Mechanisms of DiseaseMefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number
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
References (30)
- et al.
Measurement of trends in childhood malaria mortality in Africa: an assessment of progress toward targets based on verbal autopsy
Lancet Infect Dis
(2003) - et al.
Effects of artesunate-mefloquine combination on incidence of Plasmodium falciparum malaria and mefloquine resistance in western Thailand: a prospective study
Lancet
(2000) - et al.
Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand
Mol Biochem Parasitol
(1993) - et al.
Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa
Mol Biochem Parasitol
(1995) - et al.
Plasmodium falciparum: molecular characterization of multidrug-resistant Cambodian isolates
Exp Parasitol
(1996) - et al.
The epidemiology of malaria in a Karen population on the western border of Thailand
Trans R Soc Trop Med Hyg
(1996) - et al.
Plasmodium falciparum antimalarial drug susceptibility on the north-western border of Thailand during five years of extensive use of artesunate-mefloquine
Trans R Soc Trop Med Hyg
(2000) - et al.
The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin
Mol Biochem Parasitol
(2000) - et al.
Predictors of mefloquine treatment failure: a prospective study of 1590 patients with uncomplicated falciparum malaria
Trans R Soc Trop Med Hyg
(1995) - et al.
Artesunate/mefloquine treatment of multi-drug resistant falciparum malaria
Trans R Soc Trop Med Hyg
(1997)
Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P falciparum
Cell
Assessment of pfmdr1 gene copy number by tandem competitive polymerase chain reaction
Mol Biochem Parasitol
Mutations in the P falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance
Mol Cell
The mode of action and the mechanism of resistance to antimalarial drugs
Acta Tropica
Antimalarial drug resistance and combination chemotherapy
Philos Trans R Soc Lond B Biol Sci B
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