Pfcrt Mutations Research Articles

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

BackgroundWith the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations.MethodsArchived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis.ResultsThe percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×2 = 96.31, p <0.001) and pfcrt K76 (×2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (×2 = 38.52, p <0.001) and pfcrt T76 (×2 = 43.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×2 = 7.39,p=0.060; ×2 = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×2 = 20.75, p < 0.001).ConclusionIncreased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Reduced heterozygosity at intragenic and flanking microsatellites of pfcrt gene establishes natural selection based molecular evolution of chloroquine-resistant Plasmodium falciparum in India

The positive selection of a nucleotide substitution in exon 2 of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene (mutation responsible for chloroquine resistance) causes a reduction in variation of neutral loci close to the gene. This reduction in allelic diversity around flanking regions of pfcrt gene was reported in worldwide chloroquine resistant isolates and referred as selective sweep. In Plasmodium falciparum isolates of India, the selective sweep in flanking loci of pfcrt gene is well established, however, high allelic diversity observed in intragenic microsatellites of pfcrt gene implied an ongoing genetic recombination. To understand, if molecular evolution of chloroquine-resistant P. falciparum isolates in India follow a selective sweep model, we analyzed genetic diversity at both seven intragenic and seven flanking microsatellites of pfcrt (−24 to +106kb) gene in chloroquine sensitive and resistant parasites originating from high and low transmission areas. We observed low expected heterozygosity at all loci of resistant pfcrt-haplotypes (He=0–0.77) compared to the wild-type (He=0.38–0.96). Resistant SVMNT from high transmission areas showed significantly higher mean He (P=0.03, t-test) at both intragenic and pfcrt-flanking loci (−24 to +22kb) in comparison to low transmission areas. Our observation of reduction in variation at both intragenic and flanking loci of mutant pfcrt gene confirmed the selective sweep model of natural selection in chloroquine resistant P. falciparum isolates in India.

Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

ObjectiveTo investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border. MethodsDried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. ResultsMutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies. ConclusionsThe observation of pfmdr1 wild type and increasing of gene copy number may suggest declining of artesunate-mefloquine treatment efficacy in P. falciparum isolates in this border area.

Association between Prevalence of Chloroquine Resistance and Unusual Mutation in pfmdr-I and pfcrt Genes in India

This study deals with the underlying causes of failure of chloroquine in the treatment of Plasmodium falciparum infection in some malaria-endemic regions of India. Samples were collected from 141 patients in Purulia from March of 2007 to April of 2008. In vitro drug susceptibility tests, parasitic DNA isolation followed by polymerase chain reaction, and restriction fragment-length polymorphisms of different codons of the pfcrt gene (76) and pfmdr-I genes (86, 1042, and 1246) were assessed. The responses of 141 patients to chloroquine were determined. Prevalence of double pfmdr-I (58.16%) mutation (86Y+1246Y) and some (14.89%) single pfcrt mutations with triple pfmdr-I mutation (76T+86Y+1042D+1246Y) were found. Interestingly, double pfmdr-I mutation (86Y and 1246Y codons) was observed with the early treatment failure cases. These results show, for the first time in India that in vitro chloroquine resistance and in vivo chloroquine treatment failure were caused by double pfmdr-I (P < 0.001) mutation.

Prevalence of antifolate resistance mutations in Plasmodium falciparum isolates in Afghanistan

BackgroundArtesunate plus sulphadoxine-pyrimethamine (AS+SP) is now first-line treatment for Plasmodium falciparum infection in several south Asian countries, including Afghanistan. Molecular studies provide a sensitive means to investigate the current state of drug susceptibility to the SP component, and can also provide information on the likely efficacy of other potential forms of artemisinin-combination therapy.MethodsDuring the years 2007 to 2010, 120 blood spots from patients with P. falciparum malaria were obtained in four provinces of Afghanistan. PCR-based methods were used to detect drug-resistance mutations in dhfr, dhps, pfcrt and pfmdr1, as well as to determine copy number of pfmdr1.ResultsThe majority (95.5%) of infections had a double mutation in the dhfr gene (C59R, S108N); no mutations at dhfr positions 16, 51 or 164 were seen. Most isolates were wild type across the dhps gene, but five isolates from the provinces of Kunar and Nangarhar in eastern Afghanistan had the triple mutation A437G / K540E / A581G; all five cases were successfully treated with three receiving AS+SP and two receiving dihydroartemisinin-piperaquine. All isolates showed the pfcrt SVNMT chloroquine resistance haplotype. Five of 79 isolates had the pfmdr1 N86Y mutation, while 52 had pfmdr1 Y184F; positions 1034, 1042 and 1246 were wild type in all isolates. The pfmdr1 gene was not amplified in any sample.ConclusionsThis study indicates that shortly after the adoption of AS+SP as first-line treatment in Afghanistan, most parasites had a double mutation haplotype in dhfr, and a small number of isolates from eastern Afghanistan harboured a triple mutation haplotype in dhps. The impact of these mutations on the efficacy of AS+SP remains to be assessed in significant numbers of patients, but these results are clearly concerning since they suggest a higher degree of SP resistance than previously detected. Further focused molecular and clinical studies in this region are urgently required.

