Elsevier

The Lancet

Volume 364, Issue 9432, 31 July–6 August 2004, Pages 438-447
The Lancet

Mechanisms of Disease
Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

https://doi.org/10.1016/S0140-6736(04)16767-6Get rights and content

Summary

Background

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known.

Methods

The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (P falciparum) and P falciparum Ca2 ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism.

Findings

Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0–14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo.

Interpretation

Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria.

Relevance to practice

Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

Introduction

The emergence of drug resistant malaria is a serious threat to tropical communities.1, 2 Early detection of failing treatment regimens for malaria is important for guiding public health measures in areas where the disease is endemic. Frequently, such decision making relies on results of clinical studies that assess the therapeutic efficacy of antimalarials, sometimes supported by in-vitro sensitivity testing. However, clinical studies with adequate periods of patient follow-up (≥28 days) are difficult to implement, and few malaria-endemic areas have the infrastructure needed to do parasite cultures. More recently, molecular genotyping of parasites has proved useful in assessing resistance to the antifolate, sulphonamide,3 and hydroxynaphthaquinone classes4 of drugs, since point mutations in the genes that encode their drug targets cause resistance. Chloroquine (a 4-aminoquinoline) resistance in vitro and in vivo is associated with mutations in pfmdr1 (Plasmodium falciparummultidrug resistance gene 1) and pfcrt (P falciparum chloroquine resistance transporter gene) a putative transporter that modulates intraparasitic drug concentrations.5

Southeast Asia is the epicentre of P falciparum resistance to antimalarial drugs. Chloroquine resistance emerged there more than 40 years ago, and within 20 years chloroquine became largely ineffective. Chloroquine is now reserved for P vivax,P malariae, or P ovale in this region. In Thailand, mefloquine was introduced as first-line treatment for falciparum malaria in November, 1984, but despite careful regulation of its use, significant resistance developed within 6 years. Resistance has also emerged in adjacent countries, such as Burma and Cambodia. Artemisinin derivatives have since been added to mefloquine in 3-day combination regimens (ACT),6 which remain highly effective. In these areas of southeast Asia the molecular basis of drug resistance in P falciparum is multifactorial and not completely understood.

In preliminary studies, multidrug resistance in southeast Asia was linked both to mutations in pfmdr1 and to increases in this gene's copy number.7, 8, 9 Further studies have been hampered by the technical difficulties of assessing copy number in clinical samples.7, 8, 9, 10, 11, 12

We have developed a robust assay to quantitate the copy number of pfmdr1 using TaqMan Real-time PCR (TaqMan, Wellington, VA, USA), and combined this assay with analysis of single nucleotide polymorphisms in pfmdr1, pfcrt, and P falciparum Ca2+ ATPase (pfATP6) (recently identified as an encoder of the artemisinins receptor), to assess the determinants of mefloquine resistance in vitro and in vivo.

Section snippets

Study site

The field studies for this project took place in a Karen community living in malarious hill forest on the northwestern border of Thailand. Prospective studies were undertaken to determine antimalarial efficacy of different drug regimens against a backdrop of increasing drug resistance. The transmission of malaria is low and seasonal with a frequency of about one P vivax, and one P falciparuminfection every 2 years per person. In this region, almost all falciparum malaria infections result in

Results

Between 1990, and June, 1994, 1302 patients with uncomplicated falciparum malaria were enrolled into chemotherapeutic studies. They were given mefloquine monotherapy and then followed-up for between 28 and 63 days (figure 1). By June, 1994, the cumulative cure rate had fallen from 95% to 63%, with 13% high grade failures (ie, failures by 7 days after treatment). Hence, the first-line treatment was changed to mefloquine with 3 days' artesunate (MAS3). By 2002, 3322 patients had been treated with

Discussion

Molecular genotyping to identify drug resistant P falciparum parasites is of increasing importance in characterising the epidemiology of malaria and informing the choice of antimalarial treatment regimens. Genotyping for point mutations in the genes encoding dihydrofolate reductase and dihydropteroate synthase, the targets of antifols and sulphonamides respectively, is of established value in predicting susceptibility to these drugs in vivo and in vitro. More recently, single nucleotide

Glossary

pfmdr1
This gene encodes a P-glycoprotein transporter belonging to the family of ATP-dependent transporters that also mediate multidrug resistance in some human tumours. It is localised to the parasite's food vacuole
pfcrt
Mutations in this protein cause chloroquine resistance
Real-time PCR
This technique monitors products of DNA amplification by fluorescence techniques as they are generated. Comparision of results with controls allows estimation of gene copy number
PfATP6
This is an ATP-dependent

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