Dataset Information

Characteristics of Plasmodium falciparum dhfr haplotypes that confer pyrimethamine resistance, Kilifi, Kenya, 1987--2006.

ABSTRACT: Resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP) emerged in Plasmodium falciparum from Asia in the 1960s and subsequently spread to Africa. It is not known whether alleles that confer SP resistance also arose independently in Africa. We defined the coding region and microsatellite haplotypes of dhfr alleles in P. falciparum collected in Kilifi, Kenya, during 1987--2006, which spans the period when SP was first introduced. Isolates that carried a double-mutant or triple-mutant dhfr allele were detected at a low frequency, even during 1987--1988. Each of 2 double mutants carried a unique haplotype, and both were related to wild-type haplotypes from the same population. The number of isolates that carried a triple-mutant dhfr allele increased rapidly after introduction of SP and shared the haplotype of the triple mutant derived form Asia. We observed no triple-mutant alleles with haplotypes related to those of the Africa-derived wild-type and double-mutant alleles.

SUBMITTER: Certain LK

PROVIDER: S-EPMC2680814 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characteristics of Plasmodium falciparum dhfr haplotypes that confer pyrimethamine resistance, Kilifi, Kenya, 1987--2006.

Certain Laura K LK Briceño Marnie M Kiara Steven M SM Nzila Alexis M AM Watkins William M WM Sibley Carol Hopkins CH

The Journal of infectious diseases 20080601 12

Resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP) emerged in Plasmodium falciparum from Asia in the 1960s and subsequently spread to Africa. It is not known whether alleles that confer SP resistance also arose independently in Africa. We defined the coding region and microsatellite haplotypes of dhfr alleles in P. falciparum collected in Kilifi, Kenya, during 1987--2006, which spans the period when SP was first introduced. Isolates that carried a double-mutant or triple-mutant d ...[more]

PMID: 18513156

Similar Datasets

Project description:BACKGROUND: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves. METHODS: Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the south-east of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC(50s) to pyrimethamine was performed on adapted isolates. RESULTS: Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC(50)) < 10 nM. The geometric mean of IC(50) of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC(50) of the triple mutant parasite (13,804 nM). CONCLUSION: The IC(50)s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC(50)s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the Pfdhfr I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.

Project description:Sulphadoxine-pyrimethamine (SP) resistance is now widespread throughout east and southern Africa and artemisinin compounds in combination with synthetic drugs (ACT) are recommended as replacement treatments by the World Health Organization (WHO). As well as high cure rates, ACT has been shown to slow the development of resistance to the partner drug in areas of low to moderate transmission. This study looked for evidence of protection of the partner drug in a high transmission African context. The evaluation was part of large combination therapy pilot implementation programme in Tanzania, the Interdisciplinary Monitoring Programme for Antimalarial Combination Therapy (IMPACT-TZ) METHODS: The growth of resistant dhfr in a parasite population where SP Monotherapy was the first-line treatment was measured for four years (2002-2006), and compared with the development of resistant dhfr in a neighbouring population where SP + artesunate (SP+AS) was used as the first-line treatment during the same interval. The effect of the differing treatment regimes on the emergence of resistance was addressed in three ways. First, by looking at the rate of increase in frequency of pre-existing mutant dhfr alleles under monotherapy and combination therapy. Second, by examining whether de-novo mutant alleles emerged under either treatment. Finally, by measuring diversity at three dhfr flanking microsatellite loci upstream of the dhfr gene.The reduction in SP selection pressure resulting from the adoption of ACT slowed the rate of increase in the frequency of the triple mutant resistant dhfr allele. Comparing between the two populations, the higher levels of genetic diversity in sequence flanking the dhfr triple mutant allele in the population where the ACT regimen had been used indicates the reduction in SP selection pressure arising from combination therapy.The study demonstrated that, alleles containing two mutations at the dhfr have arisen at least four times independently while those containing triple mutant dhfr arose only once, and were found carrying a single unique Asian-type flanking sequence, which apparently drives the spread of pyrimethamine resistance associated dhfr alleles in east Africa. SP+AS is not recommended for use in areas where SP cure rates are less than 80% but this study reports an observed principle of combination protection from an area where pyrimethamine resistance was already high.

