PYRIMETHAMINE/SULFADOXINE COMBINATION IN THE TREATMENT OF UNCOMPLICATED FALCIPARUM MALARIA: RELATION BETWEEN DIHYDROPTEROATE SYNTHASE/DIHYDROFOLATE REDUCTASE GENOTYPES, SULFADOXINE PLASMA LEVELS, AND TREATMENT OUTCOME INSAF KHALIL, MICHAEL ALIFRANGIS, ANITA M. RØNN, HAYTHEM A. GABAR, TOMAS JELINEK, GWIRIA M. H. SATTI, AND IB C. BYGBJERG Center for Medical Parasitology, Institute of Medical Microbiology and Immunology, University of Copenhagen,Copenhagen, Denmark; Department of Biochemistry, Faculty of Medicine, University of Khartoum, Sudan; Department of Clinical Pharmacology, Copenhagen University Hospital, Copenhagen, Denmark; Department of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany; Department of Biochemistry, King Faisal University, Dammam, Saudi Arabia; Department of International Health, Institute of Public Health, University of Copenhagen, Copenhagen, Denmark Abstract. Several in vitro studies have shown the correlation between mutations in dhfr and dhps genes and resis- tance to pyrimethamine/sulfadoxine (PYR/SDX) combination, but the in vivo correlates of these mutations with PYR/ SDX efficacy have not been investigated fully. We assessed PYR/SDX efficacy in relation to the frequency of dhfr and dhps mutations in 37 Plasmodium falciparum isolates sampled before treatment. Plasma levels of SDX measured at days 0, 3, 7, and 14 ascertained drug absorption. Point mutations were detected only at codons 51 and 108 of dhfr and codon 436 of dhps. The frequency of dhfr 51/108 and dhps 436 mutations was 79% and 8%. The plasma levels of SDX indicated adequate drug absorption by all patients. The presence of Ile 51and Asn 108 mutations among parasites that cleared after treatment indicates that these mutations alone are insufficient to cause in vivo resistance. In all recrudescent parasites, however, the presence of Ile 51/Asn 108 dhfr mutations was coupled with the dhps Ala 436. The findings suggest that the presence of Ile 51/Asn 108 dhfr mutations and Ala 436 dhps confers decreased susceptibility of P. falciparum to PYR/SDX in areas of low endemicity. INTRODUCTION Some East and Central African countries have adopted the combination of pyrimethamine and sulfadoxine (PYR/SDX) as first-line treatment of acute uncomplicated falciparum ma- laria. 1 Resistance seems to develop soon after wide-scale use of the drug, mirroring events in Southeast Asia in the 1980s. 2 High multiplicity of Plasmodium falciparum infections and the drug’s strong selection pressure for mutant alleles could explain the rapid progression of resistance. 3 In Sudan, PYR/ SDX is the second-line treatment. At present, a combination of chloroquine and PYR/SDX or co-trimoxazole is recom- mended for the treatment of acute uncomplicated malaria. This situation provides a good opportunity to observe the changes in dhfr and dhps genotypes as drug pressure in- creases. Point mutations in the coding regions of dhfr and dhps genes have been implicated in in vitro resistance to PYR and SDX. 4,5 Epidemiology of mutations in these genes is increas- ingly being studied. Geographic differences have been re- ported, and the spread of dhps/dhfr mutations has been re- lated to history of PYR/SDX use and resistance in the field. 6,7 The dhfr Asn-108 mutation has been present in all antifolate resistant forms studied, but no specific set of mutations in dhfr or dhps or both is found to be predictive of therapeutic fail- ure. Prediction of PYR/SDX therapeutic failure is compli- cated because of the natural variations in host immunity, ef- ficacy of absorption and metabolism of the drugs, serum level of folates, and parasite genotypes and their efficacy in using exogenous folates. 8,9 More recent studies have indicated that parasite susceptibility to PYR is important in determining clinical outcome to PYR/SDX because synergy between PYR and SDX has been described as the abolition of folate an- tagonism of SDX inhibition by subinhibitory levels of PYR. 10 The present study assessed the in vivo outcome of treatment with PYR/SDX among patients with acute uncomplicated fal- ciparum malaria in relation to SDX plasma levels (as a marker of PYR/SDX absorption) and dhfr/dhps mutations among pretreatment parasites. PATIENTS, MATERIALS, AND METHODS Study area. The study was conducted during November 1996–February 1997 in the outpatient department of Hag Yousif hospital. It was part of a controlled, randomized, double blind, hospital-based clinical trial comparing efficacy and safety of PYR/SDX and co-trimoxazole in patients with acute uncomplicated falciparum malaria. Hag Yousif is an area of unstable malaria in Khartoum (Satti GMH, unpub- lished data). In vivo sensitivity test. The in vivo sensitivity test was con- ducted and evaluated according to 1973 and 1996 WHO guidelines. 11,12 Inclusion criteria were monoinfection with P. falciparum, temperature 37.5°C and parasitemia between 1,000 and 80,000 asexual parasites/l of blood. Pregnant women, patients with severe malaria, patients with concomi- tant nonmalarial febrile illness, patients allergic to sulfon- amide, and patients with glucose-6-phosphate dehydrogenase deficiency were excluded. PYR/SDX (Hoffman La Roche, Basel, Switzerland) was administered orally at a single dose of 25 mg/kg of SDX and 1.25 mg/kg of PYR. Therapeutic re- sponses were evaluated parasitologically 11 and clinically. 12 Parasitologically, treatment outcome is divided into sensitive (no parasites observed at day 7 and no recrudescence ob- served during the follow-up period) and 3 resistant grades: RI (parasites may disappear but reappeared within the next 28 days), RII (parasite density reduced by 75% of the initial parasite density 2 days after therapy), and RIII (parasites continue to rise or reduced by 25% 2 days after therapy). Clinically the response is divided into adequate clinical re- sponse (ACR) or treatment failures, either early or late treat- ment failures (ETF and LTF). ETF was defined as positive Am. J. Trop. Med. Hyg., 67(3), 2002, pp. 225–229 Copyright © 2002 by The American Society of Tropical Medicine and Hygiene 225