Molecular epidemiology of Plasmodium falciparum antifolate resistance in Vietnam: genotyping for resistance variants of dihydropteroate synthase and dihydrofolate reductase.

Using PCR techniques, we analysed the dihydropteroate synthase (DHPS) mutations associated with sulphonamide resistance and the dihydrofolate reductase (DHFR) mutations associated with resistance to pyrimethamine and cycloguanil in samples from Plasmodium falciparum-infected Vietnamese patients. Of the 40 samples analysed, 39 had DHFR mutations associated with high level resistance to pyrimethamine, whereas only three had mutations at position 164, which is linked to cross resistance to both DHFR inhibitors. The DHPS, 437Gly variant associated with very mild resistance to sulphadoxine was found in 38 out of the 40 samples. Of seven samples resistant to Fansidar in vivo, only two were fully explained by the currently documented DHPS mutations. The treatment failure could be due to a high level of pyrimethamine resistance caused by the detected mutations. Most patients, however, were cured with a single dose of Fansidar in spite of the high number of resistance mutations found.

Artemisinin population pharmacokinetics in children and adults with uncomplicated falciparum malaria.

AIMS: To investigate the pharmacokinetics of the antimalarial artemisinin in the field setting using sparsely collected data. METHODS: Artemisinin concentrations were determined by h.p.l.c. in a total of 107 capillary plasma samples collected on the first day and in 33 samples on the last day of a 5-day oral artemisinin regimen of 10 mg kg(-1) day(-1) in 23 paediatric (aged 2-12 years) and 31 adult (aged 16-45 years) Vietnamese patients with uncomplicated falciparum malaria. The population model was developed using NONMEM, incorporating interoccasion variability and accounting for a systematic change in artemisinin pharmacokinetics with time, modelled as a change in oral bioavailability. RESULTS: Clinical efficacy, in terms of parasite clearance and fever subsidence times, was comparable between children and adults. A one-compartment model with separate pharmacokinetic estimates for children and adults was found best to describe the disposition of artemisinin after oral administration. The population estimates for artemisinin clearance and distribution volume, respectively, were 432 1 h(-1) and 16001 for adults and 14.41 h(-1) kg(-1) and 37.91 kg(-1) for children, with an intersubject variability (collectively for both age groups) of 45% and 104%, respectively. The oral bioavailability was estimated to decrease from Day 1 to Day 5 by a factor of 6.9, a value found to be similar for children and adults. CONCLUSIONS: Artemisinin pharmacokinetic data was successfully derived in both paediatric and adult patients using 2-3 capillary blood samples taken in conjunction with parasitaemia monitoring. This study's findings advocated the dosing of artemisinin to children according to bodyweight and to adults according to a standard dose.

Artemisinin pharmacokinetics is time-dependent during repeated oral administration in healthy male adults.

The pharmacokinetics of the antimalarial artemisinin exhibited an unusual time dependency during a 7-day oral daily regimen of 500 mg in 10 healthy, male Vietnamese adults. Artemisinin areas under the plasma concentration-time curve (AUC) decreased to 34% (median) by day 4 with a further decrease by day 7 to only 24% of values obtained after the first day of administration. In seven subjects restudied after a 2-week washout period, artemisinin AUCs had almost normalized, demonstrating the reversibility of the time-dependent drug disposition. The results suggest artemisinin exhibits an auto-inductive effect on drug metabolism of an unusual magnitude. This may partly explain why some patients on standard doses, due to subparasiticidal drug levels toward the end of a standard regimen, do not completely clear parasites. Further, the possibility of drug-drug metabolic interactions during combination regimens is implicated.

Rapid detection of pyrimethamine susceptibility of Plasmodium falciparum by restriction endonuclease digestion of the dihydrofolate reductase 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.