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Table of Contents
Year : 2019  |  Volume : 56  |  Issue : 2  |  Page : 170-173

Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran

1 Department of Parasitology and Mycology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
2 Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Infectious Disease Research Center, Birjand University of Medical Sciences, Birjand, Iran
4 Department of Medical Entomology and Vector Control, School of Public Health, International Campus, Tehran University of Medical Sciences, Tehran, Iran
5 Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Submission16-Jan-2018
Date of Acceptance23-Feb-2018
Date of Web Publication31-Jul-2019

Correspondence Address:
Gholamreza Hatam
Department of Parasitology, Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.263716

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Molecular analysis of antifolate resistance-associated genes—dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) of Plasmodium vivax is important in predicting the emergence of drug resistance to sulphadoxine-pyrimethamine (SP). The present study aimed to determine the polymorphism of dhfr and dhps genes in P. vivax field isolates. Samples from 80 microscopically diagnosed vivax malaria cases were collected from endemic areas of malaria in Hormozgan Province of Iran, from June 2010 to November 2015. The two sets of codons at position 33, 57, 58, 117, 173 of dhfr and 382, 383, and 553 of dhps genes were analysed by direct sequencing of PCR products. The majority of the isolates (70%) harboured a wild-type allele for P. vivax dhfr (Pvdhfr) and P. vivax dhps (Pvdhps). Mutations were detected in three codons of Pvdhfr (P33L, S58R and S117N) and single codon in Pvdhps (A383G). Novel mutations that have not been identified previously at codon 459 (D459A) of Pvdhps were also observed. The high prevalence of point mutation as well as the rising triple mutation of Pvdhfr and Pvdhps genotypes necessitate change in programmes and guidelines to eliminate P. vivax in future.

Keywords: Dhfr; dhps; drug resistance markers; Hormozgan; Iran; mutation, Plasmodium vivax

How to cite this article:
Maghsoodloorad S, Hosseinzadeh N, Haghighi A, Solgi R, Yusuf MA, Hatam G. Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran. J Vector Borne Dis 2019;56:170-3

How to cite this URL:
Maghsoodloorad S, Hosseinzadeh N, Haghighi A, Solgi R, Yusuf MA, Hatam G. Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran. J Vector Borne Dis [serial online] 2019 [cited 2020 Jul 6];56:170-3. Available from: http://www.jvbd.org/text.asp?2019/56/2/170/263716

Among the five species of Plasmodium causing malaria in humans, Plasmodium vivax is most widely distributed in non-African endemic areas and around 2.5 billion people are at risk of infection[1]. Though, it has lower mortality and morbidity rate in comparison with P. falciparum, it accounts for substantial morbidity and economic burden in endemic countries[2]. Due to successful implementation and execution of long-time prevention and control programs in Iran, malaria has vanished from most parts of Iran, although rare cases have been observed in the southern regions[1]. The studies in Iran have reported P. vivax as the predominant malaria species[3],[4]. Chloroquine and primaquine have been used as the main drug for eradication of both asexual stages and hypnozoites of this species in endemic regions; while, sulphadoxine-pyrimethamine (SP, Fansidar®, Hofffman-La Roche, Basel, Switzerland) has been introduced as the first-line treatment for falciparum malaria. However, in recent years, resistance to this drug has been reported in some regions[5],[6]. Resistance to Fansidar occurs by specific point mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). These mutations result in change in key amino acids, in active site of the enzymes and consequently lower the affinity of the enzyme to the drug[4]. Widespread drug resistance in P. vivax has turned out to be a major public health problem in many regions and point mutations within the dhfr and dhps genes are thought to be the main cause of resistance to antimalarial drugs[7]. Moreover, more than 20 different Pvdhfr alleles have been described[7]. Finding these mutations in field-collected blood samples can be a very important tool in mapping and monitoring resistance and thus guiding malaria control measures[8].

Although, Fansidar is not commonly prescribed for the infections caused by P. vivax, it can be used to treat P. vivax-induced infections due to possibility of mixed infections and failure in true diagnosis[9]. It is noteworthy that in regions where Fansidar is not widely used, no point mutation has been reported in dhps[8]. Previous studies have shown mutations of the Pvdhfr and Pvdhps genes in south and southeast of Iran[10],[11]. However, there is a lack of complete data on Pvdhfr and Pvdhps genotypes among Iranian cases, especially in endemic provinces and neighbouring countries. Hence, the present study was formulated for genetic analysis of the dhfr and dhps genes in P. vivax in the endemic Hormozgan Province in south of Iran. The results obtained from this study can be used to predict the genes responsible for resistance to sulphadoxine in P. vivax and can be very helpful in planning for selection and substitution of new drugs, prevention of resistance to drugs, and launching effective control and preventive programs for malaria.

Statistical population

The Hormozgan Province, located in the south of Iran, is one of the warmest and driest places of Iran. It is an endemic malarious region in Iran[12]. In this study, 80 P vivax clinical isolates were collected from consented P. vivax infected patients (aged from 15 to 60 yr-old) who were seeking malaria treatment at Primary Health Care Centers, in Hormozgan Province between June 2010 and November 2015 (30 and 50 isolates from Minab and Jask towns located in Hormozgan Province, respectively). All the subjects had slide and PCR-proven infection by P. vivax[13]. Blood samples were collected in tubes containing EDTA and stored at 4 °C.

