Origins of the Recent Emergence of
            <i>Plasmodium falciparum</i>
            Pyrimethamine Resistance Alleles in Madagascar

Andriantsoanirina, Valérie; Bouchier, Christiane; Tichit, Magali; Jahevitra, Martial; Rabearimanana, Stéphane; Randrianjafy, Rogelin; Ratsimbasoa, Arsène; Mercereau‐Puijalon, Odile; Durand, Rémy; Ménard, Didier

doi:10.1128/aac.01511-09

Cited by 8 publications

(6 citation statements)

References 33 publications

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“…A 600 bp fragment of Pfdhfr gene encompassing codons 6 to 206 was amplified by nested PCR technique on both DNA extracts, as previously described [ 21 ]. Sequencing reactions were carried out with a ABI Prism BigDye Terminator cycle sequencing ready reaction kit and were run on a model 3730 xl genetic analyzer (Applied Biosystems, Courtaboeuf, France).…”

Section: Methodsmentioning

confidence: 99%

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

Andriantsoanirina

Durand

Pradines

et al. 2011

Malar J

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BackgroundRecently, 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.MethodsThirty 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 IC50s to pyrimethamine was performed on adapted isolates.ResultsFour 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 (IC50) < 10 nM. The geometric mean of IC50 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 IC50 of the triple mutant parasite (13,804 nM).ConclusionThe IC50s 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 IC50s 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.

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Section: Methodsmentioning

confidence: 99%

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

Andriantsoanirina

Durand

Pradines

et al. 2011

Malar J

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“…There is a synergistic effect in conferring resistance for SP with the quintuple mutant of pfdhfr and pfdhps , haplotype I51-R59-N108-G437-E540 [ 10 ]. Reports of increasing prevalence of these mutations have been reported in African malaria endemic areas including Ghana [ 8 , 11 – 13 ].…”

Section: Introductionmentioning

confidence: 99%

Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana

et al. 2018

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Sulfadoxine-pyrimethamine (SP) is used as malaria chemoprophylaxis for pregnant women and children in Ghana. Plasmodium falciparum resistance to SP is linked to mutations in the dihydropteroate synthase gene (pfdhps), dihydrofolate reductase gene (pfdhfr) and amplification of GTP cyclohydrolase 1 (pfgch1) gene. The pfgch1 duplication is associated with pfdhfr L164, a crucial mutant for high level pyrimethamine resistance which is rare in Ghana. The presence of amplified pfgch1 in Ghanaian isolates could be an indicator of the evolution of the L164 mutant. This study therefore determined the pfgch1 copy number variations and SP resistance mutations in clinical isolates from Ghana. One hundred and ninety-two (192) blood samples collected from children aged ≤14 years with uncomplicated malaria in 2013–14 and 2015–16 were used. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the pfgch1 copy number and nested PCR-Sanger sequencing used to detect mutations in pfdhps and pfdhfr genes. Twelve parasites (6.3%) harbored double copies of the pfgch1 gene out of the 192 samples. Of the 12, 75% had the pfdhfr I51-R59-N108, 92% had the pfdhps G437 mutant, 8% had the pfdhps E540 and 67% had the SP resistance haplotype IRNG. No L164 was detected in samples with amplified pfgch1. The rare T108 mutant associated with cycloguanil resistance showed predominance (60%) over N108 in the 2015–16 isolates. The observation of parasites with increased copy number of pfgch1 gene is indicative of the future evolution of the rare quadruple pfdhfr mutant, I51-R59-N108-L164, in Ghanaian parasites. Mutant pfdhps isolates also had increased gch1 copy number suggestive that it may also facilitate sulphadoxine resistance. The selection of parasites with pfgch1 gene amplification will enhance the sustenance and persistence of parasites with SP resistance in the country. Policy makers need to begin the search for a replacement chemoprophylaxis drug for malaria vulnerable groups in Ghana.

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“…These molecular markers for resistance are best characterized for chloroquine (9) and antifolate drugs (10)(11)(12). Antifolates are extensively used across endemic countries in intermittent preventive therapy programs for malaria in pregnant women and in children (13,14). Antifolates are also constituents of artemisinin combination therapy (ACT) regimens, such as proguanil-atovaquone-artesunate and artesunate-sulfadoxine/pyrimethamine.…”

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confidence: 99%

Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes

Mharakurwa

Kumwenda²,

Mkulama³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections-S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.

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Origins of the Recent Emergence of Plasmodium falciparum Pyrimethamine Resistance Alleles in Madagascar

Cited by 8 publications

References 33 publications

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana

Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes

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