Combined Molecular and Clinical Assessment of Plasmodium falciparum Antimalarial Drug Resistance in the Lao People’s Democratic Republic (Laos) Mayfong Mayxay, Shalini Nair, Dan Sudimack, Mallika Imwong, Naowarat Tanomsing, Tiengkham Pongvongsa, Samlane Phompida, Rattanaxay Phetsouvanh, Nicholas J. White, Tim J. C. Anderson, and Paul N. Newton* Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom Abstract. Molecular markers provide a rapid and relatively inexpensive approach for assessing antimalarial drug susceptibility. We collected 884 Plasmodium falciparum–infected blood samples from 17 Lao provinces. Each sample was genotyped for 11 codons in the chloroquine resistance transporter (pfcrt), dihydrofolate reductase (pfdhfr), and dihydropteroate synthase (pfdhps) genes. The samples included 227 collected from patients recruited to clinical trials. The pfcrt K76T mutation was an excellent predictor of treatment failure for both chloroquine and chloroquine plus sulfadoxine-pyrimethamine, and mutations in both pfdhfr and pfdhps were predictive of sulfadoxine-pyrimethamine treatment failure. In multivariate analysis, the presence of the pfdhfr triple mutation (51 + 59 + 108) was strongly and independently correlated with sulfadoxine-pyrimethamine failure (odds ratio  9.1, 95% confidence interval  1.4–60.2, P  0.017). Considerable geographic heterogeneity in allele frequencies occurred at all three loci with lower frequencies of mutant alleles in southern than in northern Laos. These findings suggest that chloroquine and sulfadoxine- pyrimethamine are no longer viable therapy in this country. INTRODUCTION Plasmodium falciparum antimalarial drug resistance has become an increasingly difficult problem in malaria-endemic countries and usually necessitates changes in national malaria treatment policy. 1 Comparative clinical trials have played a vital role in the assessment of antimalarial drug resistance. 2 However, because such trials require long patient follow-up, sufficient sample size, and are expensive, it is difficult to per- form multiple trials in one country. Therefore, there is limited information on the variability of antimalarial drug sensitivity within countries. Widespread in vitro drug sensitivity assays are impractical because of technical limitations, variable cor- relations with clinical outcomes, 3 and problems of repeatabil- ity between laboratories. The correlation of specific P. falci- parum mutations with treatment failure and the development of rapid and relatively inexpensive molecular techniques to detect these mutations 4 suggest that these methods may be appropriate and practicable for large-scale monitoring of an- timalarial drug resistance patterns 5,6 when appropriately cali- brated with clinical efficacy studies. For example, mutations in dihydrofolate reductase (pfdhfr) and dihydropteroate syn- thase (pfdhps) genes confer resistance to pyrimethamine (P) and sulfadoxine (S), respectively, both in vitro and in vivo. 7–13 Mutations in codon 76 of the P. falciparum chloroquine (CQ) resistance transporter (pfcrt) gene contributes to CQ resis- tance. 14–17 Laos is a land-locked country, with a population of approxi- mately 5.6 million that has borders with China on the north, Burma (Myanmar) on the northwest, Thailand on the west, Cambodia on the south, and Vietnam on the north and east. Malaria occurs in all 17 provinces and although prevalence varies considerably, it is more prevalent in the southern part of the country. Plasmodium falciparum resistance to CQ and SP were first noted in this country in the late 1960s, 18–20 but these drugs remained the first-line and second-line nationally recommended treatments for uncomplicated P. falciparum malaria until 2005. Clinical trials of oral CQ and SP for the treatment of uncomplicated P. falciparum malaria in five dif- ferent areas of Laos between 2000 and 2003 showed high rates of treatment failure (35–80% for CQ and 18–35% for SP). 16,21–24 Subsequent trials of artesunate-mefloquine and artemether-lumefantrine at two sites in Laos showed 42-day failure rates  6%. 25,26 Therefore, artemisinin-based combi- nation therapy (ACT) with artemether-lumefantrine was in- troduced as first-line antimalarial drug treatment of uncom- plicated P. falciparum malaria in this country in 2005. We measured the prevalence of molecular markers of an- timalarial drug resistance in P. falciparum parasites through- out the country to examine geographic variation in the preva- lence of mutations, and to provide a baseline for examining changes in drug resistance mutation frequency after the change in drug treatment policy to ACT. We also measured the prevalence of molecular markers in parasites from pa- tients recruited to efficacy trials at two sites in Laos to exam- ine the relationships between these molecular markers and therapeutic responses. MATERIALS AND METHODS Blood sample collection, clinical trials, and DNA prepara- tion. Malaria clinics and health facilities in every province in Laos (with the exception of Vientiane City where there is no malaria transmission) were identified, and patients coming to these clinics with fever were studied between 2001 and 2004. Province names are given in accordance with Sisouphanthong and Taillard. 27 Thick and thin blood smears, stained with Gi- * Address correspondence to Paul N. Newton, Microbiology Labo- ratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Re- public. E-mail: paul@tropmedres.ac Am. J. Trop. Med. Hyg., 77(1), 2007, pp. 36–43 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene 36