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:Knowledge of how malaria infections spread locally is important both for the design of targeted interventions aiming to interrupt malaria transmission and the design of trials to assess the interventions. A previous analysis of 1602 genotyped Plasmodium falciparum parasites in Kilifi, Kenya collected over 12 years found an interaction between time and geographic distance: the mean number of single nucleotide polymorphism (SNP) differences was lower for pairs of infections which were both a shorter time interval and shorter geographic distance apart. We determine whether the empiric pattern could be reproduced by a simple model, and what mean geographic distances between parent and offspring infections and hypotheses about genotype-specific immunity or a limit on the number of infections would be consistent with the data. We developed an individual-based stochastic simulation model of households, people and infections. We parameterized the model for the total number of infections, and population and household density observed in Kilifi. The acquisition of new infections, mutation, recombination, geographic location and clearance were included. We fit the model to the observed numbers of SNP differences between pairs of parasite genotypes. The patterns observed in the empiric data could be reproduced. Although we cannot rule out genotype-specific immunity or a limit on the number of infections per individual, they are not necessary to account for the observed patterns. The mean geographic distance between parent and offspring malaria infections for the base model was 0.5?km (95% CI 0.3-1.5), for a distribution with 68% of distances shorter than the mean. Very short mean distances did not fit well, but mixtures of distributions were also consistent with the data. For a pathogen which undergoes meiosis in a setting with moderate transmission and a low coverage of infections, analytic methods are limited but an individual-based model can be used with genotyping data to estimate parameter values and investigate hypotheses about underlying processes.

Project description:There is a growing concern for malaria control in the Horn of Africa region due to the spread and rise in the frequency of Plasmodium falciparum Histidine-rich Protein (hrp) 2 and 3 deletions. Parasites containing these gene deletions escape detection by the major PfHRP2-based rapid diagnostic test. In this study, the presence of Pfhrp2/3 deletions was examined in uncomplicated malaria patients in Kilifi County, from a region of moderate-high malaria transmission. 345 samples were collected from the Pingilikani dispensary in 2019/2020 during routine malaria care for patients attending this primary health care facility. The Carestart™ RDT and microscopy were used to test for malaria. In addition, qPCR was used to confirm the presence of parasites. In total, 249 individuals tested positive for malaria by RDT, 242 by qPCR, and 170 by microscopy. 11 samples that were RDT-negative and microscopy positive and 25 samples that were qPCR-positive and RDT-negative were considered false negative tests and were examined further for Pfhrp2/3 deletions. Pfhrp2/3-negative PCR samples were further genotyped at the dihydrofolate reductase (Pfdhfr) gene which served to further confirm that parasite DNA was present in the samples. The 242 qPCR-positive samples (confirmed the presence of DNA) were also selected for Pfhrp2/3 genotyping. To determine the frequency of false negative results in low parasitemia samples, the RDT- and qPCR-negative samples were genotyped for Pfdhfr before testing for Pfhrp2/3. There were no Pfhrp2 and Pfhrp3 negative but positive for dhfr parasites in the 11 (RDT negative and microscopy positive) and 25 samples (qPCR-positive and RDT-negative). In the larger qPCR-positive sample set, only 5 samples (2.1%) were negative for both hrp2 and hrp3, but positive for dhfr. Of the 5 samples, there were 4 with more than 100 parasites/µl, suggesting true hrp2/3 deletions. These findings revealed that there is currently a low prevalence of Pfhrp2 and Pfhrp3 deletions in the health facility in Kilifi. However, routine monitoring in other primary health care facilities across the different malaria endemicities in Kenya is urgently required to ensure appropriate use of malaria RDTs.

Project description:BackgroundResistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited.MethodsTemporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed.ResultsThe prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7% in the first study (1996-2000) to 88% in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4% in 1998 to 44.4% three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008-2009 study, 5.3% of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples.ConclusionsThere was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.

Dataset Information

Characteristics of Plasmodium falciparum dhfr haplotypes that confer pyrimethamine resistance, Kilifi, Kenya, 1987--2006.

Publications

Characteristics of Plasmodium falciparum dhfr haplotypes that confer pyrimethamine resistance, Kilifi, Kenya, 1987--2006.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets