Keywords

Plasmodium falciparum; Polymorphism; Chloroquine resistance; Sulfadoxine/pyrimethamine (SP) resistance; Artemisinin combination therapy (ACT)

Introduction

The emergence of anti-malarial drug resistance is dependent on the occurrence that causes spontaneous change genetically (mutation or gene amplification) in a malaria parasite. These genetic alterations of the parasitic gene cause susceptibility to a particular drug [1]. The resistance property of Plasmodium falciparum against available anti-malarial drugs is a major challenge towards malaria control in different endemic areas of the globe [2]. Combinations of different antimalarial drugs having different molecular targets, ultimately helps to delay the emergence of resistance [3]. Therefore recent malaria control programmes precisely focus on the adaption of different combination medicine against plasmodium infection [4]. Single use of antimalarial drugs now has been banned by the WHO, especially against P. falciparum. Mutations in different target genes can be used as molecular markers to detect the drugresistant parasite [5].

Onset of chloroquine (CQ) resistant falciparum malaria was reported from Thailand in 1957 [6] and from Cambodia in 1960, followed by sub-Saharan Africa and gradually it emerged in different parts the Southeast Asia and Africa [7]. In 1980s, CQresistant parasite rapidly increased in South and Central America [8]. Without replacing a particular drug having such low cost and reliability as CQ, the morbidity and motility resurged among the children in Africa [9]. The substitution of lysine to threonine (K76T) at the codon position 76 of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene is associated with in vivo as well as in vitro CQ resistance in Africa, South America as well as in Southeast Asia [10, 11]. Further it was proved that the point mutations associated with the entire pfcrt gene were found to be highly co related to in vitro CQ resistance among P. falciparum isolates from Africa, South America, and Southeast Asia. In India, CQ resistant P. falciparum was reported for first time in the North-Eastern state of Assam, in 1973 at Karbi-anglang subdivision [12]. Soon after it spread to many other areas in India [13].

The point mutation in different position of a gene on a chromosome (locus) (C72S, M741, N75E, K76T, A220S, Q271E, N326S, 1456T, R371I) of pfcrt gene, a transporter that is located at chromosome 7, may distinguish between CQ-resistance and CQ-sensitive strain. Various molecular analyses confer CQ resistance by allowing enhanced efflux of CQ from the digestive vacuole (DV) due to the genetic alteration in pfcrt gene [14]. The analysis of K76T mutation in pfcrt gene reveals its 100% association with the in vitro CQ resistance. Various nucleotide mutation that alters the amino acid sequence of a protein (non-synonymous) polymorphisms at codon 76 of pfcrt gene are found (K76I, K76N or K76T), with greater CQ efflux. A specific combination of pfcrt and pfmdrl alleles, resulting in varying responses to CQ, seems geographically restricted, which may explain why some field studies reported that there is an association between Plasmodium falciparum multidrugresistance gene (pfmdr1) polymorphisms and CQ resistance. Significant association between the pfmdr1 86Y and the EC of CQ among clones with the pfcrt 76T allele suggests the possible role of both mutations in development of CQ resistance.

Pfmdr1 is one of the principal genes that associate CQ resistance [15]. Mutation in the pfmdr-1 gene, located on chromosome 5 has some significant role in P. falciparum resistance towards various antimalarials, such as CQ, mefloquine, quinine, and artemisinin derivatives. In an earlier study, significant association (linkage disequilibrium, LD) between the alleles pfmdr1 86Y and pfcrt 76T has been observed [15]. Studies conducted in different geographical areas of the world have suggested that the point mutation of tyrosine at codon 86 (N-86 to 86-Y) is related to CQ resistance [16]. Several other pfmdr1 polymorphisms 184-phe, 1034-cys, 1042- asp and 1246-tyr are being implicated to vary the degrees in CQ resistance [16, 17]. In India pfmdr1 86-tyr and 184-phe are commonly observed mutant allele with high in vitro IC50 for CQ [18]. In some parts of the world, CQ resistance occurs due to polymorphisms in both pfcrt and pfmdr1 gene [19]. In India in vivo, CQ treatment failure and in vitro CQ resistance are linked with pfcrt 76-thr but not for pfmdr1 gene [18].

