The antifolate combination sulfadoxine /pyrimethamine (SP) is currently the first line treatment for uncomplicated falciparum malaria in several African countries. Pyrimethamine (PM) and sulfadoxine (SD) are inhibitors of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) respectively. The selection and rapid spread of resistance to SP, which in some parts of East Africa has reached a prevalence of more than 30% , has already seriously compromised the therapeutic usefulness of SP. A new antifolate combination of chlorproguanil (CPG) and dapsone (DDS), known as Lapdap™ [2, 3], has now been developed. CPG is metabolised in vivo to its active triazine metabolite, chlorcycloguanil (CCG), which, like PM, inhibits the DHFR enzyme. DDS, like SD, inhibits the activity of dhps. LapDap™ is effective in vivo and retains efficacy against SP resistant infections , justifying the use of this drug as a replacement for SP.
Resistance to SP in Africa is attributable to parasites that carry point mutations at codons 108, 51 and 59 of dhfr, and resistance is augmented by point mutations at codon 437 and/or 540 of the dhps gene . Experience in South East Asia and South America shows that the continued use of SP will eventually select for the mutation at codon 164 (from isoleucine to leucine). Once this occurs in Africa, parasites will become highly resistant to SP. This study shows that the selection of this Leu-164 mutation will also render the new antifolate combination CPG/DDS ineffective . Therefore, the occurrence in Africa of this mutation would compromise the useful therapeutic life (UTL) of the new antifolate combination CPG/DDS.
Although SP has been widely used in Africa, the Leu-164 mutation has not yet been reported using standard protocols for the detection of this point mutation in dhfr (PCR-RFLP and sequencing). For instance, a study carried out on isolates collected during CPG/DDS trial in 1999, in Usambara Mountains, Tanzania, did not detect the 164-Leu using PCR-RFLP . However, by using the yeast complementation approach, which is based on the expression of plasmodium dhfr genes in yeast cells followed by the selection of cells expressing highly resistant alleles, Hastings and co-workers  have reported the presence of the Leu-164 mutation in three SP resistant isolates collected between 1998–1999, from Muheza, Tanzania – an area with a high level of resistance resistance to SP . This finding indicates that parasites carrying dhfr alleles with the Leu-164 mutation exist in Africa. As a consequence, the UTL of the new combination CPG/DDS could be very short.
Although the level of SP resistance is high in Muheza, this drug is still used as the first line treatment because of the lack of affordable alternative antimalarials. Under these conditions, one would expect the selection and spread of parasites carrying the Leu-164 mutation under SP pressure, if this mutation conferred a biological advantage. Treatment-mediated selection of the mutation would be expected to raise the gene frequency to the point where the dhfr alleles can be detected in Muheza using standard protocols.
With this in mind, the presence of the Leu-164 mutation are being monitored in isolates collected at Muheza from 1999, the year the Leu-164 mutation was detected in this area, using the yeast cell complementation technique. In this paper, are presented the results of the analysis of 85 recent isolates collected randomly from children suffering from uncomplicated malaria at Muheza Designated District Hospital, Tanzania, between October 2002 to January 2003.