Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique.
ABSTRACT: In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108?+?dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108?+?dhps G437E540 and sextuple dhfr I51R59N108?+?dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.
Project description:The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.
Project description:In 2007, Malawi replaced sulfadoxine-pyrimethamine (SP) with an artemisinin-based combination therapy as the first-line treatment for uncomplicated Plasmodium falciparum malaria in response to failing SP efficacy. Here we estimate the effect of reduced SP pressure on the prevalence of SP-resistant parasites and the characteristics of the associated selective sweeps flanking the resistance loci.Samples obtained from individuals with clinical malaria during a period of high SP use (1999-2001), a transitional period (2007-2008), and a period of low SP use (2012) were genotyped for resistance markers at pfdhfr-ts codons 51, 59, and 108 and pfdhps codons 437, 540, and 581. Expected heterozygosity was estimated to evaluate the genetic diversity flanking pfdhfr-ts and pfdhps.An increase in the prevalence of the resistance haplotypes DHFR 51I/59R/108N and DHPS 437G/540E occurred under sustained drug pressure, with no change in haplotype prevalence 5 years after reduction in SP pressure. The DHPS 437G/540E/581G haplotype was observed in 2007 and increased in prevalence during a period of reduced SP pressure. Changes to the sweep characteristics flanking pfdhfr-ts and pfdhps were minimal.In contrast to the rapid and complete return of chloroquine-susceptible falciparum malaria after chloroquine was withdrawn from Malawi, a reemergence of SP efficacy is unlikely in the near future.
Project description:There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd ?-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015.
Project description:Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti.DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum.Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch's T-test p-value = 0.53 and permutations p-value = 0.59).This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.
Project description:Sulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended for intermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment of malaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated with mutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6-59 month children with uncomplicated malaria and in asymptomatic 2-10 month old infants.An open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacy protocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 10 months old asymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to the high failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and a review of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-corrected early treatment failure was 38.8% (95% CI 26.8-50.8) and total failures by day 28 were 82.2% (95% CI 72.5-92.0). There was no significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carried parasites with mutations at codons 51, 59 and 108 in the dhfr gene and 63% carried a double mutation at codons 437 and 540. 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotype maybe associated with earlier treatment failure.In northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study found a new combination of parasite mutations that maybe associated with increased and earlier failure.ClinicalTrials.gov NCT00361114.
Project description:Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively.Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing.In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted.pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.
Project description: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 (76 T), while 68% had mutant pfmdr-1 genotype (86 Y). 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 (51 I/59 R/108 N/164 L) 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.
Project description:Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen.Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540.A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540).High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the potential of the emergence of AS resistance. Study findings necessitate the continuous monitoring of the efficacy of the national anti-malarial drugs policy in Yemen. In addition, monitoring SP efficacy using molecular markers that has shown to be a practical and informative method for monitoring the partner drug of AS.
Project description:Haiti is striving for zero local malaria transmission by the year 2025. Chloroquine remains the first-line treatment, and sulfadoxine/pyrimethamine (SP) has been used for mass drug-administration pilot programs. In March 2016, nationwide molecular surveillance was initiated to assess molecular resistance signatures for chloroquine and SP. For 778 samples collected through December 2017, we used Sanger sequencing to investigate putative resistance markers to chloroquine (Pfcrt codons 72, 74, 75, and 76), sulfadoxine (Pfdhps codons 436, 437, 540, 581, 613), and pyrimethamine (Pfdhfr codons 50, 51, 59, 108, 164). No parasites harbored Pfcrt point mutations. Prevalence of the Pfdhfr S108N single mutation was 47%, and we found the triple mutant Pfdhfr haplotype (108N, 51I, and 59R) in a single isolate. We observed no Pfdhps variants except in 1 isolate (A437G mutation). These data confirm the lack of highly resistant chloroquine and SP alleles in Haiti and support the continued use of chloroquine and SP.
Project description:BACKGROUND:Plasmodium falciparum-resistance to sulphadoxine-pyrimethamine (SP) has been largely reported among pregnant women. However, the profile of resistance markers to SP dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) in the general population are varied and not frequently monitored. Currently, SP is used as partner drug for artemisinin combination therapy (SP-artesunate) in some sub-Saharan African countries or as a prophylactic drug in intermittent preventive treatment of malaria during pregnancy and infants and in seasonal malaria chemoprevention (SMC). Profiling of P. falciparum-resistant genotypes to SP is dynamic and critical in providing data that would be useful for malaria control programmes. This study assessed the profile of dhfr and dhps genes genotypes among individuals with malaria in Lagos, Nigeria. METHODS:Molecular markers of SP resistance were identified by nested PCR and sequenced among malaria positive dried blood spots (DBS) that were collected from individuals attending health facilities from January 2013 to February 2014 and during community surveys from October 2010 to September 2011 across different Local Government Areas of Lagos State, Nigeria. RESULTS:A total of 242 and 167 samples were sequenced for dhfr and dhps, respectively. Sequence analysis of dhfr showed that 95.5% (231/242), 96.3% (233/242) and 96.7% (234/242) of the samples had N51I, C59R and S108N mutant alleles, respectively. The prevalence of dhps mutation at codons A437G, A613S, S436A, A581G, I431V and K540E were 95.8% (160/167), 41.9% (70/167), 41.3% (69/167), 31.1% (52/167), 25.1% (42/167), and 1.2% (2/167) respectively. The prevalence of triple mutations (CIRNI) in dhfr was 93.8% and 44.3% for the single dhps haplotype mutation (SGKAA). Partial SP-resistance due to quadruple dhfr-dhps haplotype mutations (CIRNI-SGKAA) and octuple haplotype mutations (CIRNI-VAGKGS) with rate of 42.6% and 22.0%, respectively has been reported. CONCLUSIONS:There was increased prevalence in dhfr triple haplotype mutations when compared with previous reports in the same environment but aligned with high prevalence in other locations in Nigeria and other countries in Africa. Also, high prevalence of dhfr and dhps mutant alleles occurred in the study areas in Lagos, Nigeria five to eight years after the introduction of artemisinin combination therapy underscores the need for continuous monitoring.