Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal

ABSTRACT: BACKGROUND: As a result of the widespread resistance to chloroquine and sulphadoxinepyrimethamine, artemisinin-based combination therapy (ACT) (including artemetherlumefantrine and artesunate-amodiaquine) has been recommended as a first-line antimalarial regimen in Senegal since 2006. Intermittent preventive treatments with antimalarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to antimalarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar. METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., Pfcrt, Pfdhfr, Pfdhps and Pfmdr1, and the copy number of Pfmdr1 were evaluated for a panel of 174 isolates collected from patients recruited at the military hospital of Dakar from 14 October 2009 to 19 January 2010. RESULTS: The Pfcrt 76 T mutation was identified in 37.2% of the samples. The Pfmdr1 86Y and 184 F mutations were found in 16.6% and 67.6% of the tested samples, respectively. Twenty-eight of the 29 isolates with the 86Y mutation were also mutated at codon 184. Only one isolate (0.6%) had two copies of Pfmdr1. The Pfdhfr 108 N/T, 51I and 59R mutations were identified in 82.4%, 83.5% and 74.1% of the samples, respectively. The double mutant (108 N and 51I) was detected in 83.5% of the isolates, and the triple mutant (108 N, 51I and 59R) was detected in 75.3%. The Pfdhps 437 G, 436 F/A and 613 S mutations were found in 40.2%, 35.1% and 1.8% of the samples, respectively. There was no double mutant (437 G and 540E) or no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E). The prevalence of the quadruple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G) was 36.5%. CONCLUSIONS: Since 2004, the prevalence of chloroquine resistance had decreased. The prevalence of isolates with high-level pyrimethamine resistance is 83.5%. The prevalence of isolates resistant to sulphadoxine is 40.2%. However, no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E), which is associated with a high level of sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate.     

In vitro sensitivity of Plasmodium falciparum to amodiaquine compared with other major antimalarials in Madagascar

Chloroquine has been used in Madagascar since 1945 and remains the first-line treatment for uncomplicated cases of malaria. Low-grades of resistance type R1 and R2 have been reported. Thus, in vitro tests were performed in order to monitor the drug sensitivity of Plasmodium falciparum from different study sites, with the aim of identifying alternatives to chloroquine. Chloroquine IC50 values ranged from 0.2 nM to 283.4 nM (n = 190, mean IC50 = 52.6 nM; 95% CI = 46.1-59.1 nM). Fifteen isolates (7.9%) were chloroquine-resistant. One mefloquine-resistant isolate was detected (1/139). The test isolates were sensitive to amodiaquine (n = 118), quinine (n = 212), pyrimethamine (n = 86) and cycloguanil (n = 79). The median IC50 for amodiaquine was 12.3 nM (mean IC50 = 15.3 nM, 95% CI = 13.3-17.3 nM). Amodiaquine was 3.4 times as active as chloroquine in vitro and 7 times as active as quinine against P. falciparum. These results indicate that amodiaquine may be a potent alternative to chloroquine in Madagascar. There was positive correlation between tested quinoline-containing drugs activities, which suggests in vitro cross-susceptibility.     

Therapeutic efficacy of artesunate in the treatment of uncomplicated Plasmodium falciparum malaria and anti-malarial, drug-resistance marker polymorphisms in populations near the China- Myanmar border

ABSTRACT: BACKGROUND: The aim of this study was to evaluate the clinical outcome after seven-day artesunate monotherapy for uncomplicated Plasmodium falciparum malaria in Yingjiang County along the China-Myanmar border and investigate genetic polymorphisms in the P. falciparum chloroquine-resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes. METHODS: Patients [greater than or equal to] one year of age with fever (axillary temperature [greater than or equal to]37.5 degreesC) or history of fever and P. falciparum mono-infection were included. Patients received anti-malarial treatment with artesunate (total dose of 16 mg/kg over seven days) by directly observed therapy. After a 28- day follow-up, treatment efficacy and effectiveness were assessed based on clinical and parasitological outcomes. Treatment failure was defined as recrudescence of the original parasite and distinguished with new infection confirmed by PCR. Analysis of gene mutation and amplification were performed by nested polymerase chain reaction. RESULTS: Sixty-five patients were enrolled; 10 withdrew from the study, and six were lost to follow-up. All but two patients demonstrated adequate clinical and parasitological response; 12 had detectable parasitaemia on day 3. These two patients were confirmed to be new infection by PCR. The efficacy of artesunate was 95.9 %. The pfcrt mutation in codon 76 was found in all isolates (100 %), and mutations in codons 71 and 72 were found in 4.8 % of parasite isolates. No mutation of pfmdr1 (codons 86 or 1246) was found. Among all samples, 5.1 % were wild type for pfdhfr, whereas the other samples had mutations in four codons (51, 59, 108 and 164), and mutations in pfdhps (codons 436, 437, 540 and 581) were found in all isolates. No samples had mutations in pfatp6 codons 623 or 769, but two new mutations (N683K and R756K) were found in 4.6 % and 9.2 % of parasite isolates, respectively. CONCLUSION: Plasmodium falciparum infection was associated with slow parasite clearance and suspected artemisinin resistance at the China-Myanmar border area. The prevalence of pfcrt 76 T and markers for SP resistance are still high. It should be strengthened further on parasite clearance time or clearance half life to confirm the resistance status, and molecular epidemiology should provide complementary information to assess the appropriateness of current policies based on artemisinin derivatives.     