Dihydrofolatereductase (DHFR) is an important enzyme that converts Dihydrofolate (DHF) to Tetrahydrofolate (THF) at the presence of NADPH, which is used as H+ donor [20]. Folate biosynthesis is very important for P. falciparum parasite because it is a precursor of purine essential for DNA replication. To prevent parasite DNA replication antifolate drug is used which can inhibit these proteins. Though, sulfadoxine and pyrimethamine (SP) is combined drugs but the mode of action of each component is different. Sulfadoxine actively inhibits dihydropteroate synthase (DHPS), the enzyme is involved in folate synthesis, mediating the synthesis of dihydropteroate which results reduction of dihydropteroate. The reduction of dihydropteroate subsequently decreases dyhydrofolate synthesis, which lowers the substrate of dhfr and the activity of dhfr-inhibitor increases. In this way, concomitant blockage of dihydropteroate synthase (dhps) and the inhibition of Dihydro folate reductase (dhfr) results in the synergistic action and interruption of the DNA replication process. Pyrimethamine is a potent inhibitor of DHFR, which plays three main roles in the folate pathway in P. falciparum. It controls de novo folate synthesis by catalyzing the synthesis of tetrahydrofolate (THF). It also mediates the salvage of exogenous folate derivatives, 7, 8- dihydrofolate (DHF) and the completely oxidized folate, by reducing them to tetrahydrofolate. Due to lack of folic acid, parasite multiplication is stopped and gradually parasites are destroyed [21]. In vitro resistance to pyrimethamine is known to be associated with the key dhfr mutation at S108N codon. Additional mutations in dhfr N51I, C59R and I164L confer higher levels of resistance [22]. Mutation of dhfr profile demonstrated several varients over wild type allele. dhfr 108-asn is very common followed by mutation polymorphism at 51-Ile and 59- arg.

Sulfadoxine competes with the substrate that binds to the parasite enzyme P. falciparum dihydropteroate synthetase (pfdhps). A mutation at several amino acid positions of this enzyme reduces binding efficiency with the drug. For that reason, a higher amount of drug is required to inhibit the mutated pfdhps enzyme and the growth of parasite [23]. There are five different amino acid positions (436, 437, 540, 580 and 613) which undergo polymorphism in its mutated form and highly associates sulfadoxine resistance. The amino acid sequence of wild type pfdhps allele at these positions is the SAKAA haplotype. Mutation in pfdhps may start settling amino acid positions at 436 or 437, followed by the mutations at other amino acid positions. The higher the number of mutations in pfdhps gene is proportional to the higher level of drug resistance property shown by the parasite [24]. In vitro resistance to sulpfadoxine has been associated with the key mutation of dhps A437G; additional mutations in dhps S436A, K540E, A581G and A613T⁄S confer higher levels of resistance [25]. Previous studies reported the presence of 11 different genotypic variant of pfdhps in the Indian P. falciparum population [24]. The wild-type SAKAA allele of pfdhps gene was highly leading in parasite populations of all regions of India except Andaman and Nicobar Island [26].

In Artemesinin Combination Therapy (ACT), the artemisinin compounds rapidly reduce the parasite number whereas the typically long half-life drugs clear the remaining parasite population [27]. If the parasite becomes resistant to any of these compounds, both Parasite clearance time (PCT) and median Parasite clearance half-life (PCHL) increase leading to ACT failure [28]. Early ACT Failure indicates the parasite line is less susceptible to AS, while Delayed Parasite Clearance is usually associated with the reduced activity of fast-acting drug (artemisinins) and recrudescence is associated with the less efficacy of the long acting drugs (Anti-folate drugs). Artemisinin resistance is characterized by slow parasite clearance in Thailand and Cambodia [29, 30]. Clearance (assessed by microscopy) of sensitive P. falciparum parasite is achieved within 2 days in 95% of patients whereas artemisinin-resistant infections remain slide-positive for 3 or more days; treatment failure is more common in such infections after ACT [31]. Another cause of great concern is this artemisinin compounds are regarded as the last and final order of antimalarials.

