Prevalence of <i>Plasmodium falciparum</i> Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia

<div><p>Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of <i>Plasmodium falciparum</i> for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on <i>P</i>. <i>falciparum</i> isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (<i>pfcrt</i> and <i>pfmdr1</i>) and SP (<i>pfdhps</i> and <i>pfdhfr</i>) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both <i>pfcrt</i> K76T and <i>pfmdr1</i> N86Y mutations. All <i>P</i>. <i>falciparum</i> isolates harboured the <i>pfdhps</i> A437G mutation. Prevalence of <i>pfdhfr</i> gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of <i>pfcrt</i> and <i>pfmdr1</i> together with quintuple <i>pfdhps/pfdhfr</i> mutation (combination of <i>pfdhps</i> A437G+A581G and <i>pfdhfr</i> C59R+S108N+I164L). All <i>P</i>. <i>falciparum</i> isolates carried single copy number of <i>pfmdr1</i> and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control.</p></div

Frequency of combined <i>pfdhfr</i> /<i>pfdhps</i> mutants in isolates from Kota Marudu.

<p>Frequency of combined <i>pfdhfr</i> /<i>pfdhps</i> mutants in isolates from Kota Marudu.</p

Distribution of essential mutations for CQ, PYR and SDX resistance in Malaysia.

<p>The map shows the distribution of common SNPs for <i>P</i>. <i>falciparum</i> CQ, PYR and SDX resistant previously reported in Pahang [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref009" target="_blank">9</a>], Lundu, Sarawak [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref008" target="_blank">8</a>] and Sabah: Kalabakan (Tawau Division) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref030" target="_blank">30</a>], Keningau & Nabawan (Interior Division) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref039" target="_blank">39</a>], Kota Kinabalu (West Coast Division) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref035" target="_blank">35</a>], ^aKota Marudu—present study, ^bKudat [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref035" target="_blank">35</a>] (Kudat Division). This map was generated by using SimpleMappr online software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165515#pone.0165515.ref040" target="_blank">40</a>]. <i>Pfcrt</i>, <i>Plasmodium falciparum</i> chloroquine resistant gene; <i>Pfdhfr</i>, <i>Plasmodium falciparum</i> dihydrofolate reductase gene; <i>Pfdhps</i>, <i>Plasmodium falciparum</i> dihydropteroate synthase gene; CQ, chloroquine; SDX, sulphadoxine; PYR, pyrimethamine. Amino acids: A, alanine; G, glycine; I, isoleucine; K, lysine; L, leucine N, asparagine; S, serine; T, threonine.</p

Prevalence of CQ, PYR and SDX resistant marker genotypes in Malaysia reported from year 2003 to 2014.

<p>Prevalence of CQ, PYR and SDX resistant marker genotypes in Malaysia reported from year 2003 to 2014.</p

Frequency of wild type and mutant alleles of <i>pfcrt</i>, <i>pfmdr1</i>, <i>pfdhfr</i> and <i>pfdhps</i> in <i>P</i>. <i>falciparum</i> isolates from Kota Marudu, Sabah.

<p>Frequency of wild type and mutant alleles of <i>pfcrt</i>, <i>pfmdr1</i>, <i>pfdhfr</i> and <i>pfdhps</i> in <i>P</i>. <i>falciparum</i> isolates from Kota Marudu, Sabah.</p