Role of pfmdr1 mutations on chloroquine resistance in Plasmodium falciparum isolates with pfcrt K76T from Papua New Guinea

Abstract

The N86Y mutation in pfmdr1 is reported to play an additional role for the chloroquine resistance in Plasmodium falciparum isolates. However, not much has been done to clarify whether this mutation augments the level of chloroquine resistance in the isolates harboring pfcrt K76T mutation. We compared the in vitro chloroquine efficacy between pfcrt K76T mutant parasites with or without N86Y mutation from Papua New Guinea. A total of 57 isolates (4% sensitive, 14% borderline, and 82% resistant) were successfully tested in vitro for chloroquine sensitivity. We found a slightly higher effective concentration of chloroquine needed to inhibit P. falciparum by 50% (mean EC₅₀ = 107 nM) in isolates with the pfcrt K76T + pfmdr1 N86Y than that in isolates with the pfcrt K76T + pfmdr1 N86 (EC₅₀ = 88 nM), but this difference was not statistically significant. A significant non-random association was observed between the pfcrt K76T and pfmdr1 N86Y alleles. Our results suggest that the pfmdr1 N86Y mutation plays a compensatory role to chloroquine-resistant isolates under a chloroquine pressure while it may also augment the level of chloroquine resistance in the K76T parasites to a small extent.

Introduction

The alarming increase of the Plasmodium falciparum resistance to commonly used antimalarial drugs represents a major public health threat (Bjorkman and Bhattarai, 2005). Chloroquine-resistant P. falciparum first emerged in the late 1950s in both Southeast Asia and South America and have since spread rapidly to the most endemic regions (Wellems and Plowe, 2001).

In 2000, the P. falciparum chloroquine-resistant transporter (pfcrt) gene was identified (Fidock et al., 2000). The resistance was associated with a reduced accumulation of chloroquine in the parasite digestive vacuole (Saliba et al., 1998) but how the pfcrt gene exerts such an effect on the digestive vacuole is largely unclear. An acquired efflux system of chloroquine (Sanchez et al., 2005) and/or an increased acidity in the digestive vacuole (Bennett et al., 2004) is assumed to play a role for the reduced accumulation. A lysine to threonine change at position 76 (K76T) has been found in every in vitro chloroquine-resistant parasite from around the world (Fidock et al., 2000, Babiker et al., 2001, Wootton et al., 2002). Many studies have confirmed the presence of K76T mutations in the in vivo chloroquine resistance but this mutation was not the sole requirement (Babiker et al., 2001, Djimde et al., 2001a, Pillai et al., 2001, Jelinek et al., 2002, Vinayak et al., 2003), suggesting that host factors affect the clearance of chloroquine-resistant parasites (Djimde et al., 2001a, Djimde et al., 2001b).

Another extensively investigated gene is the P. falciparum multiple drug resistance 1 (pfmdr1), a homologue of the mammalian multiple drug resistance gene encoding a P-glycoprotein on the chromosome 5 of the P. falciparum. It is a typical member of the ATP-binding cassette transporter superfamily localized to the parasite vacuole, where it may regulate intracellular drug concentrations (Duraisingh and Cowman, 2005). Mutation was observed at the 86, 184, 1034, 1042, and 1246 positions, which were strongly linked to the chloroquine resistance in laboratory clones obtained from various regions (Foote et al., 1990). Several field studies indicated the positive association between the asparagine to tyrosine change at position 86 (N86Y) and the chloroquine resistance both in vitro (Basco et al., 1995, von Seidlein et al., 1997, Grobusch et al., 1998, Duraisingh et al., 2000a) and in vivo (Gomez-Saladin et al., 1999, Nagesha et al., 2001). However, other studies have cast doubts about this association (Pillai et al., 2001, Thomas et al., 2002).

Currently, pfmdr1 mutations are said to assist the chloroquine-resistant parasites by augmenting the level of resistance. Studies have shown that an introduction of the wild allele at positions 1034, 1042, and 1246 in pfmdr1 into the chloroquine-resistant 7G8 clone resulted in the reduction of the drug concentration needed to inhibit parasite growth by 50%, EC₅₀, from 389 nM to 204 nM (Reed et al., 2000). Nevertheless, the mutant allele at these three positions introduced into the chloroquine-sensitive D10 clone was unable to confer chloroquine resistance. For the N86Y mutation, however, this association has not been fully confirmed in both experimental and field works.

In Papua New Guinea, oral 4-aminoquinoline (chloroquine for adults and amodiaquine for children) has been used for many years as the first-line treatment for uncomplicated malaria. However, the therapeutic efficacy of chloroquine decreased from 50–70% in the 1980s to 20–30% in the 1990s (Muller et al., 2003). The Department of Health and Welfare of Papua New Guinea, therefore, added sulfadoxine/pyrimethamine (SP) to the 4-aminoquinolines as their treatment policy for uncomplicated malaria in 2000. In this study, we determined the pfcrt and pfmdr1 polymorphisms and in vitro sensitivity to chloroquine in P. falciparum isolates from East Sepik Province, Papua New Guinea, to explore the roles of pfmdr1 gene mutations in chloroquine resistance.