Plasmodium falciparum kelch 13 (pfk13) polymorphisms after 441 codon has been accounted for reduced susceptibility to artemisinin in vitro (ring stage survivality>10% and increase IC50 of artemisinin) as well as with increase in parasite clearance halflife (PCHL>5 h) thereby resulting in vivo ACT treatment failure in different parts of the world [32]. In African rodent malaria, polymorphisms at 739F and 770F codon of ubiquitin binding protein 1 (ubp-1) gene associated reduce susceptibility of artemisinin. Although ubp-1 polymorphism have not been observed in human beings yet. Recent study revealed that ubiquitin binding protein plays together with Kelch protein. Pfnhe1 another gene, located on chromosome 13, contains a predicted Na⁺-H⁺ exchanger. It is one of the new implicated putative transporters that modulate parasite response to antimalarials [33]. Analysis of microsatellite variations provides a significant association between DNNND repeats in the Cterminal cytoplasmic domain of pfnhe1 and in vitro response to quinine (QN). Sometime cg2, a putative transporter that modulates intraparasitic drug concentrations [34]. Recently it has been proved that polymorphism in COXI, pfmrp and cytochrome P450 partially modulates the different antimalarial drug activity in vitro as well as in vivo.

Malaria parasite changes its genetic arrangement (polymorphism) of different candidate gene (pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13) which lead to inactivation of antimalarial drug. This phenomenon makes a major problem to eradicate malaria globally.

