Dihydrofolate-reductase mutations in plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia

Matthew Grigg, Bridget Barber, Jutta Marfurt, Mallika Imwong, Timothy William, Elspeth Bird, Kim Piera, Ammar Aziz, U Boonyuen, Chris Drakeley, J Cox, Nicholas J White, Q Cheng, Tsin Yeo, Sarah Auburn, Nicholas Anstey

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Abstract

Background: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (HH) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission.

Methods: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.

Results:
Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild- type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had doublemutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.

Conclusion: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans. � 2016 Grigg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Original languageEnglish
Article numbere0149519
Pages (from-to)1-19
Number of pages19
JournalPLoS One
Volume11
Issue number3
DOIs
Publication statusPublished - 2016

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Plasmodium knowlesi
dihydrofolate reductase
Tetrahydrofolate Dehydrogenase
Malaysia
Borneo
Oxidoreductases
mutation
Pressure
drugs
Mutation
Pharmaceutical Preparations
pyrimethamine
Pyrimethamine
mutants
Genotype
malaria
Malaria
Genes
genotype
Folic Acid Antagonists

Cite this

Grigg, Matthew ; Barber, Bridget ; Marfurt, Jutta ; Imwong, Mallika ; William, Timothy ; Bird, Elspeth ; Piera, Kim ; Aziz, Ammar ; Boonyuen, U ; Drakeley, Chris ; Cox, J ; White, Nicholas J ; Cheng, Q ; Yeo, Tsin ; Auburn, Sarah ; Anstey, Nicholas. / Dihydrofolate-reductase mutations in plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia. In: PLoS One. 2016 ; Vol. 11, No. 3. pp. 1-19.
@article{bf3b724f52244e96b478b00342d71f56,
title = "Dihydrofolate-reductase mutations in plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia",
abstract = "Background: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (HH) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. Methods: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Results: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2{\%}) and R34L (10.0{\%}), resulting in 21 different genotypes, including the wild- type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32{\%}) patients had single mutants and 14 (3{\%}) had doublemutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4{\%} and 8{\%}, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Conclusion: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans. � 2016 Grigg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",
author = "Matthew Grigg and Bridget Barber and Jutta Marfurt and Mallika Imwong and Timothy William and Elspeth Bird and Kim Piera and Ammar Aziz and U Boonyuen and Chris Drakeley and J Cox and White, {Nicholas J} and Q Cheng and Tsin Yeo and Sarah Auburn and Nicholas Anstey",
note = "Australian NHMRC (Grants 1037304 and 1045156)",
year = "2016",
doi = "10.1371/journal.pone.0149519",
language = "English",
volume = "11",
pages = "1--19",
journal = "PLoS One",
issn = "1932-6203",
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}

Dihydrofolate-reductase mutations in plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia. / Grigg, Matthew; Barber, Bridget; Marfurt, Jutta; Imwong, Mallika; William, Timothy; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Boonyuen, U; Drakeley, Chris; Cox, J; White, Nicholas J; Cheng, Q; Yeo, Tsin; Auburn, Sarah; Anstey, Nicholas.

In: PLoS One, Vol. 11, No. 3, e0149519, 2016, p. 1-19.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Dihydrofolate-reductase mutations in plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia

AU - Grigg, Matthew

AU - Barber, Bridget

AU - Marfurt, Jutta

AU - Imwong, Mallika

AU - William, Timothy

AU - Bird, Elspeth

AU - Piera, Kim

AU - Aziz, Ammar

AU - Boonyuen, U

AU - Drakeley, Chris

AU - Cox, J

AU - White, Nicholas J

AU - Cheng, Q

AU - Yeo, Tsin

AU - Auburn, Sarah

AU - Anstey, Nicholas

N1 - Australian NHMRC (Grants 1037304 and 1045156)

PY - 2016

Y1 - 2016

N2 - Background: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (HH) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. Methods: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Results: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild- type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had doublemutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Conclusion: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans. � 2016 Grigg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AB - Background: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (HH) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. Methods: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Results: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild- type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had doublemutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Conclusion: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans. � 2016 Grigg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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