The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin
Introduction
Chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for the most serious form of malaria, is increasing throughout the world, particularly in Africa where the burden of disease is the greatest. This has been associated with an increase in severe malaria mortality and morbidity [1]. Alternative therapies have been introduced including new arylaminoalcohol drugs, mefloquine (MEF) and halofantrine (HAL) and more recently the endoperoxide drugs, artemisinin (QHS) and its derivatives. High levels of resistance to MEF already exist in parts of S.E. Asia where recently the therapeutic efficacy has dropped to <50% despite the doubling of the therapeutic dose [2], while an ‘innate resistance’ has been described in west Africa, with in vitro and in vivo resistance reported from areas where the drug has not previously been used [3]. In various field studies in vitro cross-resistance was observed between MEF, HAL and QHS, with an inverse relationship with CQ [4], [5], [6].
The molecular basis of resistance to these anti-malarials is beginning to be unravelled. Two genetic elements for CQR have been identified. Firstly, by dissection of a genetic cross between a parasite clone resistant to CQ (Dd2) and one sensitive to CQ (HB3), a complex polymorphism on chromosome 7 has been identified which appears to be essential but not sufficient for CQR in a range of isolates [7], [8] Within this region is the cg2 gene, which encodes an ≈330 kDa protein which has been localised to the parasite plasma membrane and food vacuole [8]. Cg2 contains several non silent point polymorphisms, as well as size variations in three repeat regions (kappa, gamma and omega) and a polyasparagine tract [8]. The Dd2-like omega repeat allele was singly the most discriminating polymorphism between sensitive and resistant isolates [8].
Secondly, the role of the pfmdr1 gene, a plasmodial homologue of human multidrug resistance transporter genes, is still controversial. Several alleles of pfmdr1 have repeatedly been associated with CQR, in vivo and in vitro, in some [9], [10], [11], [12], [13], [14], [15] but not in all [16], [17], [18] studies. The reason for this remains unclear but may reflect genuine differences in the genetic basis of resistance in parasites from different geographical areas.
In laboratory studies, MEF and HAL resistance [19], [20], [21] and in one study QHS resistance [21], have been associated with amplification of the pfmdr1 gene. Mutation of pfmdr1 was associated with a higher level of resistance to MEF [21]. In Thailand, amplification of pfmdr1 was found in isolates obtained from areas of high-level resistance to MEF [17], [22]. In the present study we have analysed sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the omega repeat region of the cg2 gene in isolates from the Gambia, which were characterised for drug sensitivity to MEF, QHS and CQ, as well as to several other anti-malarials.
Section snippets
Field isolates
The Gambian field isolates were obtained following consent from patients with uncomplicated malaria at the Outpatients Clinic of the Medical Research Council Laboratories, Fajara, The Gambia (September–October 1996). These were directly used for the in vitro drug tests or spotted onto filter paper. The study was approved by the MRC Laboratories Scientific Coordinating Committee and the Gambia Government/MRC Ethical Committee.
Nucleic acid extraction
Parasite DNA from the field samples was extracted from filter paper by
Sequence polymorphisms in pfmdr1 and genetic associations
PCR-typing revealed variation at only two of the five point polymorphisms in the pfmdr1 gene, codon-86 and codon-184 (Table 3, Fig. 2B). A large degree of polymorphism was observed in the linker region with the general pattern of seven or eight asn codons followed by 0–2 asp codons and then between zero and eleven asn codons (Table 3). Interestingly, there is a significant association between having eight asn codons followed by two asp codons and the tyr-86 allele in these samples (P=0.006,
Discussion
In this study we have investigated the role of genetic polymorphisms in mediating the anti-malarial sensitivity of P. falciparum isolates from The Gambia, west Africa, an area of emerging CQR and where MEF is rarely used. A low prevalence of conventional in vitro resistance to CQ (7.4%) and QUIN (0%), MEF (14.8%) and HAL (32.1%) was previously observed [24]. Cross-resistance was observed between CQ and QUIN and between QHS, DHA, MEF and HAL [24]. We have observed an incomplete association
Acknowledgements
MTD is funded by a Wellcome Trust Prize Studentship. DCW is funded by the PHLS of the UK. We would like to acknowledge all our friends and colleagues in The Gambia. Special thanks to Shabbar Jaffar for help with the statistics.
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