Glutathione Transport: A New Role for PfCRT in Chloroquine Resistance

Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.

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

BackgroundMalaria 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.MethodsA 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.ResultsMutations 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.ConclusionThe 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.

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

BackgroundThe 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.ResultsA 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.ConclusionsThis 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.

Evolution of the pfcrt T76 and pfmdr1 Y86 markers and chloroquine susceptibility 8 years after cessation of chloroquine use in Pikine, Senegal

The goal of the present study was to assess the evolution of the in vitro chloroquine resistance and also the prevalence of pfcrt T76 and pfmdr1 Y86 mutations in Pikine from 2000 while chloroquine (CQ) was the first-line treatment of malaria to 2009 when artemisinin-based combination therapies (ACTs) are in use. We genotyped pfcrt K76T and pfmdr1 N86Y polymorphisms by PCR-RFLP and assessed in vitro CQ susceptibility by double-site enzyme-linked pLDH immunodetection (DELI) assay in Plasmodium falciparum isolates collected in Pikine, Senegal. The proportions of the pfcrt T76 allele in the light of the three different treatment policies were 72.4 % before CQ withdrawal (2000 to 2003), 47.2% while amodiaquine plus Fansidar was the first-line treatment (2004 to 2005), and 59.5 % since the ACT use was implemented (2006 to 2009). The prevalence of pfcrt T76 decreased significantly after CQ was stopped [X (2) = 6.54, P = 0.01 (2000-2003 versus 2004-2005)] and then slightly since ACTs have been implemented [X(2) = 1.12, P = 0.28 (2000-2003 versus 2006-2009)]. There were no significant differences on the prevalence of pfmdr1 Y86 throughout the three treatment policies. The DELI assay was carried out episodically in 2000 (n = 36), 2001 (n = 47), and 2009 (n = 37). The mean IC(50)s of the isolates to CQ in 2000 versus 2009 and 2001 versus 2009 are significantly different (P < 0.05). The Fisher exact test found a significant association between the presence of the pfcrt T76 mutant allele and in vitro resistance in 2000/2001 (P = 0.023), while in 2009 there were no association between both variables (P = 0.274). Mutant pfcrt T76 and pfmdr1 Y86 alleles and in vitro CQ-resistant strains are still circulating in Pikine. The official discontinuation of CQ use is not completely followed by its total withdrawal from private drug sellers, and the molecule still exerts pressure on local P. falciparum populations.

Distribution of Drug Resistance Genotypes in Plasmodium falciparum in an Area of Limited Parasite Diversity in Saudi Arabia

Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent.

Molecular monitoring of plasmodium falciparum drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaoundé, Cameroon: Implications for the future

BackgroundRegular monitoring of the levels of anti-malarial resistance of Plasmodium falciparum is an essential policy to adapt therapy and improve malaria control. This monitoring can be facilitated by using molecular tools, which are easier to implement than the classical determination of the resistance phenotype. In Cameroon, chloroquine (CQ), previously the first-line therapy for uncomplicated malaria was officially withdrawn in 2002 and replaced initially by amodiaquine (AQ) monotherapy. Then, artemisinin-based combination therapy (ACT), notably artesunate-amodiaquine (AS-AQ) or artemether-lumefantrine (AL), was gradually introduced in 2004. This situation raised the question of the evolution of P. falciparum resistance molecular markers in Yaoundé, a highly urbanized Cameroonian city.MethodsThe genotype of pfcrt 72 and 76 and pfmdr1 86 alleles and pfmdr1 copy number were determined using real-time PCR in 447 P. falciparum samples collected between 2005 and 2009.ResultsThis study showed a high prevalence of parasites with mutant pfcrt 76 (83%) and pfmdr1 86 (93%) codons. On the contrary, no mutations in the pfcrt 72 codon and no samples with duplication of the pfmdr1 gene were observed.ConclusionThe high prevalence of mutant pfcrt 76T and pfmdr1 86Y alleles might be due to the choice of alternative drugs (AQ and AS-AQ) known to select such genotypes. Mutant pfcrt 72 codon was not detected despite the prolonged use of AQ either as monotherapy or combined with artesunate. The absence of pfmdr1 multicopies suggests that AL would still remain efficient. The limited use of mefloquine or the predominance of mutant pfmdr1 86Y codon could explain the lack of pfmdr1 amplification. Indeed, this mutant codon is rarely associated with duplication of pfmdr1 gene. In Cameroon, the changes of therapeutic strategies and the simultaneous use of several formulations of ACT or other anti-malarials that are not officially recommended result in a complex selective pressure, rendering the prediction of the evolution of P. falciparum resistance difficult. This public health problem should lead to increased vigilance and regular monitoring.

Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections

BackgroundIn 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.MethodsBetween 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.ResultsThe 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%.ConclusionThis 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.

Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene pfcrt

BackgroundIn Plasmodium falciparum, resistance to chloroquine (CQ) is conferred by a K to T mutation at amino acid position 76 (K76T) in the P. falciparum CQ transporter (PfCRT). To date, at least 15 pfcrt genotypes, which are represented by combinations of five amino acids at positions 72-76, have been described in field isolates from various endemic regions. To identify novel mutant pfcrt genotypes and to reveal the genetic relatedness of pfcrt genotypes, a large-scale survey over a wide geographic area was performed.MethodsSequences for exon 2 in pfcrt, including known polymorphic sites at amino acid positions 72, 74, 75 and 76, were obtained from 256 P. falciparum isolates collected from eight endemic countries in Asia (Bangladesh, Cambodia, Lao P.D.R., the Philippines and Thailand), Melanesia (Papua New Guinea and Vanuatu) and Africa (Ghana). A haplotype network was constructed based on six microsatellite markers located -29 kb to 24 kb from pfcrt in order to examine the genetic relatedness among mutant pfcrt genotypes.ResultsIn addition to wild type (CVMNK at positions 72-76), four mutant pfcrt were identified; CVIET, CVIDT, SVMNT and CVMNT (mutated amino acids underlined). Haplotype network revealed that there were only three mutant pfcrt lineages, originating in Indochina, Philippines and Melanesia. Importantly, the Indochina lineage contained two mutant pfcrt genotypes, CVIET (n = 95) and CVIDT (n = 14), indicating that CVIDT shares a common origin with CVIET. Similarly, one major haplotype in the Melanesian lineage contained two pfcrt genotypes; SVMNT (n = 71) and CVMNT (n = 3). In Africa, all mutant pfcrt genotypes were the CVIET of the Indochina lineage, probably resulting from the intercontinental migration of CQ resistance from Southeast Asia.ConclusionsThe number of CQ-mutant lineages observed in this study was identical to that found in previous studies. This supports the hypothesis that the emergence of novel CQ resistance is rare. However, in the mutant pfcrt genotypes, amino acid changes at positions 72, 74 and 75 appear to have recently been generated at least several times, producing distinct pfcrt mutant genotypes. The occurrence of new mutations flanking K76T may yield stronger resistance to CQ and/or a higher fitness than the original pfcrt mutant.

Efficacy of artemether-lumefantrine in treatment of malaria among under-fives and prevalence of drug resistance markers in Igombe-Mwanza, north-western Tanzania

BackgroundDrug resistance to anti-malarials is a major public health problem worldwide. This study aimed at establishing the efficacy of artemether-lumefantrine (ACT) in Igombe-Mwanza, north-western Tanzania after a few years of ACT use, and establish the prevalence of mutations in key targets for artemisinin, chloroquine and sulphadoxine/pyrimetamine (SP) drugs.MethodsA prospective single cohort study was conducted at Igombe health centre using artemether-lumefantrine combination therapy between February 2010 and March 2011. The follow-up period was 28 days and outcome measures were according to WHO guidelines. Blood was collected on Whatman filter paper for DNA analysis. DNA extraction was done using TRIS-EDTA method, and mutations in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Pfatp6 were detected using PCR-RFLP methods established previously.ResultsA total of 103 patients completed the 28 days follow-up. The mean haemoglobin was 8.9 g/dl (range 5.0 to 14.5 g/dl) and mean parasite density was 5,608 parasites/μl. Average parasite clearance time was 34.7 hours and all patients cleared the parasites by day 3. There was no early treatment failure in this study. Late clinical failure was seen in three (2.9%) patients and late parasitological failure (LPF) was seen in two (1.9%). PCR-corrected LPF was 1% and adequate clinical and parasitological response was 96%. The majority of parasites have wild type alleles on pfcrt 76 and pfmdr1 86 positions being 87.8% and 93.7% respectively. Mutant parasites predominated at pfdhfr gene at the main three positions 108, 51 and 59 with prevalence of 94.8%, 75.3% and 82.5% respectively. Post-treatment parasites had more wild types of pfdhps at position 437 and 540 than pre-treatment parasites. No mutation was seen in pfatp6 769 in re-infecting or recrudescing parasites.ConclusionThe efficacy of artemether-lumefantrine for treatment of uncomplicated malaria is still high in the study area although the rate of re-infection is higher than previously reported. Parasite clearance after 48 hours was lower compared to previous studies. The prevalence of wild type allele pfcrt 76 K and pfmdr1 86 N was high in the study area while markers for SP resistance is still high. Artemether-lumefantrine may be selecting for wild type alleles on both positions (437 and 540) of pfdhps.

