% (26/93) of samples. A total of 8.6% of isolates (8/93) carried the S436A/A437G/A581G triple mutation. No mutations were found in pfatp6 codons 623 or 769, but another two mutations (N683K and R756K) were found in 4.6% (3/97) and 9.2% (6/97) of parasite isolates, respectively. CONCLUSIONS: This study identified a high frequency of mutations in pfcrt, pfdhfr and pfdhps associated with CQ and SP resistance in P. falciparum and no mutations linked to artemisinin resistance (pfatp6). Molecular epidemiology should be included in routine surveillance protocols and used to provide complementary information to assess the appropriateness of the current national anti-malarial drug policy.     

Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum remains controversial. By investigating the kinetics of chloroquine accumulation under varying-trans conditions, we recently presented evidence for a saturable and energy-dependent chloroquine efflux system present in chloroquine resistant P. falciparum strains. Here, we further characterize the putative chloroquine efflux system by investigating its substrate specificity using a broad range of different antimalarial drugs. Our data show that preloading cells with amodiaquine, primaquine, quinacrine, quinine, and quinidine stimulates labeled chloroquine accumulation under varying-trans conditions, while mefloquine, halofantrine, artemisinin, and pyrimethamine do not induce this effect. In the reverse of the varying-trans procedure, we show that preloaded cold chloroquine can stimulate quinine accumulation. On the basis of these findings, we propose that the putative chloroquine efflux system is capable of transporting, in addition to chloroquine, structurally related quinoline and methoxyacridine antimalarial drugs. Verapamil and the calcium/calmodulin antagonist W7 abrogate stimulated chloroquine accumulation and energy-dependent chloroquine extrusion. Our data are consistent with a substrate specific and inhibitible drug efflux system being present in chloroquine resistant P. falciparum strains.     

Mutations in transmembrane domains 1, 4 and 9 of the Plasmodium falciparum chloroquine resistance transporter alter susceptibility to chloroquine, quinine and quinidine

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV.     

Association of molecular markers in Plasmodium falciparum crt and mdr1 with in vitro chloroquine resistance: A Philippine study

Specific mutations in the pfcrt and pfmdr1 genes have been reported to be associated with chloroquine-resistant falciparum malaria parasites worldwide. These genetic markers are considered to be useful tools for the elucidation of several aspects of the epidemiology of drug resistant malaria. In this study, Plasmodium falciparum isolates from three distinct areas of the Philippines were analyzed for drug-resistance-associated genetic mutations, and their association with the in vitro chloroquine (CQ) response. Two novel pfcrt 72–76 allelic types, CVMDT and SVMDT, were detected. The frequency of the pfcrt K76T mutation in the isolates that were successfully tested for in vitro CQ susceptibility was found to be 100% in Kalinga, 80% in Palawan, and 87% in Mindanao. The frequency of the pfmdr1 N86Y mutation was 39% in Kalinga, 35% in Palawan, and 93% in Mindanao isolates. No mutations were found at positions 1042 and 1246 of pfmdr1. However, there were no significant associations found between polymorphisms in these genes and in vitro CQ susceptibility. The results of this study indicate that mutations in pfcrt and pfmdr1 are not predictive of in vitro CQ resistance in Philippine isolates and may therefore not be suitable as molecular markers for surveillance.     

In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast as the target of azithromycin

Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.     

Sequential treatment with quinine and mefloquine or quinine and pyrimethamine-sulfadoxine for falciparum malaria.

Patients with falciparum malaria were studied in Thailand, an area of known chloroquine resistance. The patients were unselected and some had severe malaria, and they were randomly assigned to one of two sequential regimes. A short course of quinine (average 4 doses, equivalent to 2 g base) followed by a single dose of pyrimethamine-sulfadoxine (Fansidar) cured 92% of patients (36 out of 39), while a short course of quinine followed by a single 1-5-dose of mefloquine cured all of the 35 patients who could be followed up. Gastrointestinal side effects were minimal if at least 12 hours elapsed between the last dose of quinine and the mefloquine. Sequential quinine and mefloquine is the most effective treatment for patients with chloroquine-resistant falciparum malaria, including those with severe or complicated disease. Mefloquine, however, is not commercially available, and the similar regimen using Fansidar is almost as effective.     

In vitro sensitivity of Plasmodium falciparum from China-Myanmar border area to major ACT drugs and polymorphisms in potential target genes

Drug resistance has always been one of the most important impediments to global malaria control. Artemisinin resistance has recently been confirmed in the Greater Mekong Subregion (GMS) and efforts for surveillance and containment are intensified. To determine potential mechanisms of artemisinin resistance and monitor the emergence and spread of resistance in other regions of the GMS, we investigated the in vitro sensitivity of 51 culture-adapted parasite isolates from the China-Myanmar border area to four drugs. The 50% inhibitory concentrations (IC₅₀s) of dihydroartemisinin, mefloquine and lumefantrine were clustered in a relatively narrow, 3- to 6-fold range, whereas the IC₅₀ range of artesunate was 12-fold. We assessed the polymorphisms of candidate resistance genes pfcrt, pfmdr1, pfATP6, pfmdr6 and pfMT (a putative metabolite/drug transporter). The K76T mutation in pfcrt reached fixation in the study parasite population, whereas point mutations in pfmdr1 and pfATP6 had low levels of prevalence. In addition, pfmdr1 gene amplification was not detected. None of the mutations in pfmdr1 and pfATP6 was associated significantly with in vitro sensitivity to artemisinin derivatives. The ABC transporter gene pfmdr6 harbored two point mutations, two indels, and number variations in three simple repeats. Only the length variation in a microsatellite repeat appeared associated with altered sensitivity to dihydroartemisinin. The PfMT gene had two point mutations and one codon deletion; the I30N and N496– both reached high levels of prevalence. However, none of the SNPs or haplotypes in PfMT were correlated significantly with resistance to the four tested drugs. Compared with other parasite populations from the GMS, our studies revealed drastically different genotype and drug sensitivity profiles in parasites from the China-Myanmar border area, where artemisinins have been deployed extensively for over 30 years.     

Multiple transporters associated with malaria parasite responses to chloroquine and quinine

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.