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.     

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.     

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.     

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.     

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.     

Decreased prevalence of Plasmodium falciparum resistance markers to amodiaquine despite its wide scale use as ACT partner drug in Zanzibar

ABSTRACT: BACKGROUND: Zanzibar has recently undergone a rapid decline in Plasmodium falciparum transmission following combined malaria control interventions with artemisinin-based combination therapy (ACT) and integrated vector control. Artesunate-amodiaquine (ASAQ) was implemented as first-line treatment for uncomplicated P. falciparum malaria in Zanzibar in 2003. Resistance to amodiaquine has been associated with the single nucleotide polymorphism (SNP) alleles pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y. An accumulation of these SNP alleles in the parasite population over time might threaten ASAQ efficacy. The aim of this study was to assess whether prolonged use of ASAQ as first-line anti-malarial treatment selects for P. falciparum SNPs associated with resistance to the ACT partner drug amodiaquine. METHODS: The individual as well as the combined SNP allele prevalence were compared in pretreatment blood samples from patients with uncomplicated P. falciparum malaria enrolled in clinical trials conducted just prior to the introduction of ASAQ in 2002-2003 (n = 208) and seven years after wide scale use of ASAQ in 2010 (n = 122). RESULTS: There was a statistically significant decrease of pfcrt 76T (96-63%), pfmdr1 86Y (75-52%), 184Y (83-72%), 1246Y (28-16%) and the most common haplotypes pfcrt/pfmdr1 TYYD (46-26%) and TYYY (17-8%), while an increase of pfcrt/pfmdr1 KNFD (0.4-14%) and KNYD (1-12%). CONCLUSIONS: This is the first observation of a decreased prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y in an African setting after several years of extensive ASAQ use as first-line treatment for uncomplicated malaria. This may support sustained efficacy of ASAQ on Zanzibar, although it was unexpected considering that all these SNPs have previously been associated with amodiaquine resistance. The underlying factors of these results are unclear. Genetic dilution by imported P. falciparum parasites from mainland Tanzania, a de-selection by artesunate per se and/or an associated fitness cost might represent contributing factors. More detailed studies on temporal trends of molecular markers associated with amodiaquine resistance are required to improve the understanding of this observation.     

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.     

Application of a multi-faceted approach for the assessment of treatment response in falciparum malaria: a study from Malaysian Borneo

Thirty-two patients reporting to the Lundu District Hospital, Sarawak, Malaysian Borneo, with uncomplicated falciparum malaria were recruited into a multifaceted study to assess treatment response. Following combined chloroquine and sulphadoxine/pyrimethamine treatment the patients were followed for 28 days according to the World Health Organisation in vivo drug response protocol. The in vivo study revealed that 13 (41%) of the patients had a sensitive response to treatment, five (16%) cleared asexual stage parasites but had persistent gametocytes, 11 (34%) had RI type resistance and three (9%) had RII type resistance requiring quinine intervention before day 7 for parasite clearance. Although clinically insignificant, patients with persistent gametocytes, surviving chloroquine and sulphadoxine/pyrimethamine treatment during maturation, were placed in the reduced response to treatment group for analysis. Allelic typing detected 100% prevalence of the pfcrt K76T marker associated with chloroquine resistance and 78% prevalence of the pfdhfr NRNL haplotype associated with sulphadoxine/pyrimethamine treatment failure. High serum chloroquine levels and pfdhfr haplotypes with 相似文献   

pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum

The emergence and spread of multidrug resistant Plasmodium falciparum has severely limited the therapeutic options for the treatment of malaria. With ever-increasing failure rates associated with chloroquine or sulphadoxine-pyrimethamine treatment, attention has turned to the few alternatives, which include quinine and mefloquine. Here, we have investigated the role of pfmdr1 3' coding region point mutations in antimalarial drug susceptibility by allelic exchange in the GC03 and 3BA6 parasite lines. Results with pfmdr1-recombinant clones indicate a significant role for the N1042D mutation in contributing to resistance to quinine and its diastereomer quinidine. The triple mutations S1034C/N1042D/D1246Y, highly prevalent in South America, were also found to enhance parasite susceptibility to mefloquine, halofantrine and artemisinin. pfmdr1 3' mutations showed minimal effect on P. falciparum resistance to chloroquine or its metabolite mono-desethylchloroquine in these parasite lines, in contrast to previously published results obtained with 7G8 parasites. This study supports the hypothesis that pfmdr1 3' point mutations can significantly affect parasite susceptibility to a wide range of antimalarials in a strain-specific manner that depends on the parasite genetic background.     