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.

Mutant pfcrt "SVMNT" haplotype and wild type pfmdr1 "N86" are endemic in Plasmodium vivax dominated areas of India under high chloroquine exposure

BackgroundChloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated.MethodsFinger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme.ResultsIn 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%).ConclusionsThe wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Differential genetic hitchhiking around mutant pfcrt alleles in the Indian Plasmodium falciparum population

To study the origin and spread of the chloroquine-resistant Plasmodium falciparum population in the Indian subcontinent. Fourteen microsatellites spanning a ∼120 kb region, flanking the P. falciparum chloroquine resistance transporter (pfcrt) gene, were analysed in 185 parasite isolates. The Indian P. falciparum population exhibited a selective valley of reduced genetic variation in the flanking microsatellites of the mutant pfcrt alleles (up to ±29 kb) as compared with the wild-type allele. This valley is much narrower than the ±200 kb valley reported from African and South-East Asian countries. The majority of the isolates showed asymmetry in the selective valley, where upstream microsatellites showed less genetic variation than the downstream microsatellites. Regional variation in the width and symmetry of the selective valley was noticed, which seems to be related to the number of pfcrt alleles present in the parasite population of a region. Forty-six different microsatellite haplotypes were observed among the P. falciparum isolates containing mutant pfcrt alleles. Parasite populations from different regions of mainland India shared microsatellite haplotypes between them, but they shared none with the isolates from the Andaman and Nicobar Islands, and vice versa. Indian isolates shared microsatellite haplotypes with the isolates from Papua New Guinea and Thailand. With regard to chloroquine there is regional variation in the selection pressure on the P. falciparum population in India. These findings will help the regional implementation of drug policy in India's malaria control programme.

Large differences in prevalence of Pfcrt and Pfmdr1 mutations between Mwanza, Tanzania and Iganga, Uganda—A reflection of differences in policies regarding withdrawal of chloroquine?

BackgroundMalaria is still a major public health problem in the world and sub-Saharan Africa is one of the most affected areas. Efforts to control malaria are highly affected by drug resistance to commonly used antimalarials. The introduction of artemisinin based combination therapy (ACT) as a first line drug seems to be a major step in treatment of uncomplicated malaria, though search for drugs to combine with artemisinins still continues. There have been reports on increased prevalence of the wild type markers Pfcrt 76K and Pfmdr1 86N in some African countries and ideas of using chloroquine (CQ) in intermittent presumptive treatment for adults (IPTa) is coming up. The common combination of artemether and lumefantrine even selects for parasites that are wild type at these positions. This study is comparing prevalence of mutation at these two positions in two East African countries with ACT as their first line drug but following somewhat different drug policies regarding CQ. In Tanzania CQ was stopped in 2001 but in Uganda CQ was retained in combination with sulfadoxine–pyrimethamine (SP) and used in home based management of fever for some time. SP is still used in IPT for pregnant women. MethodsBlood smears and dried blood spots on Whatman filter papers were collected from 100 patients with uncomplicated malaria in Mwanza, Tanzania and 100 patients from Iganga, Uganda. DNA was extracted from all samples using Tris EDTA method. PCR and RFLP were performed and sequencing done on Pfcrt amplification products. ResultsThe prevalence of K76T mutations at Pfcrt in samples from Mwanza, Tanzania was 40.5% (34/84) and 100% (100/100) in samples from Iganga, Uganda. Prevalence of N86Y mutations in Pfmdr1 was 16.9% (13/77) and 77.7% (63/81) in samples from Mwanza and Iganga, respectively. The re-emergence of CQ sensitive isolates in Mwanza, Tanzania showed the haplotype CVMNK typical for wild type isolates. ConclusionsThe prevalence of CQ resistant parasites in Mwanza, Tanzania is low compared to the existing high level of resistant parasites in Iganga, Uganda. This could be an indication that CQ may become useful in the future in Tanzania. This study shows clearly that there is a difference in mutations at these positions in these two countries implementing similar but somewhat different drug policies. In Uganda the drug resistance has reached fixation while in Tanzania the prevalence is going down.

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.