ANALYSIS OF PFCRT, PFMDR1, DHFR, AND DHPS MUTATIONS AND DRUG SENSITIVITIES IN PLASMODIUM FALCIPARUM ISOLATES FROM PATIENTS IN VIETNAM BEFORE AND AFTER TREATMENT WITH ARTEMISININ THANH NGO, MANOJ DURAISINGH, MICHAEL REED, DAVID HIPGRAVE, BEVERLEY BIGGS, AND ALAN F. COWMAN Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; University of Melbourne, Parkville; MacFarlane Burnet Centre for Medical Research, Melbourne, Australia; National Institute of Malariology, Parasitology, and Entomology, Hanoi, Vietnam Abstract. We have analyzed artemisinin sensitivity in Plasmodium falciparum isolates obtained from patients in South Vietnam and show that artemisinin sensitivity does not differ before and after drug treatment. There was an increase in the level of mefloquine resistance in the isolates after drug treatment that was concomitant with a decrease in chloroquine resistance, suggesting that treatment with artemisinin has selected for increased mefloquine resistance. Mutations in the pfmdr1 gene, previously shown to be associated with sensitivity to mefloquine, were selected against. All isolates resistant to chloroquine encoded Thr-76 in the pfcrt gene consistent with an essential role in the mechanism of chloroquine resistance. Mutations in pfmdr1 also were linked to chloroquine resistance. High levels of mutation in dhfr and dhps genes, which have previously been associated with Fansidar resistance, also were found, suggesting that this drug would not be useful for malaria control in this part of Vietnam. INTRODUCTION The development and spread of drug resistance in Plasmo- dium falciparum is a major problem for the treatment and control of malaria in many endemic countries. In Southeast Asia, 21.9 million cases of malaria were reported in 1995 alone. 1 In Vietnam, chloroquine resistance was first reported in 1961 and reached a peak in 1990, ranging in incidence from 60–80% in some regions. 2 Chloroquine resistance has now spread to most regions of Vietnam where malaria is endemic 3 and has led to the widespread use of alternative antimalarial drugs (mefloquine, halofantrine, artemisinin derivatives, and pyrimethamine/sulfadoxine). Use of Fansidar (pyrimeth- amine + sulfadoxine) has increased rapidly over the last 30 years. 4 As a consequence, reported levels of resistance to the drug have increased from approximately 20% to over 80%. 5 Since 1991, artemisinin and its derivatives have been used to treat malarial patients in Vietnam. These drugs have become increasingly important as antimalarial therapy in this setting because of their rapid mode of action and efficacy against multidrug-resistant forms of P. falciparum malaria. Resistance of P. falciparum to chloroquine has been asso- ciated with lower drug accumulation. 6 Mutations in the pfcrt gene have been strongly linked to the mechanism of chloro- quine resistance. 7 The presence of Tyr-76 residue within the Pfcrt protein is linked to chloroquine resistance, suggesting that it plays an important role in the mechanism of resistance to this antimalarial. Additionally, mutations within the pfmdr 1 gene have been shown to confer increased resistance to chloroquine, suggesting that they play a role in modulating higher levels of chloroquine resistance. 8,9 The same mutations also have been shown to confer quinine resistance and alter the level of resistance and sensitivity to mefloquine and arte- misinin. 8 The combination of pyrimethamine and sulfadoxine was effective when it was introduced in the late 1960s as a single- dose treatment of acute P. falciparum malaria. 10,11 Over the years, however, resistance to Fansidar has been reported, mainly in areas of intense use, particularly areas of chloro- quine resistance. Variant sequences of P. falciparum dihydro- folate reductase (DHFR), the target enzyme of pyrimeth- amine, were first described in 1988. 12,13 Resistance to pyri- methamine has been shown to result from a mutation in the DHFR enzyme, changing Ser-108 to Asn-108, and subsequent mutations can greatly increase the level of resistance to this drug. 14,15 Ten mutant genotypes for DHFR have been re- ported from a large number of field samples. 4,16–18 Resistance to sulfonamide and sulfones has been shown to result from mutations within dihydropteroate synthetase (DHPS). 19,20–22 The amino acid changes at four positions (Ser-436, Gly-437, Ala-581, and Ala-613) have been shown to confer resistance to sulfadoxine and also cross-resistance to sulfones and sul- fonamides. 21,22 Distribution of mutations in the dhfr and dhps genes and their association with Fansidar resistance have been assessed in different geographic areas, and it has been found that a greater number of mutations in both dhfr and dhps is a good predictor of likely drug failure. 4,17,18,23 Vietnam has well-developed systems for malaria surveil- lance, with recent data showing that more than half the popu- lation lives in forests and mountainous areas where they may be at risk of malaria infection. Drug-resistant malaria has been an increasing problem in the last two decades, and al- though alternative drugs such as artemisinin and its deriva- tives have become widely used as first-line therapy for ma- laria, they have been associated with a high frequency of recrudescence. 24 In this 1998 study, we have further catego- rized malaria drug resistance in Vietnam by analyzing muta- tions in the pfcrt, pfmdr1, dhfr, and dhps genes in P. falci- parum primary and recrudescent isolates collected from pa- tients in an area of Binh Phuoc Province highly endemic for malaria. MATERIALS AND METHODS Study area and collection of blood samples. The subjects of the study lived on the Phu Rieng rubber plantation in south- ern Vietnam. The area is hyperendemic for malaria, which is aggravated by multidrug-resistant P. falciparum. The peak seasons of infection are March–June and September– December (annual main peak of transmission). The annual parasite rate/slide is 10–30% for both P. falciparum and P. vivax. Blood samples were collected from all P. falciparum- positive cases before administering artemisinin (20 mg/kg on Am. J. Trop. Med. Hyg., 68(3), 2003, pp. 350–356 Copyright © 2003 by The American Society of Tropical Medicine and Hygiene 350