References

1. Peterson DS, Miller LH, Wellems TE (1995) Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins. Proceedings of the National Academy of Sciences 92: 7100-7104.
2. Trape JF, Tall A, Diagne N, Ndiath O, Ly AB, et al. (2011) Malaria morbidity and pyrethroid resistance after the introduction of insecticide-treated bednets and artemisinin-based combination therapies: a longitudinal study. Lancet Infect Dis 11: 925-932.
3. Phillips M, Phillips-Howard PA (1996) Economic implications of resistance to antimalarial drugs. Pharmacoeconomics 10: 225-238.
4. World Health Organization (WHO) (2003) Assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria. Geneva.
5. Chaijaroenkul W, Wisedpanichkij R, Na-Bangchang K (2010) Monitoring of in vitro susceptibilities and molecular markers of resistance of Plasmodium falciparum isolates from Thai-Myanmar border to chloroquine, quinine, mefloquine and artesunate. Acta Tropica 113: 190-194.
6. Payne D (1987) Spread of chloroquine resistance in Plasmodium falciparum. Parasitology Today 3: 241-246.
7. Awad-el-Kariem FM, Miles MA, Warhurst DC (1992) Chloroquine-resistant Plasmodium falciparum isolates from the Sudan lack two mutations in the pfmdr1 gene thought to be associated with chloroquine resistance. Trans R Soc Trop Med Hyg 86: 587-589.
8. Lopes D, Rungsihirunrat K, Nogueira F, Seugorn A, Gil JP, et al. (2002) Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand. Malar J 1: 1.
9. Trape JF, Pison G, Preziosi MP, Enel C, du Loû AD, et al. (1998) Impact of chloroquine resistance on malaria mortality. Comptes Rendus de l'Académie des Sciences-Series III-Sciences de la Vie 321: 689-697.
10. Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, et al. (2001) A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257-263.
11. Durand R, Jafari S, Vauzelle J, Delabre JF, Jesic Z, et al. (2001) Analysis of pfcrt point mutations and chloroquine susceptibility in isolates of Plasmodium falciparum. Mol Biochem Parasitol 114: 95-102.
12. Sehgal PN, Sharma MI, Sharma SL, Gogai S (1973) Resistance to chloroquine in falciparum malaria in Assam State, India. Journal of Communicable Diseases 5: 175-180.
13. Clyde DF, Herrington DA, Losonsky G, Cortesia M, Murphy JR, et al. (1987) Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 328: 257-259.
14. Bray PG, Mungthin M, Hastings IM, Biagini GA, Saidu DK, et al. (2006) PfCRT and the trans-vacuolar proton electrochemical gradient: Regulating the access of chloroquine to ferriprotoporphyrin IX. Mol Microbiol 62: 238-251.
15. Ghanchi NK, Ursing J, Beg MA, Veiga MI, Jafri S, et al. (2011) Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan. Malar J 10: 1.
16. Andriantsoanirina V, Ratsimbasoa A, Bouchier C, Jahevitra M, et al. (2009) Plasmodium falciparum drug resistance in Madagascar: facing the spread of unusual pfdhfr and pfmdr-1 haplotypes and the decrease of dihydroartemisinin susceptibility. Antimicrob Agents Chemother 53: 4588-4597.
17. Ngo T, Duraisingh M, Reed M, Hipgrave D, Biggs B, et al. (2003) Analysis of pfcrt, pfmdr1, dhfr and dhps mutations and drug sensitivities in Plasmodium falciparum isolates from patients in Vietnam before and after treatment with artemisinin. Am J Trop Med Hyg 68: 350-356.
18. Valecha N, Joshi H, Mallick PK, Sharma SK, Kumar A, et al. (2009) Low efficacy of chloroquine: Time to switchover to artemisinin-based combination therapy for falciparum malaria in India. Acta Tropica 111: 21-28.
19. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, et al. (2001) High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183: 1535-1538.
20. Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, et al. (1984) The functional human dihydrofolate reductase gene. J Biol Chem 259: 3933-3943.
21. Marfurt J Drug resistant malaria in Papua New Guinea and molecular monitoring of parasite resistance (Doctoral dissertation, University of Basel).
22. Aubouy A, Jafari S, Huart V, Migot-Nabias F, Mayombo J, et al. (2003) P. DHFR and DHPS genotypes of Plasmodium falciparum isolates from Gabon correlate with in vitro activity of pyrimethamine and cycloguanil, but not with sulfadoxine–pyrimethamine treatment efficacy. J Antimicrob Chemother 52: 43-49.
23. Triglia T, Menting JG, Wilson C, Cowman AF (1997) Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proceedings of the National Academy of Sciences 94: 13944-13949.
24. Sharma YD (2012) Molecular surveillance of drug-resistant malaria in India. Curr Sci 102: 696-703.
25. Wernsdorfer WH and Noedl H. Molecular markers for drug resistance in malaria: Use in treatment, diagnosis and epidemiology. Curr Opin Infect Dis 16: 553-558.
26. Das MK, Lumb V, Mittra P, Singh SS, Dash AP, et al. (2010) High chloroquine treatment failure rates and predominance of mutant genotypes associated with chloroquine and antifolate resistance among falciparum malaria patients from the island of Car Nicobar, India. J Antimicrob Chemother 65: 1258-1261.
27. Li J, Zhou B (2010) Biological actions of artemisinin: Insights from medicinal chemistry studies. Molecules 15: 1378-1397.
28. Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, et al. (2012) Emergence of artemisinin-resistant malaria on the western border of Thailand: A longitudinal study. Lancet 379: 1960-1966.
29. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, et al. (2008) Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med 359: 2619-2620.
30. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, et al. (2009) Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361: 455-467.
31. White NJ (2008) Qinghaosu (artemisinin): The price of success. Science 320: 330-334.
32. Mbengue A, Bhattacharjee S, Pandharkar T, Liu H, Estiu G, et al. (2015) A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria. Nature 520: 683-687.
33. Mu J, Joy DA, Feng X, Furuya T, Chotivanich K, et al. (2003) Early origin and recent expansion of Plasmodium falciparum. Science 300: 318-321.
34. Viana GM, Machado RL, Calvosa VS, Póvoa MM (2006) Mutations in the pfmdr1, cg2 and pfcrt genes in Plasmodium falciparum samples from endemic malaria areas in Rondonia and Pará State, Brazilian Amazon Region. Cadernos de Saúde Pública 22: 2703-2711.