Prevalence of polymorphisms at position 86 of the Pfmdr1 gene in Plasmodium falciparum parasites

Deepak Kumar; Vidyut Prakash; Gopal Nath

doi:10.4103/1687-7942.205167

ORIGINAL ARTICLE

Year : 2016 | Volume : 9 | Issue : 2 | Page : 103-105

Prevalence of polymorphisms at position 86 of the Pfmdr1 gene in Plasmodium falciparum parasites

Deepak Kumar, Vidyut Prakash, Gopal Nath
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

Date of Submission	19-Nov-2016
Date of Acceptance	14-Dec-2016
Date of Web Publication	25-Apr-2017

Correspondence Address:
Deepak Kumar
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1687-7942.205167

Abstract

Background Malaria is one of the major public health problems in India. The majority of cases are because of Plasmodium falciparum. A sudden increase in chloroquine (CQ) resistance in P. falciparum cases has been noted. Of the various genetic alteration genes known, Pfmdr1 has been shown to be associated with CQ resistance. Point mutations in the Pfmdr1 gene at several positions result in amino acid changes associated with CQ resistance. The mutation in codon 86 (from asparagine to tyrosine, N86Y) appears to be the most important.
Objective The aim of this study is to determine the prevalence of polymorphisms at position 86 of the Pfmdr1 gene among patients not responding to CQ therapy.
Materials and methods Blood samples of known P. falciparum cases not responding to CQ treatment were collected. DNA was isolated according to the manufacturer’s instructions. Nested PCR was performed to amplify the Pfmdr1 gene using commercially obtained primers. The finally amplified product was subjected to restriction digestion with AflIII (mutational allele) and ApoI (wild-type allele). The digests were resolved on a 3% agarose gel and stained with ethidium bromide.
Results A total of 25 P. falciparum isolates from patients not responding to CQ therapy were used in the study. Polymorphism was determined successfully in 16 isolates and classified as mutant-type Y86 (16) and wild-type N86 (9).
Conclusion A strong association was observed between point mutations in the Pfmdr1 gene, codon 86, and in vivo CQ resistance in these isolates.

Keywords: chloroquine resistance, pfmdr1 gene, Plasmodium falciparum

How to cite this article:
Kumar D, Prakash V, Nath G. Prevalence of polymorphisms at position 86 of the Pfmdr1 gene in Plasmodium falciparum parasites. Parasitol United J 2016;9:103-5

How to cite this URL:
Kumar D, Prakash V, Nath G. Prevalence of polymorphisms at position 86 of the Pfmdr1 gene in Plasmodium falciparum parasites. Parasitol United J [serial online] 2016 [cited 2023 Mar 30];9:103-5. Available from: http://www.new.puj.eg.net/text.asp?2016/9/2/103/205167

Introduction

Malaria remains a major public health problem in india, with plasmodium falciparum being the predominant species [1]. Chloroquine (CQ), the previously used drug in the treatment of uncomplicated P. falciparum malaria, is no longer the drug of choice in most malaria-endemic countries as a result of the emergence and spread of resistance [2]. Three complementary approaches were used to monitor drug resistance: follow-up of therapeutic failures by clinical trials, phenotypic determination of parasite sensitivity by a short-term in-vitro culture assay, and study of the prevalence of molecular markers associated with drug resistance [3]. Today, the molecular basis of antimalarial drugs is the method of choice to monitor drug resistance.

Current molecular studies of P. falciparum isolates suggest that few gene loci named as Pfmdr1 and Pfmdr2, and Pfcrt are associated with CQ resistance to P. falciparum [2]. The Pfmdr1 gene located on chromosome 5, which codes for Pgh1, a P-glycoprotein homologue, has generated interest in resistance to CQ and other antimalarials. It is a typical member of the ATP-binding cassette transporter superfamily localized to the parasite vacuole, where it may regulate intracellular drug concentrations [4].

Point mutations at various loci and polymorphisms in the Pfmdr1 gene have been implicated to varying degrees in CQ resistance. Of the various genetic alterations, point mutations in the Pfmdr1 gene at codon 86 (from asparagine to tyrosine, N86Y) appear to be the most extensively studied in different geographical areas [5],[6]. Only a few studies from India have assessed the prevalence of molecular markers associated with CQ resistance [7]. The main aim of this study is to determine the prevalence of polymorphisms at position 86 of the Pfmdr1 gene among the patients not responding to CQ therapy.

