Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in <it>Plasmodium falciparum</it> in Haiti

<p>Abstract</p> <p>Background</p> <p>Malaria caused by <it>Plasmodium falciparum</it> infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (<it>dhfr</it>) and dihydropteroate synthetase (<it>dhps</it>) genes that confer PYR and SDX resistance, respectively, in <it>P. falciparum</it> to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in <it>P. falciparum</it> in Haiti.</p> <p>Methods</p> <p>DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in <it>P. falciparum</it> using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the <it>dhfr</it> gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the <it>dhps</it> gene in <it>P. falciparum</it>.</p> <p>Results</p> <p>Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the <it>dhfr</it> gene. No mutations in <it>dhfr</it> at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in <it>dhps</it> at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban <it>vs</it> rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59).</p> <p>Conclusion</p> <p>This study has shown the presence of the S108N mutation in <it>P. falciparum</it> that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti.</p

Plot of the first two principal components of principal component analysis, color coded by study site.

<p>Plot of the first two principal components of principal component analysis, color coded by study site.</p

Allele frequency distribution in Haiti compared to high and low malaria transmission countries. Non-Haitian data taken from Anderson et al.[20]. Mebat and Buksak are sites within Papua New Guinea.

<p>Allele frequency distribution in Haiti compared to high and low malaria transmission countries. Non-Haitian data taken from Anderson et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140416#pone.0140416.ref020" target="_blank">20</a>]. Mebat and Buksak are sites within Papua New Guinea.</p

Map of study sites in Haiti. Study sites are indicated by the colored stars. Blue stars indicate the study sites where the majority of samples were collected.

<p>Map of study sites in Haiti. Study sites are indicated by the colored stars. Blue stars indicate the study sites where the majority of samples were collected.</p

Genetic Diversity of <i>Plasmodium falciparum</i> in Haiti: Insights from Microsatellite Markers

<div><p>Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of <i>Plasmodium falciparum</i> populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of <i>P</i>. <i>falciparum</i> samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global <i>P</i>. <i>falciparum</i> populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium (defined as non-random association of alleles from different loci). For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%), moderate to high levels of genetic diversity (mean number of alleles per locus = 4.9, heterozygosity = 0.61), low levels of population structure (highest pairwise F_st = 0.09 and no clustering in principal components analysis), and moderate linkage disequilibrium (ISA = 0.05, P<0.0001). In addition, population bottleneck analysis revealed no evidence for a reduction in the <i>P</i>. <i>falciparum</i> population size in Haiti. We conclude that the high level of genetic diversity and lack of evidence for a population bottleneck may suggest that Haiti’s <i>P</i>. <i>falciparum</i> population has been stable and discuss the implications of our results for understanding the impact of malaria control interventions. We also discuss the relevance of parasite population history and other host and vector factors when assessing transmission intensity from genetic diversity data.</p></div

Multiplicity of infection, mean number of alleles, and heterozygosity in Haiti compared to ranges observed in high and low malaria transmission populations.

<p>^¥ Data reported from Anderson et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140416#pone.0140416.ref020" target="_blank">20</a>].</p><p>Multiplicity of infection, mean number of alleles, and heterozygosity in Haiti compared to ranges observed in high and low malaria transmission populations.</p