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.     

Conservation of a novel vacuolar transporter in Plasmodium species and its central role in chloroquine resistance of P. falciparum

Chloroquine resistance in Plasmodium falciparum has recently been shown to result from mutations in the novel vacuolar transporter, PfCRT. Field studies have demonstrated the importance of these mutations in clinical resistance. Although a pfcrt ortholog has been identified in Plasmodiumvivax, there is no association between in vivo chloroquine resistance and codon mutations in the P. vivax gene. This is consistent with lines of evidence that suggest alternative mechanisms of chloroquine resistance among various malaria parasite species.     

Enhancement of the antimalarial efficacy of amodiaquine by chlorpheniramine in vivo

Resistance in Plasmodium falciparum to amodiaquine (AQ) can be reversed in vitro with with antihistaminic and tricyclic antidepressant compounds, but its significance in vivo is unclear. The present report presents the enhancement of the antimalarial efficacy of AQ by chlorpheniramine, an H1 receptor antagonist that reverses chloroquine (CQ) resistance in vitro and enhances its efficacy in vivo, in five children who failed CQ and/or AQ treatment, and who were subsequently retreated and cured with a combination of AQ plus CP, despite the fact that parasites infecting the children harboured mutant pfcrtT76 and pfmdr1Y86 alleles associated with AQ resistance. This suggests a potential clinical application of the reversal phenomenon.     

Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests

The dissemination of mutant and resistant strains of Plasmodium falciparum makes a considerable contribution to the spread of drug-resistant malaria. Populations around harbours and airports could be particularly exposed to Plasmodium isolates introduced with imported cases of malaria. The use of chloroquine as well as the use of and sulfadoxine/pyrimethamine is currently an effective method for treating uncomplicated cases of malaria in Madagascar. As part of a monitoring programme, in vitro methods were used to assess the sensitivity of P. falciparum isolates in two coastal towns in Madagascar: Mahajanga on the west coast and Toamasina on the east coast. All of the isolates from both sites were sensitive to amodiaquine, quinine, pyrimethamine and cycloguanil. All of the isolates from Mahajanga were sensitive to chloroquine (n = 25; mean IC50 = 22.6 nM, 95% confidence interval: 16.8-28.7 nM), whereas three of the isolates from Toamasina were resistant to chloroquine (n = 18; mean IC50 = 66.3 nM; 95% confidence interval: 42.6-90 nM). The frequency of the Pfcrt Thr-76 and the dhfr Asn-108 mutations was estimated by PCR/RFLP. The 43 P. falciparum isolates examined, including the three in vitro chloroquine-resistant isolates from Toamasina were all wild-type (Lys-76). Phenotyping and genotyping studies suggested that the prevalence of chloroquine- and pyrimethamine-resistant isolates and of mutant strains of P. falciparum is very low. These results showed that in vitro test and genotyping of resistance markers approaches could be successfully used to monitor the emergence of drug-resistant malaria and to try to alleviate the lack of medical teams able to carry out in vivo test. The possible hazard/risk associated with imported cases of malaria is discussed.     

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.     

Evolutionary paradigm of chloroquine-resistant malaria in India

Drug pressure in the field is believed to be responsible for the emergence of drug-resistant Plasmodium falciparum, the parasite that causes malaria. Variants of the P. falciparum chloroquine resistance transporter (pfcrt) gene have been shown to be responsible for conferring resistance to the commonly used drug chloroquine. In particular, an amino acid mutation, K76T, was shown to have a strong positive correlation with the chloroquine-resistant varieties of malaria parasites. Global studies have reported highly reduced genetic diversity surrounding K76T in the pfcrt gene, which indicates that the mutation has been a target of positive Darwinian natural selection. However, two recent studies of P. falciparum in India found high genetic diversity in the pfcrt gene, which, at first sight, do not support the role of natural selection in the evolution of chloroquine resistance in India.     

