Dataset Information

Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

ABSTRACT: 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.

SUBMITTER: Alker AP

PROVIDER: S-EPMC478490 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

Alker Alisa P AP Mwapasa Victor V Meshnick Steven R SR

Antimicrobial agents and chemotherapy 20040801 8

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, ...[more]

PMID: 15273102

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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:BACKGROUND:In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan. METHODS:A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing. RESULTS:The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R?+?108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R?+?108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance. CONCLUSION:The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS?+?SP being effective in Pakistan.

Dataset Information

Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

Publications

Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

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