IN THE PLASMODIUM FALCIPARUM DMYDROFOLATE =DUCTASE-THYhUDYLATE SYNTHASE GENE AND IN VITRO RESISTANCE TO PYRTMETHAMINE A N D CYCLOGUANIL

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium fakiparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. fakiparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC,,,s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ue-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC,,, and, to a much lesser extent, cycloguanil IC5,,. These results support a partial crossresistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance. Pyrimethamine and proguanil are dihydrofolate reductase @HFR) inhibitors that continue to play a major role in the chemotherapy of chloroquine-resistant Plasniodiurn falcipar u m 1 Initially used as a monotherapy in the 1950s and 1960s, both of these DHFR inhibitors are currently used in combination with another antimalarial drug to delay the emergence of resistant strains and/or to enhance their specific activity against the malaria Sulfadoxinepyrimethamine is indicated for the treatment of acute, uncomplicated malarial attacks due to chloroquine-resistant P. falciparunz infections in areas where this drug combination remains generally effective, such as in most of the African continent. F’roguanil, in combination with chloroquine, is recommended for chemoprophylaxis in nonimmune individuals traveling to certain areas where malaria is endemic and in pregnant women residing in malaria-endemic zones? Proguanil is also used for the treatment of multidrug-resistant P. fak iparum infections in combination with a new antiprotozoan drug, ato~aquone.6*~ Pyrimethamine and cycloguanil, the biologically active human metabolite of proguanil, share similar chemical structures and inhibit the same molecular target.8-g These two features suggest a potential for cross-resistance between the drugs. However, earlier in vivo and in vitro studies have drawn contradictory conclusions? Another approach based on the analysis of the genetic mechanism of antifolate resistance may help resolve this question. Comparison of the P. falciparum dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene sequences from several reference clones has suggested genetic profìles that are associated with pyrimethamine resistance and cycloguanil resistance.10*’ According to these studies, a Ser-to-Asn substitution at position 108 confers resistance to pyrimethamine, while a Ser-to-Thr substitution at position 108, associated with a Ala-to-Val substitution at position 16, confers resistance to cycloguanil. Ancillary mutations at positions 51, 59, and/or 164 are associated with elevated levels of antifolate resistance. These molecular criteria for antifolate resistance have not been fully confirmed in naturally occurring isolates of P. fakiparum. In our previous studies on Cameroonian isolates, the key dhfr codon was compared with the phenotype defined by the in vitro sensitivity or resistance to pyrimethamine.I2 In the present study, the in vitro responses to both pyrimethamine and cycloguanil were determined for clinical isolates obtained in Cameroon and the DHFR domain of the dhfr-ts gene was fully sequenced, with the aim to establish whether there is a particular genotype that defines pyrimethamine and cycloguanil resistance. PATIENTS, MATERIALS, AND METHODS Patients. Most of the patients participated in the clinical trials conducted in Yaounde, Cameroon between 1996 and 1998. Inclusion criteria included an age 2 5 years old, fever at consultation (or history of fever within the past 24 hr), a monoinfection with P. falciparum based on the microscopic examination of Giemsa-stained thin and thick blood smears, a parasite density >5,000 asexual parasites/pl of blood; and no recent history of self-medication with antimalarial drugs, as confirmed by a negative Saker-Solomons urine test rest11t.I~ Patients with signs and symptoms of severe and complicated malaria, as defined by the World Health Organizawere excluded. Depending on the clinical conditions, the patients were keated with the first-line (amodiaquine),