Materials and methods

Study design

The present study is an observational study, carried out in the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. The total duration of the study was 2 years: from July 2014 to June 2016. From the patients, a written consent was obtained and the study was approved by the Institute Ethical Committee. A total of 25 nonduplicate blood samples positive for P. falciparum by the immunochromatographic test (J. Mitra and Co. Pvt Ltd, New Delhi, India) from patients admitted to SS Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, and not responding to CQ treatment were collected and tested. These samples were stored at −20°C for further use.

DNA isolation

DNA was extracted from P. falciparum-positive blood samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). A volume of 200 μl of blood sample was used for extraction and DNA was eluted in 100 μl of elution buffer.

Primers

The amplification of the Pfmdr1 gene was carried out using a nested protocol. During primary reaction, primers P1 (5′-ATGGGTAAAGAGCAGAAAGA-3′) and P2 (5′-AACGCAAGTAATACATAAAGTCA-3′) were used to amplify the region flanking codon 86. Nested primers P3 (5′-TGGTAACCTCAGTATCAAAGAA-3′) and P4 (5′-ATAAACCTAAAAAGGAACTGG-3′) were used to amplify the PCR product in the secondary reaction [7].

Amplification of DNA

A PCR reaction was carried out in 25 µl volume. The reaction mix contained 10× reaction buffer (5 µl/sample), dNTPs (2 µl/sample), forward and reverse primers, and Taq polymerase enzyme (0.66 pmol). Amplification was carried out on a Bio Rad Thermal cycler (Bio-Rad Laboratories, Hercules, California, USA) with a heated lid. The hot-start method was used by heating at 94°C for 5 min initially. Thereafter, amplification was carried out for 35 cycles at 90°C for 1 min (denaturation), 65°C for 1 min (annealing), and 72°C for 1 min (extension). An extra extension was carried out at 72°C for 7 min.

The amplification products of primary PCR were again amplified with nested primers following the same protocol. The finally amplified product was subjected to restriction digestion with AflIII (mutational allele) and ApoI (wild-type allele). The digests were resolved on a 3% agarose gel and stained with ethidium bromide. Positive and negative controls were run with each batch of samples analyzed. Documentation of gel was performed using Gel Doc System (Bio-Rad Laboratories, Hercules, California, USA). DNA fragments were compared by size and the results obtained were analyzed.

Results

[Table 1] shows the results of nested PCR, followed by a restriction analysis to determine polymorphisms at codon 86 of the Pfmdr1 gene. On nested PCR for Pfmdr1, all the isolates showed the Pfmdr1 codon 86 region amplicon with a product size of 500 bp ([Figure 1]).

Table 1 Prevalence of polymorphisms at codon 86 of the Pfmdr1 gene in 25 isolates of Plasmodium falciparum

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Figure 1 Results of the nested PCR-amplified product for the Pfmdr1 gene in chloroquine treatment failure in 16 Plasmodium falciparum isolates. Lanes: L, molecular weight ladder; C, negative control; S1–S16, blood samples; Pfmdr1 codon 86 region amplicon with a product size of 500 bp.

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On digestion with the AflIII in CQ-resistant isolates, two fragments of 279 and 222 bp were generated, indicating a mutant allele at codon 86 (86T) ([Figure 2]; lanes S1, S4, S6, S7, S8, S10, S11, S12, and S14). ApoI did not digest the amplicon of the CQ-resistant strains at this site. Polymorphism was thus determined successfully in the 16 isolates ([Figure 2]) and classified as wild-type AsnN86 (9) and mutant-type TyrY86 (16). None of the isolates showed a mixed genotype of AsnN86+TyrY86 (0).

Figure 2 Results of restriction fragment length polymorphism after digestion of a PCR-amplified product with endonuclease, AflIII and ApoI for Pfmdr1 gene (86Y). Lanes: L, molecular weight ladder; C, negative control; S1, S4, S6, S7, S8, S10, S11, S12, and S14, two fragments of 279 and 222 bp, indicating a mutant allele at codon 86 (86T); S2, S3, S5, S9, and S13, a single band of 500 bp indicating no mutation in the Pfmdr1 gene.

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Discussion

Drug-resistant malaria has become a major problem in malaria control. Resistance to CQ in P. falciparum first emerged almost simultaneously in Southeast Asia (Thai–Cambodian border) and South America (Colombia) in the late 1950s [8],[9],[10]. Since then, CQ resistance has spread to all parts of the world where malaria is endemic. In India, CQ resistance was first reported in 1973 in Karbi-Anglong district and in 1974 in Nowgong district (Assam) [11]. Gradually, it has spread toward the west and south, covering almost the entire country [12].

