|SHORT RESEARCH COMMUNICATION
|Year : 2019 | Volume
| Issue : 2 | Page : 170-173
Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran
Somayeh Maghsoodloorad1, Nahid Hosseinzadeh2, Ali Haghighi2, Rahmat Solgi3, Mustapha Ahmed Yusuf4, Gholamreza Hatam5
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 Submission||16-Jan-2018|
|Date of Acceptance||23-Feb-2018|
|Date of Web Publication||31-Jul-2019|
Department of Parasitology, Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz
Source of Support: None, Conflict of Interest: None
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 Mar 31];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. Though, it has lower mortality and morbidity rate in comparison with P. falciparum, it accounts for substantial morbidity and economic burden in endemic countries. 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. The studies in Iran have reported P. vivax as the predominant malaria species,. 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,. 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. 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. Moreover, more than 20 different Pvdhfr alleles have been described. 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.
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. It is noteworthy that in regions where Fansidar is not widely used, no point mutation has been reported in dhps. Previous studies have shown mutations of the Pvdhfr and Pvdhps genes in south and southeast of Iran,. 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.
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. 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. 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. 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. The sequences were translated through a translation tool, available online at the Expert Protein Analysis System (ExPASy) proteomic server. Translated sequences were aligned using multiple sequence alignment tool, ClustalW2 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|
Click here to view
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. The prevalence and distribution of the resistant allelic types in this study are similar to those reported earlier from Malaysia and Iran, while it was different with those isolated from Thailand and Madagascar. 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 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. 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, 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. 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. 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.
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.
| References|| |
Howes RE, Battle KE, Mendis KN, Smith DL, Cibulskis RE, Baird JK, et al
. Global epidemiology of Plasmodium vivax. Am J Trop Med Hyg
Gholizadeh S, Naseri N, Zakeri S, Djadid ND. The role of molecular techniques on malaria control and elimination programs in iran: A review article. Iran J Parasitol
Raeisi A, Nikpoor F, Ranjbar Kahkha M, Faraji L. The trend of malaria in Iran from 2002 to 2007. Hakim Res J
Reza YM, Taghi RM. Prevalence of malaria infection in Sarbaz, Sistan and Baluchistan Provinces. Asian Pac J Trop Biomed
2011; 1(6): 491-2.
Sharifi-Sarasiabi K, Haghighi A, Kazemi B, Taghipour N, Nazemalhosseini E, Gachkar L. Molecular surveillance of Plasmodium vivax
and Plasmodium falciparum dhfr
mutations in isolates from southern Iran. Rev Inst Med Trop Sao Paulo
Imwong M, Pukrittakayamee S, Looareesuwan S, Pasvol G, Poirreiz J, White NJ, et al
. Association of genetic mutations in Plasmodium vivax dhfr
with resistance to sulfadoxine-pyri-methamine: Geographical and clinical correlates. Antimicrob Agents Chemother
Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH. Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol
2007; 23(5): 213-22.
Imwong M, Pukrittayakamee S, Cheng Q, Moore C, Looa-reesuwan S, Snounou G, et al
. Limited polymorphism in the dihydropteroate synthetase (dhps)
gene of Plasmodium vivax
isolates from Thailand. Antimicrob Agents Chemother
Snounou G, White NJ. The co-existence of Plasmodium:
Sidelights from falciparum and vivax malaria in Thailand. Trends Parasitol
2004; 20(7): 333.
Mirahmadi H, Rafee M, Zaman J, Mehravaran A, Shafiei R. Mutational analysis of Plasmodium vivax dhfr
gene among cases in southeast of Iran. DNA
Maghsoodloorad S, Haghighi A, Sharifi Sarasiabi K, Taghipoor N, Hosseinzadeh N, Gachkar L, et al
. Genetic diversity of dihydropteroate synthetase (dhps)
gene of Plasmodium vivax
in Hormozgan Province, Iran. Iran J Parasitol
Hatam GR, Nejati F, Mohammadzadeh T, Shahriari R, Sarkari B. Population based seroprevalence of malaria in Hormozgan Province, southestern Iran: A low transmission area. Malar Res Treat
Snounou G, Singh B. Nested PCR analysis of Plasmodium
parasites. Methods Mol Med
Das S, Banik A, Hati AK, Roy S. Low prevalence of dihydro- folate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax
isolates of West Bengal, India. Malar J
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The proteomics server for indepth protein knowledge and analysis. Nucleic Acids Res
2003; 31(13): 3784-8.
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al
. Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics
2012; 28(12): 1647-9.
Mula P, Fernandez-Martinez A, de Lucio A, Ramos JM, Reyes F, Gonzalez V, et al
. Detection of high levels of mutations involved in antimalarial drug resistance in Plasmodium falci-parum
and Plasmodium vivax
at a rural hospital in southern Ethiopia. Malar J
Sastu UR, Abdullah NR, Norahmad NR, Saat MNF, Muniandy PK, Jelip J. Mutations of pvdhfr
genes in vivax
endemic-malaria areas in Kota Marudu and Kalabakan, Sabah. Malar J
Rungsihirunrat K, Sibley CH, Mungthin M, Na-Bangchang K. Geographical distribution of amino acid mutations in Plasmodium vivax
dhfr and dhps from malaria endemic areas of Thailand. Am J Trop Med Hyg
2008; 78(3): 462-7.
Barnadas C, Tichit M, Bouchier C, Ratsimbasoa A, Randriana-solo L, Raherinjafy R, et al. Plasmodium vivax
dhfr and dhps mutations in isolates from Madagascar and therapeutic response to sulphadoxine-pyrimethamine. Malar J
Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Safi N, Butt W, et al
. Molecular surveillance of Plasmodium vivax
dhfr and dhps mutations in isolates from Afghanistan. Malar J
Afsharpad M , Zakeri S, Pirahmadi S, Djadid ND. Molecular assessment of dhfr/dhps mutations among Plasmodium vivax
clinical isolates after introduction of sulfadoxine-pyrimethamine in combination with artesunate in Iran. Infect Genet Evol
2012; 12(1): 38-44.
Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutan-to I, Ruckert P, et al
. Real-time PCR for dihydrofolate reductase
gene single-nucleotide polymorphisms in Plasmodium vivax
isolates. Antimicrob Agents Chemother
Ganguly S, Saha P, Chatterjee M, Maji AK. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr
in clinical isolates of Plasmodium vivax
from Kolkata, India. Antimicrob Agents Chemother
2014; 58(1): 196-200.
Sharifi K, Haghighi A, Gachkar L, Kazemi B, Taghipour N, Hosseinzadeh N. Molecular characterization of dihydrofolate reductase-thymidylate synthase
gene concerning antifolate resistance of Plasmodium vivax. Iran J Parasitol
2009; 4(4): 10-8.