Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.
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 (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism.


RESULTS
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.


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


CONCLUSIONS
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.
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Ric N
RN
Price
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Anne-Catrin
AC
Uhlemann
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Alan
A
Brockman
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Rose
R
McGready
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Elizabeth
E
Ashley
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Lucy
L
Phaipun
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Rina
R
Patel
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Kenneth
K
Laing
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Sornchai
S
Looareesuwan
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Nicholas J
NJ
White
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
François
F
Nosten
Department of Cellular and Molecular Medicine-Infectious Diseases, St George's Hospital Medical School, London, UK (S Krishna DPhil, K Laing PhD, Rina Patel BSc, A-C Uhlemann MD); Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, UK (E Ashley MD, R McGready PhD, F Nosten PhD, RN Price MD, NJ White DSc); Shoklo Malaria Research Unit, Mae Sod, Tak Province, Thailand (E Ashley MD, A Brockman BSc, R McGready PhD, F Nosten PhD, L Phaipun); Menzies School of Health Research Charles Darwin University, Darwin, Australia (RN Price MD); Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (S Looareesuwan MD, NJ White DSc).
Sanjeev
S
Krishna
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000962:Antimalarials / Q000008:administration & dosage / Q000494:pharmacology*; D019306:Artemisia; D037621:Artemisinins / Q000008:administration & dosage; D000077332:Artesunate; D000252:Calcium-Transporting ATPases / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D003062:Codon / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004359:Drug Therapy, Combination; D005260:Female; D018628:Gene Dosage; D019450:Genes, MDR; D005838:Genotype; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology; D008297:Male; D015767:Mefloquine / Q000008:administration & dosage / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D008875:Middle Aged; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics*; D012717:Sesquiterpenes / Q000008:administration & dosage; D013785:Thailand
D018528:ATP-Binding Cassette Transporters; D000962:Antimalarials; D037621:Artemisinins; D003062:Codon; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D012717:Sesquiterpenes; C059987:mdr gene protein, Plasmodium; D000077332:Artesunate; D000252:Calcium-Transporting ATPases; D015767:Mefloquine