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A high-level pyrimethamine resistance Plasmodium falciparum lineage with triple dihydrofolate reductase (dhfr) mutations prevails across Africa. However, additional minority lineages were seen. We examined transmission success of mutant dhfr haplotypes among 22 children in The Gambia and 60 infected Anopheles gambiae mosquitoes fed on their blood. Additional polymorphic genes of the gametocyte-specific protein (pfg377) and merozoite surface protein-1 (MSP-1) were examined. Similarities were seen between pfg377 and MSP-1 alleles in children and mosquitoes and evidence of cross-mating between different parasite genotypes was seen in some infected mosquitoes, reflecting high transmission success of existing clones. With regard to dhfr, 16 haplotypes were seen among the children: 2 carried double mutations and 14 carried triple mutations. However, only nine haplotypes, all with triple mutations, were detected among mosquitoes. A single triple-mutant dhfr haplotype, similar to that in other countries in Africa, predominated among children (42%) and mosquitoes (60%), supporting the hypothesis of migration of this haplotype across Africa. However, evidence of cross-mating between the above haplotypes signifies the role of local evolution.

The polymorphism of malaria parasites will greatly influence the efficiency of antimalarial drugs and vaccines. This study determined the genetic diversity of Plasmodium falciparum infections in 107 travelers and estimated the importance of mutations in the parasite dihydrofolate reductase (dhfr) gene for clinical breakthrough during proguanil prophylaxis. Genotyping with regards to the three highly polymorphic antigen-coding regions (merozoite surface protein-1 [msp-1], msp-2, and the glutamate-rich protein [glurp]) revealed multiple genotypes (up to five) in 64% of the patients. Single genotype infections were mainly associated with prior intake of antimalarial drugs, but also with a shorter stay in a malaria-endemic area and low parasite density. Malaria breakthrough despite proguanil prophylaxis was always associated with mutations in the dhfr gene; always the Asn-108 mutation and often the Ile-51 and Arg-59 mutations. The Leu-164 mutation was found in four travelers from Africa. Travelers with limited time in an endemic area were often infected with polyclonal P. falciparum infections, which suggests that single mosquito inoculations are often composed of several genetically diverse parasites. Chemoprophylaxis reduces the number of infecting clones and selects for resistant parasites as shown for proguanil through mutations in the dhfr gene.

A rapid and simple method to detect pyrimethamine susceptibility of Plasmodium falciparum by analyzing DNA from whole blood is presented. Samples from cultured isolates and from patients infected with P. falciparum were spotted onto filter paper disks, dried, and stored for subsequent analyses. After extracting the P. falciparum DNA using Chelex-100 ion-chelating resin (Bio-Rad, Richmond, CA), the polymerase chain reaction (PCR) was used to amplify the dihydrofolate reductase (dhfr) gene. The PCR product of 727 basepairs was digested with the Alu I restriction endonuclease to detect whether the isolates were sensitive or resistant to the antimalarial drugs pyrimethamine and cycloguanil. This reaction endonuclease digests only DNA from pyrimethamine-sensitive parasites because the recognition locus of Alu I is changed by mutations giving rise to pyrimethamine and cycloguanil resistance. This method is simple and sensitive and could therefore bu used to study the epidemiology of pyrimethamine resistant in P. falciparum. The DHFR gene of pyrimethamine-resistance clones from Vietnamese patients showed three amino acid changes that were previously found in pyrimethamine-resistant isolates. Two other clones, T9/94 and T9/96, originally isolated from a single malaria patient from Thailand, had different DHFR gene sequences. The nucleotide sequence of the DHFR gene from T9/96 was identical to the wild-type DHFR sequence, whereas T9/94 possessed amino acid substitutions at positions 16 and 108 that have been described in cycloguanil-resistant parasites.