MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment

MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment

Genetic Diversity of Dihydropteroate synthetase Gene (dhps) Of Plasmodium vivax in Hormozgan Province, Iran

Background: The present study was formulated in order to determine pol­ymorphism of dihydropteroate synthetase gene (dhps) of Plasmodium vivax (P. vivax) in Hormozgan Province, southern Iran and mutations at codons 382, 383, 512, 553, and 585 associated with resistance of P. vivax to sulfadoxine. Method: One-hundred eighteen isolates of P. vivax were prepared within 2007-2008 to determine dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene. The isolates were determined in the study of genetic diversity of dihy­dropteroate synthetase gene (dhps) of P. vivax. The study was performed via PCR test and nucleotide sequencing. Results: Of 118 blood samples infected by P. vivax, 46 and 72 samples be­longed to Minab and Jask, respectively. No mutation was detected at 5 target codons. However, among these 118 samples, three isolates (2.54%) were found to have a mutation at the new codon 421. Conclusion: Since mutation was detected in dihydrofolate reductase (Pvdhfr) gene in the same samples but no mutation was found at five main codons of Pvdhps gene, it can be concluded that P. vivax, considering their mutations in Pvdhfr, is still susceptible to sulfadoxine and therefore, to fansidar in Hor­mozgan Province, Southern Iran

Patient sampling details.

<p>Patient sampling details.</p

Neighbour-joining tree illustrating the genetic relatedness between the <i>P</i>. <i>vivax</i> isolates across 9 loci.

<p>Only isolates with complete genotyping data across all 9 loci are presented. The two isolates highlighted with black dots, 6J and 9J, exhibited differing <i>pvmdr1</i> multi-locus genotypes from the other isolates: L953-Y976-N1010-<u>F</u>0176 in 6J and 9J versus L953-Y976-N1010-<u>L</u>1076 in the other isolates. The dotted grey outlines illustrate the isolates with high ancestry to <i>K</i>1 and <i>K</i>2 at <i>K</i> = 2.</p

<i>P</i>. <i>vivax</i> prevalence maps.

<p>These maps were generated by the Malaria Atlas Project, University of Oxford. The colour scales reflect the model-based geostatistical point estimates of the annual mean <i>P</i>. <i>vivax</i> parasite rate in the 1–99 year age range (<i>Pv</i>PR_1-99) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166124#pone.0166124.ref017" target="_blank">17</a>] within the stable spatial limits of transmission in 2010. The approximate locations of the study sites described here are indicated with numbered open circles in the Iran panel: Bandar Lengeh County (1), Qeshm County (2), Minab County (3), Rudan County (4) and Jask County (5). All MAP maps are available to users under the CCAL 3.0. <a href="http://www.map.ox.ac.uk/about-map/open-access/" target="_blank">http://www.map.ox.ac.uk/about-map/open-access/</a>.</p

Summary of Pvmdr1 haplotypes.

<p>Summary of Pvmdr1 haplotypes.</p

Population structure.

<p>Bar plots illustrating the population structure at <i>K</i> = 2, <i>K</i> = 3 and <i>K</i> = 4. Each vertical bar represents an individual sample and each colour represents one of the <i>K</i> clusters (sub-populations) defined by STRUCTURE. For each sample, the predicted ancestry to each of the <i>K</i> sub-populations is represented by the colour-coded bars. <i>K</i>1 = red, <i>K</i>2 = orange, <i>K</i>3 = light green and <i>K</i>4 = dark green. Samples are ordered by date of collection (oldest to newest) within each of the Autochthonous, Imported and Unknown sample groups.</p

Complexity of infection and population diversity.

<p>Complexity of infection and population diversity.</p