Molecular Evidence of Increased Resistance to Anti-Folate Drugs in Plasmodium falciparum in North-East India: A Signal for Potential Failure of Artemisinin Plus Sulphadoxine-Pyrimethamine Combination Therapy

North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.     

Electrocardiographic study in Ghanaian children with uncomplicated malaria,treated with artesunate-amodiaquine or artemether-lumefantrine

Background

In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined.

Methods

Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion.

Results

The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%.

Conclusion

This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.     

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.     

A molecular map of chloroquine resistance in Mali

Plasmodium falciparum chloroquine resistance (CQR) transporter point mutation (PfCRT 76T) is known to be the key determinant of CQR. Molecular detection of PfCRT 76T in field samples may be used for the surveillance of CQR in malaria-endemic countries. The genotype-resistance index (GRI), which is obtained as the ratio of the prevalence of PfCRT 76T to the incidence of CQR in a clinical trial, was proposed as a simple and practical molecular-based addition to the tools currently available for monitoring CQR in the field. In order to validate the GRI model across populations, time, and resistance patterns, we compiled data from the literature and generated new data from 12 sites across Mali. We found a mean PfCRT 76T mutation prevalence of 84.5% (range 60.9–95.1%) across all sites. CQR rates predicted from the GRI model were extrapolated onto a map of Mali to show the patterns of resistance throughout the participating regions. We present a comprehensive map of CQR in Mali, which strongly supports recent changes in drug policy away from chloroquine.     

Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of crossresistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.     

Effect of chloroquine phosphate and toxic concentrations of lead acetate on Ca2+-ATPase activity in isolates and clones of Plasmodium Falciparum

The basal activity of Ca2+-ATPase in two isolates (NL56, UNC) and two clones (D6, W2) of P.falciparum was assessed. The effects of various concentrations of chloroquine phosphate and toxic concentrations of lead acetate were also evaluated in the clones and strains of P.falciparum. The Ca2+-ATPase activity was measured by monitoring the rate of release of inorganic phosphate from the gamma-position of ATP on spectrophotometer at 820nm wavelength. The various concentrations of chloroquine (3, 6, 9, 12, 18μg/ml) and lead acetate (5, 10, 20, 30, 40μg/ml) on Ca2+-ATPase activity were measured respectively. Chloroquine phosphate inhibited Ca2+-ATPase activity in both the isolates and the cloned strains of P.falciparum in concentration dependent manner. Median Inhibitory concentration of chloroquine (MIC50) estimated from the plot of activity against chloroquine concentration was found to be 2.6mg/ml at pH 7.4 for both the isolates and cloned strains examined. Lead acetate at concentrations 5-20μg/ml inhibited Ca2+-ATPase activity in concentration dependent manner in clone W2 (Chloroquine resistant strain) while the same range of concentrations of lead acetate stimulated the activity of the enzyme in clone D6 (Chloroquine sensitive strain).The inhibitory effect of lead acetate on the enzyme in clone D6 was observed at concentrations above 20μg/ml. The result also suggests that lead ions could modulate and moderate calcium ion homeostasis in P. falciparum via its effect on Ca2+-ATPase activity. Also sufficient influx of lead ions into P. falciparum may transform the biochemical or bioenergetics nature of chloroquine sensitive strain of P. falciparum (D6) to that similar to chloroquine resistant strain (W2). In conclusion, inhibition of Ca2+-ATPase activity of P.falciparum may be part of the mechanism of action of chloroquine in its use as chemotherapy for malaria. The study implies that populations simultaneously exposed to lead pollution and malaria infection may experience failure in chloroquine therapy.     

Chloroquine clinical failures in P. falciparum malaria are associated with mutant Pfmdr-1, not Pfcrt in Madagascar

Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important, particularly regarding the evolution and spread of Pfmdr-1 alleles in P. falciparum populations under changing drug pressure which may have important consequences in terms of antimalarial use management.     

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C₇₂M₇₄N₇₅K₇₆ (42.9%) and mutant C₇₂I₇₄E₇₅T₇₆ (53.8%) were observed. Also, wildtype N₈₆Y₁₈₄ (62.9%) and mutant N₈₆F₁₈₄ (21.1%), Y₈₆Y₁₈₄ (6.4%), and Y₈₆F₁₈₄ (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C₇₂I₇₄E₇₅T₇₆ and C₇₂M₇₄N₇₅K₇₆) and major mdr-1 haplotypes (N₈₆Y₁₈₄, N₈₆F₁₈₄ and Y₈₆Y₁₈₄). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C₇₂I₇₄E₇₅T₇₆ haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.     

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.     

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.     

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.