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.     

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.     

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.     

Identification of polymorphisms in genes associated with drug resistance in Plasmodium falciparum isolates from school-age children in Kinshasa,Democratic Republic of Congo

BackgroundThe emergence and spread of Plasmodium falciparum parasites resistant to antimalarial drugs constitutes an obstacle to malaria control and elimination. This study aimed to identify the prevalence of polymorphisms in pfk13, pfmdr1, pfdhfr, pfdhps and pfcrt genes in isolates from asymptomatic and symptomatic school-age children in Kinshasa.MethodsNested-PCR followed by sequencing was performed for the detection of pfk13, pfmdr1, pfdhfr, pfdhps and pfcrt polymorphisms.ResultsTwo mutations in pfk13, C532S and Q613E were identified in the Democratic Republic of Congo for the first time. The prevalence of the drug-resistance associated mutations pfcrt K76T, pfdhps K540E and pfmdr1 N86Y was low, being 27%, 20% and 9%, respectively.ConclusionWe found a low prevalence of genetic markers associated with chloroquine and sulfadoxine-pyrimethamine resistance in Kinshasa. Furthermore, no mutations previously associated with resistance against artemisinin and its derivatives were observed in the pfK13 gene. These findings support the continued use of ACTs and IPTp-SP. Continuous molecular monitoring of antimalarial resistance markers is recommended.     

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.     

Evidence for a pfcrt-associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.     

Reduced Susceptibility of Plasmodium falciparum to Artesunate in Southern Myanmar

Background

Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.

Methods

A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.

Results

The median (range) parasite clearance half-life and time were 4.8 (2.1–9.7) and 60 (24–96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.

Conclusions

A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12610000896077     

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.     

Field-based evidence for the linkage of pfcrt and pfdhfr drug-resistant malaria genotypes and clinical profiles of severe malaria in Niger

Drug resistance has been shown to increase malaria mortality and morbidity in both community- and hospital-based studies. We investigated the association between two Plasmodium falciparum drug resistance-related molecular markers and clinical profiles of severe malaria in children hospitalised in Niger. PCR-RFLP analysis showed that the codon 108 mutation of the pfdhfr gene was positively linked to severe malarial anaemia. These findings are consistent with persistent parasite infection leading to unbalanced anaemia in young children. No significant relationship was found between the molecular markers and hypoglycaemia or hyperparasitaemia. Conversely, the pfcrt T76 mutation was found to be negatively associated with cerebral malaria and neurological symptoms, such as convulsions and coma. These results have implications for the strain-specific virulence hypothesis and for parasite fitness and evolution. Our findings are discussed in regard to the local malaria transmission level.     

Diversity of Plasmodium falciparum chloroquine resistance transporter (pfcrt) exon 2 haplotypes in the Pacific from 1959 to 1979

Nearly one million deaths are attributed to malaria every year. Recent reports of multi-drug treatment failure of falciparum malaria underscore the need to understand the molecular basis of drug resistance. Multiple mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) are involved in chloroquine resistance, but the evolution of complex haplotypes is not yet well understood. Using over 4,500 archival human serum specimens collected from 19 Pacific populations between 1959 and 1979, the period including and just prior to the appearance of chloroquine treatment failure in the Pacific, we PCR-amplified and sequenced a portion of the pfcrt exon 2 from 771 P. falciparum-infected individuals to explore the spatial and temporal variation in falciparum malaria prevalence and the evolution of chloroquine resistance. In the Pacific, the prevalence of P. falciparum varied considerably across ecological zones. On the island of New Guinea, the decreases in prevalence of P. falciparum in coastal, high-transmission areas over time were contrasted by the increase in prevalence during the same period in the highlands, where transmission was intermittent. We found 78 unique pfcrt haplotypes consisting of 34 amino acid substitutions and 28 synonymous mutations. More importantly, two pfcrt mutations (N75D and K76T) implicated in chloroquine resistance were present in parasites from New Hebrides (now Vanuatu) eight years before the first report of treatment failure. Our results also revealed unexpectedly high levels of genetic diversity in pfcrt exon 2 prior to the historical chloroquine resistance selective sweep, particularly in areas where disease burden was relatively low. In the Pacific, parasite genetic isolation, as well as host acquired immune status and genetic resistance to malaria, were important contributors to the evolution of chloroquine resistance in P. falciparum.