DNA extraction and nested PCR

Parasite DNA was extracted from 250 μl infected whole blood using a QIAamp DNA extraction mini-kit (QIAGEN) and used as template for PCR amplification. Point mutations in different variants of Pvdhfr and Pvdhps genes were investigated in all P. vivax isolates with specific primers by nested PCR reactions, followed by sequencing analysis as described previously[14]. Primers were designed on the basis of complete P. vivax strain sequence (Accession No. X98123 for Pfdhfr and AY186730 for Pvdhps) available in the GenBank. Amplification was performed in a final volume of 25 μl PCR reaction, containing 125 μM each dNTP, 2 mM MgCl2, 250 nM each primer, 1 mM spermidine, 1 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), and 2 μl template from either genomic DNA or the primary reaction. The amplicon produced by NEST I reaction was used as the template DNA in NEST II reaction. All the PCR products obtained from the nested PCR were subjected to electrophoresis on 1.5% agarose gel. For detection of point mutation at residues P33L, F57I/L, S58R, S117 N/T and I173L of Pvdhfr as well as S382A, A383G, A553G of Pvdhps, all the samples were sequenced in Digestive Disease Research Institute, Iran. The mutation at these codons were found to be involved in clinical antifolate resistance[12]. The sequences were translated through a translation tool, available online at the Expert Protein Analysis System (ExPASy) proteomic server[15]. Translated sequences were aligned using multiple sequence alignment tool, ClustalW2[16] and compared with the wild-type allele sequences (Accession No. X98123 and AY186730 for Pvdhfr and Pvdhps, respectively) in order to determine possible mutations. Polymorphisms of these two genes were confirmed by reading both the forward and reverse strands.

Detection of mutations in Pvdhfr and Pvdhps genes

All the 80 samples were found to be infected with P. vivax as mono-infection in both the microscopy and PCR methods and successfully analysed for targeted single nucleotide polymorphisms (SNPs) in both the Pvdhfr and Pvdhps genes. Sequence comparison revealed that 56 isolates carried the wild-type allele for Pvdhfr; the remaining isolates (24) carried mutant Pvdhfr genotypes. In Pvdhfr, polymorphisms at positions 33L, 58R, 117N were found in 2.5, 16.2 and 21.2% of isolates, respectively. In case of Pvdhps gene, polymorphisms were found only at position 383G in 6.2% of the isolates [Table 1].
Table 1: The distribution of SNPs combinations of Pvdhfr and Pvdhps alleles associated with sulphadoxine- pyrimethamine in P. vivax isolates

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Distribution of Pvdhfr-Pvdhps haplotypes in Iran

The combination of Pvdhfr and Pvdhps genes among all the 80 samples in this study demonstrated six identical haplotypes. The two most prevalent haplotypes among all examined samples were P33F57 S58S117I173/S382A383A553D459 (70%); and P33F57S58N117I173/S382A383A553D459 (11.2%). The double mutants P33F57R58S117I173/S382G383A553D459; and P33F57R58N117I173/S382A383A553D459; as well as triple mutants L33F57R58N117I173/S382A383A553D459 (mutations in boldface) were found in 6.2, 7.5 and 2.5% samples, respectively. The single mutation P33F57S58S117I173/S382 A383A553A459 was the lowest prevalent sequence (2.5%).

The present study was performed in order to analyse the Pvdhfr and Pvdhps genes mutation in P. vivax in the Hormozgan Province of Iran for recognition of mutation at codons 33, 57, 58, 117, 173 and 382, 383, 553 related to antifolate drug resistance. Genetic diversity of Pvdhfr and Pvdhps is well-known in some parts of Iran where P. vivax usually co-exists with P. falciparum. So, the choice of effective treatment is crucial to prevent the emergence and spread of the resistance[17]. The prevalence and distribution of the resistant allelic types in this study are similar to those reported earlier from Malaysia[18] and Iran[5], while it was different with those isolated from Thailand[19] and Madagascar[20]. The most common haplo- types of Pvdhfr were the wild type and double mutants. Quadruple mutants were not detected in any of the examined isolates. An earlier study[21] in Afghanistan reported similar frequencies of Pvdhfr mutant alleles in codons 58 and 117.

The mutation in both the related genes was seen in 30% of the isolates. The study did not find mutations at codon F57I/L which have been reported from different parts of the country by various researchers[22]. It has been hypothesized that the S117N mutation is the first step in the drug resistance selection process, and S117T has been strongly associated with SP resistance in areas with extensive use of SP[23], but S117T mutation was not found in the present study. Mutations in codons 382, 383 and 553 of Pvdhps were more prevalent in areas with extensive use of SP than in those with low SP use[24]. In the present study, the mutation was seen only at codon 383 among key codons related to sulfadoxine resistance of Pvdhps gene. Furthermore, two patients (2.5%) showed mutations at codon 459 of Pvdhps for the first time.

This is the first study to report that pvdhps mutant genes are associated with the SP resistance among the positive Iranian P. vivax patients. In the present study, the most common haplotypes of Pvdhfr were wild type and single mutant (117N); the double and triple mutant were seen in 11 and 2 patients, respectively but quadruple mutants were not detected among the isolates examined. These results were inconsistent with an earlier study in Iran[25]. In case of Pvdhfr/Pvdhps haplotypes, double mutation (R58-G383) and triple mutation (R58-N117-L33) were reported, with low prevalence. The findings suggest that P. vivax parasites in Hormozgan may still be susceptible to SP, but additional caution should be taken for treatment of P. vivax malaria in Iran. Consequently, permanent surveillance ofP vivax molecular markers is necessary to check the expansion of SP resistance.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

Written informed consent was obtained by all the cases before inclusion in the study. The protocol of this study was reviewed and received Ethical clearance (357; March 16, 2010) from the Shahid Beheshti University of Medical Sciences, Tehran.

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