The development of molecular techniques for the rapid identification of drug-resistant parasites is of immense importance for the epidemiology and information on the choice of antimalarial treatment regimens. In our study, by nested PCR, a strong association was observed between codon 86 mutations in all isolates and CQ resistance, hence confirming the role of this mutation in the CQ resistance. This finding corroborates well with other findings reported from different areas of the world [5],[13]. The Southeast Asian CQ-resistant isolates (K1 genotype) have shown the N86Y mutation [14]. A few studies have reported contrasting observations; the CQ-resistant South American isolates (7G8 genotype) were found to be negative for N86Y mutation [15]. In our study, 16 (64%) isolates were of mutant-type TyrY86, whereas nine (36%) failed to show polymorphisms at codon wild-type AsnN86 of the Pfmdr1 gene. An unidentified gene or mutation in the same gene at another locus is believed to play a role in our CQ resistance cases. Many studies based on gene knockout and transfection proposed that the Pfmdr1 gene alone is not sufficient to confer CQ resistance and other genes are also involved in the developmental process of drug resistance [16],[17]. More information on the genetics of drug resistance is needed to help design novel and improved molecular-based tools for early detection and to prevent the emergence of new foci of drug resistance.

Conclusion

Point mutation in Pfmdr1, at codon 86, has a strong association with CQ treatment failure in P. falciparum. Thus N86Y mutation in Pfmdr1 can be a good molecular marker of CQ resistance in P. falciparum.

Acknowledgements

Authors would like to acknowledge their thanks to Satyendra Kumar Kausal for helping in collection of samples.

Author contribution: All authors contributed equally.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Shah N, Dhillon G, Dash A, Arora U, Meshnick S, Valecha N. Antimalarial drug resistance of Plasmodium falciparum in India: changes over time and space. Lancet Infect Dis 2011; 11:57–64.
2.	White N. Antimalarial drug resistance. J Clin Invest 2004; 113:1084–1092.
3.	Andriantsoanirina V, Lascombes V, Ratsimbasoa A, Menard D. Rapid detection of point mutations in Plasmodium falciparum genes associated with antimalarial drugs resistance by using high-resolution melting analysis. J Microbiol Methods 2009; 78:165–170.
4.	Duraisingh MT, Cowman AF. Contribution of the Pfmdr1 gene to antimalarial drug-resistance. Acta Trop 2005; 94:181–190.
5.	Grobusch M, Adagu I, Kremsner P, Warhurst D. Plasmodium falciparum: in vitro chloroquine susceptibility and allele-specific PCR detection of Pfmdr1 Asn86Tyr polymorphism in Lambarene, Gabon. Parasitology 1998; 116:211–217.
6.	von Seidlein L, Duraisingh M, Drakeley C, Bailey R, Greenwood B, Pinder M. Polymorphism of the Pfmdr1 gene and chloroquine resistance in Plasmodium falciparum in the Gambia. Trans R Soc Trop Med Hyg 1997; 91:450–453.
7.	Shrivastava S, Gupta R, Mahanta J, Dubey M. Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India. PLoS One 2014; 9:e103848.
8.	Spencer H. Drug-resistant malaria: changing patterns mean difficult decisions. Trans R Soc Trop Med Hyg 1985; 79:748–758.
9.	Young MD, Moore DV. Chloroquine resistance in Plasmodium falciparum. Am J Trop Med Hyg 1961; 10:317–320.
10.	Wernsdorfer WH, Payne D. The dynamics of drug resistance in Plasmodium falciparum. Pharmacol Ther 1991; 50:95–121.
11.	Sehgal PN, Sharma MID, Sharma SL, Gopal S. Resistance to chloroquine in falciparum malaria in Assam state, India. J Com Dis 1973; 5:175–180.
12.	Clyde DF. Variations in response of malaria parasites to drugs in Asia and Oceania. La Med Tropic 1987; 3:3–21.
13.	Adagu IS, Warhurst DC. Association of cg2 and Pfmdr1 genotype with chloroquine resistance in field samples of Plasmodium falciparum from Nigeria. Parasitology 1999; 119:343–348.
14.	Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C et al. Resistance to antimalarials in Southeast Asia and genetic polymorphisms in Pfmdr1. Antimicrob Agents Chemother 2003; 47:2418–2423.
15.	Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ et al. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 1990; 345:255–258.
16.	Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000; 403:906–909.
17.	Sharma YD. Genetic alteration in drug resistance markers of Plasmodium falciparum. Indian J Med Res 2005; 121:13–22.