Drug resistant malaria is a major problem in malaria control. At the present, there are few antimalarial drugs available in endemic areas which are both cheap and save. Since resistance to chloroquine has spread across sub-Saharan Africa, several countries have switched their first-line drug to the antifolate sulfadoxine-pyrimethamine 1.

Soon after sulfadoxine-pyrimethamine was introduced to malaria-control programs in the late 1960s, resistance to this drug was noted 2. The efficacy of sulfadoxine-pyrimethamine primarily depends on the sensitivity of the parasite to pyrimethamine 3. The dihydrofolate reductase domain of Plasmodium falciparum (pfDHFR) is the target of this drug. DHFR catalyzes the reduction of dihydrofolate to regenerate tetrahydrofolate which is required for one-carbon transfer reactions and deoxythymidylate synthesis of the parasites.

The discovery of changes in codons of the pfDHFR-gene strongly indicated that single amino acid changes lead to observed resistance. Mutations at amino acid position 51, 59, 108 and 164 have been shown to be linked with resistance of P. falciparum to antifolate antimalarials. A scheme for evolution of resistance could be derived as a result of stepwise mutations starting with the S108N mutation, which was shown to be the optimal mutation in leading to both the decreased binding affinity for inhibitors and the retention of enzyme activity 45. Absolute resistance is conferred by the addition of I164L mutation in the quadruple mutant form (N108/I51/R59/L164). In this fourth mutation the enzyme is about 1,000-fold less sensitive to pyrimethamine 6.

The I164L mutation also plays an important role in the development of resistance to the more potent antifolate combination chlorproguanil-dapsone: while the triple mutant allele (N108/I51/R59) has no great impact on the sensitivity of this drug, parasites that carry the quadruple mutant allele are resistant 78. This highly potent quadruple mutation was described first in South East Asia, later also in South America 91011. There is a concern that continued sulfadoxine-pyrimethamine pressure, as well as the widespread use of trimethoprim-sulfamethoxazole for prophylaxis against opportunistic infections in patients with AIDS in Africa, is going to select these highly resistant alleles 12. This would rapidly make sulfadoxine-pyrimethamine, as well as chlorproguanil-dapsone, ineffective in this region. For this reason continued surveillance of this mutation is needed to evaluate the prevalence, the distribution and the speed with which these populations might be selected.

TropNetEurop is a European surveillance system that covers approximately 12% of all imported malaria cases in Europe 13. Travellers can be used as a highly sensitive surveillance tool to detect development of drug-resistance in endemic areas, as it is suggested that most of them carry a monoclonal Plasmodium strain. Initial data on molecular surveillance, not including the I164L mutation, have already been published elsewhere 14.

Since there are only limited data available on the prevalence of the I164L mutation throughout Africa, the purpose of this survey was to screen a sample of P. falciparum imported from Africa for this particular antifolate mutation.

Over the 18-month time period we collected 277 blood samples from patients with a microscopically confirmed P. falciparum infection acquired in Africa. The DNA from all blood samples was amplified by nested PCR systems as described above. 271 samples showed the expected bands at 522 bp for the DHFR gene, while six samples turned out to be negative for P. falciparum DNA.

Among the 271 observed samples of P. falciparum, 170 (62.7%) were imported from West Africa, 46 (16.6%) from East Africa, 36 (13%) from Central Africa, 17 (6.1%) from South Africa and two from Madagascar (Tab 1).

The burden of falciparum malaria is carried mainly by tropical Africa. Since resistance to Chloroquine is now widespread in Africa, the antifolate combination sulfadoxine-pyrimethamine (SP) is often the only affordable alternative.

Unfortunately, SP is particularly prone to the rapid emergence of resistance 18. There is evidence, that in vitro pyrimethamine chemosensitivity will be predictive of in vivo SP efficacy, and that parasitological resistance in Africa is primarily due to the triple mutant in DHFR: S108N, N51I and C59R 192021.

At the moment, the only drug available in Africa to deal with SP failure is often further treatment with SP. Researchers are, therefore, seeking new, safe and inexpensive, second line drugs or combination regimens. Among these regimes chlorproguanil-dapsone is considered a potential replacement for SP in Africa because of its effectiveness against infections associated with triple mutants in DHFR, its short half-life and its low price 212223. However, the efficacy of combinations involving old drugs may be short-lived, since resistance-conferring mutations already exist. In the case of chlorproguanil-dapsone, the change in DHFR enzyme structure resulting from the I164L mutation is known to be associated with resistance to this drug 1920.

Experience with SP in South East Asia and South America has shown that continued use of SP will select for the quadruple mutant DHFR, including I164L. So far, mutations of this potency have not been observed in Africa in any study using standard PCR analyses 1017212324252627. The I164L mutation was also not found in any of the isolates we analyzed within this survey.

Recently, Hastings et al. 28 developed a yeast-based system that is sensitive enough to identify point mutations in the DHFR gene even if they are rare. With the help of this technique alleles that encoded the I164L mutations were isolated from 3 of 6 patient samples with SP treatment failure from Tanzania. However, mutations of all kinds occur during DNA replication, but not all establish themselves and are functional.

Overall, efficient surveillance systems to detect and monitor resistance to SP are required, since malaria infections will become fully resistant to SP and chlorproguanil-dapsone with the selection of the DHFR mutation I164L in Africa. For this reason, several national and regional networks of sentinel sites have been set up in recent years and collaborate in order to monitor antimalarial drug resistance. The East African Network for Monitoring Antimalarial Treatment (EANMAT), for example, started in 1998 to monitor drug resistance with standard in vivo tests 29. TropNetEurop, as the largest network for infectious diseases acquired by travellers worldwide, can function as an additional sensitive tool in the surveillance of antimalarial drug resistance.