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Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins

by Wang P, Lee CS, Bayoumi R, Djimde A, Doumbo O, Swedberg G, Dao LD, Mshinda H, Tanner M, Watkins WM, Sims P, Hyde JE

Published in 1997

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance

by Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avila JC, Cespedes JL, Carter D, Doumbo OK

Published in 1997

To assess the relationship between mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and clinical pyrimethamine-sulfadoxine resistance, polymerase chain reaction surveys and analyses for new mutations were conducted in four countries with increasing levels of pyrimethamine-sulfadoxine resistance: Mali, Kenya, Malawi, and Bolivia. Prevalence of mutations at DHFR codon 108 and a new mutation at DHPS 540 correlated with increased pyrimethamine-sulfadoxine resistance (P < .05). Mutations at DHFR 51, DHFR 59, and DHPS 437 correlated with resistance without achieving statistical significance. Mutations at DHFR 164 and DHPS 581 were common in Bolivia, where pyrimethamine-sulfadoxine resistance is widespread, but absent in African sites. Two new DHFR mutations, a point mutation at codon 50 and an insert at codon 30, were found only in Bolivia. DHFR and DHPS mutations occur in a progressive, stepwise fashion. Identification of specific sets of mutations causing in vivo drug failure may lead to the development of molecular surveillance methods for pyrimethamine-sulfadoxine resistance.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins

by Wang P, Lee CS, Bayoumi R, Djimde A, Doumbo O, Swedberg G, Dao LD, Mshinda H, Tanner M, Watkins WM, Sims P, Hyde JE

Published in 1997

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance

by Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avila JC, Cespedes JL, Carter D, Doumbo OK

Published in 1997

To assess the relationship between mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and clinical pyrimethamine-sulfadoxine resistance, polymerase chain reaction surveys and analyses for new mutations were conducted in four countries with increasing levels of pyrimethamine-sulfadoxine resistance: Mali, Kenya, Malawi, and Bolivia. Prevalence of mutations at DHFR codon 108 and a new mutation at DHPS 540 correlated with increased pyrimethamine-sulfadoxine resistance (P < .05). Mutations at DHFR 51, DHFR 59, and DHPS 437 correlated with resistance without achieving statistical significance. Mutations at DHFR 164 and DHPS 581 were common in Bolivia, where pyrimethamine-sulfadoxine resistance is widespread, but absent in African sites. Two new DHFR mutations, a point mutation at codon 50 and an insert at codon 30, were found only in Bolivia. DHFR and DHPS mutations occur in a progressive, stepwise fashion. Identification of specific sets of mutations causing in vivo drug failure may lead to the development of molecular surveillance methods for pyrimethamine-sulfadoxine resistance.

Molecular basis of in vivo resistance to sulfadoxine-pyrimethamine in African adult patients infected with Plasmodium falciparum malaria parasites

by Basco LK, Tahar R, Ringwald P

Published in 1998

In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

Molecular epidemiology of malaria in Yaounde, Cameroon II. Baseline frequency of point mutations in the dihydropteroate synthase gene of Plasmodium falciparum

by Basco LK, Ringwald P

Published in 1998

Sulfadoxine-pyrimethamine is one of the alternative antimalarial drugs used to treat chloroquine-resistant Plasmodium falciparum malaria. The molecular target of sulfadoxine, an analog of p-aminobenzoic acid that inhibits the folate biosynthetic pathway, is dihydropteroate synthase (DHPS). The nucleotide sequence of the DHPS gene was determined in 32 clinical isolates obtained in Yaounde, Cameroon, and compared with the sequence of reference clones and Cambodian strains of P. falciparum. Of the 32 Cameroonian isolates, 31 displayed one of the sulfadoxine-sensitive mutation patterns: Ala-436/Ala-437/Ala-581/Ala-613 (n = 20), Ser-436/Gly-437/Ala-581/Ala-613 (n = 6), Ser-436/Ala-437/Ala-581/Ala-613 (n = 4), and Ala-436/Gly-437/Ala-581/Ala-613 (n = 1). One isolate had a sulfadoxine-resistant profile characterized by a double mutation: Phe-436/Ala-437/Ala-581/Ser-613. Although the majority of the isolates had a sulfadoxine-sensitive genetic profile, further studies are needed to correlate the mutation patterns and in vitro and in vivo sulfadoxine sensitivity.

Molecular basis of in vivo resistance to sulfadoxine-pyrimethamine in African adult patients infected with Plasmodium falciparum malaria parasites

by Basco LK, Tahar R, Ringwald P

Published in 1998

In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

Molecular epidemiology of malaria in Yaounde, Cameroon II. Baseline frequency of point mutations in the dihydropteroate synthase gene of Plasmodium falciparum

by Basco LK, Ringwald P

Published in 1998

Sulfadoxine-pyrimethamine is one of the alternative antimalarial drugs used to treat chloroquine-resistant Plasmodium falciparum malaria. The molecular target of sulfadoxine, an analog of p-aminobenzoic acid that inhibits the folate biosynthetic pathway, is dihydropteroate synthase (DHPS). The nucleotide sequence of the DHPS gene was determined in 32 clinical isolates obtained in Yaounde, Cameroon, and compared with the sequence of reference clones and Cambodian strains of P. falciparum. Of the 32 Cameroonian isolates, 31 displayed one of the sulfadoxine-sensitive mutation patterns: Ala-436/Ala-437/Ala-581/Ala-613 (n = 20), Ser-436/Gly-437/Ala-581/Ala-613 (n = 6), Ser-436/Ala-437/Ala-581/Ala-613 (n = 4), and Ala-436/Gly-437/Ala-581/Ala-613 (n = 1). One isolate had a sulfadoxine-resistant profile characterized by a double mutation: Phe-436/Ala-437/Ala-581/Ser-613. Although the majority of the isolates had a sulfadoxine-sensitive genetic profile, further studies are needed to correlate the mutation patterns and in vitro and in vivo sulfadoxine sensitivity.

Low-dose treatment with sulfadoxine-pyrimethamine combinations selects for drug-resistant Plasmodium falciparum strains

by Kun JF, Lehman LG, Lell B, Schmidt-Ott R, Kremsner PG

Published in 1999

A total of 252 children were enrolled in a drug trial to assess the effect of minimal doses of sulfadoxine (Sdx) and pyrimethamine (Pyr). Parasite samples isolated from these patients were analyzed before and after treatment to investigate the level of drug-resistant strains. The parasite genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were assayed for point mutations that are associated with resistance against drugs. Before treatment, Pyr(r) genotypes of the DHFR gene were found in 42% of all samples, 8% of the patients harbored a mixed parasite population and 50% had a sensitive DHFR genotype. In terms of the DHPS gene, we found mutations in 45% of the parasites. Twenty-four percent had a Ser(436) mutation, and 26% had a Gly(437) mutation. Recrudescent parasites were highly enriched for both Pyr(r) and Sdx(r) strains after treatment (P < 0.001 and P = 0.029, respectively).

Low-dose treatment with sulfadoxine-pyrimethamine combinations selects for drug-resistant Plasmodium falciparum strains

by Kun JF, Lehman LG, Lell B, Schmidt-Ott R, Kremsner PG

Published in 1999

A total of 252 children were enrolled in a drug trial to assess the effect of minimal doses of sulfadoxine (Sdx) and pyrimethamine (Pyr). Parasite samples isolated from these patients were analyzed before and after treatment to investigate the level of drug-resistant strains. The parasite genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) were assayed for point mutations that are associated with resistance against drugs. Before treatment, Pyr(r) genotypes of the DHFR gene were found in 42% of all samples, 8% of the patients harbored a mixed parasite population and 50% had a sensitive DHFR genotype. In terms of the DHPS gene, we found mutations in 45% of the parasites. Twenty-four percent had a Ser(436) mutation, and 26% had a Gly(437) mutation. Recrudescent parasites were highly enriched for both Pyr(r) and Sdx(r) strains after treatment (P < 0.001 and P = 0.029, respectively).

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