Analysis of Point Mutations Ln the Dihydrofolate Reductase- Thymidylate Synthase Gene of Plasmodium Falciparum

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of pioguanil, and pyrimethamine) is associated with a Serto Asn108 point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparunt. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment length polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolateresistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 of 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-IO8 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC,,] < 100 nM) and cycloguanil (ICso < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC,, (78 nM), which was within the sensitive range. This study provides further evidence that anfifolateresistant P. falciparum isolates are already present in Yaounde, Cameroon. Malaria is a major public health problem and is associated with high mortality and morbidity rates in Cameroon. Transmission is seasonal in the northern, drier region and occurs throughout the year in the southern half of the country, which is densely covered with tropical rain forest.' Yaounde, the capital city of Cameroon, is situated in the southern plateau at an average altitude of 800 meters above sea level. The first-line drug for the treatment of acute, uncomplicated Plasmodium falciparunz malaria is chloroquine (or amodiaquine) for the indigenous population, and halofantrine (or sometimes mefloquine or mefloquine-sulfadoxine-pyrimethamine) is often indicated for the noninimune nonindigenous population. Secondand third-line drugs for resistant cases are sulfadoxine-pyrimethamine and quinine, respectively. In our previous in vitro study conducted in 1994-1995, it was shown that about 42% of the clinical isolates obtained from symptomatic Cameroonian patients in Yaounde are resistant to pyrimethamine andlor cycloguanil, a biologically active major metabolite of proguanil.2 Although recent clinical data on the efficacy of sulfadoxine-pyrimethamine are lacking in Cameroon, the in vitro results are alarming. If in vivo evaluation confirms the decreasing efficacy of the drug, the use of this drug combination as the second-line antimalarial drug to treat chloroquine-resistant P. falciparunt malaria may be compromised. Since an increasing number of cases are expected to be treated with sulfadoxine-pyrimethamine in Cameroon, it is important to establish the baseline data on the sensitivity of P. f a k i p a r u m isolates and monitor the evolution of drug sensitivity. In addition to clinical evaluation, which is usually timeconsuming to conduct, in vitro assay is generally accepted as a valuable tool to monitor the drug sensitivity of malaria laria.3 An altemative, novel approach is based on nucleic acid detection of genes that are associated with drug resistance. -The bifunctional -enzyme dihydrofolate reductasethymidylate synthase (DHFR-TS) is one of the few known molecular targets of antimalarial agents classified as antifolates, otherwise known as DHFR in hi bit or^.^ Antimalarial antifolates include pyrimethamine and cycloguanil. Previous studies based on reference clones as well as clinical isolates of P. falciparunz from various geographic origins have provided strong evidence that in vitro antifolate resistance is associated with point mutations in the DHFR domain of the DHFR-TS gene?-* Among several point mutations that may occur in the gene, a Serto Asn-108 point mutation is considered to be the key mutation that confers resistance to antifolate This mutation can be detected with ease by polymerase chain reaction and restriction fragment length polymorphism.", Due to the unavailability of restriction enzymes, the other point mutations associated with high levels of pyrimethamine resistance (codons 51,59, and 164 of the DHFR-TS gene) were not studied. We have conducted the present study with the aim of confiiming our previous observations, which were based on in vitro assays, using a simple and rapid molecular technique that is suitable for field use in Africa and to establish the correlation between in vitro antifolate resistance and DHFR-TS gene mutations that occur in clinical isolates obtained in Yaounde,