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Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.

To assess the level of resistance to chloroquine (CQ) of Plasmodium falciparum in The Gambia in 1995-1996 we measured susceptibility in vivo by quantifying parasitaemia of children with mild malaria on days 4 and 8 after treatment. Pretreatment blood samples were used for susceptibility testing in vitro by the World Health Organization microtest and the prevalence of the tyrosine (tyr)86 allele of the Pfmdr1 gene was assessed by the polymerase chain reaction and restriction fragment length polymorphism analysis. Seven of 42 children (17%) treated with CQ remained parasitaemic on day 4 and required a change of antimalarial treatment. Susceptibility assays in vitro were performed on 50 P. falciparum isolates obtained from eligible children before treatment; 36 (72%) were resistant to CQ (> or = 1.6 mumol/L). The median minimum inhibitory concentration (MIC) of artemether was 3.38 nmol/L (range 0.42-13.51 nmol/L) and the median MIC of dihydroartemisinin was 0.88 nmol/L (range 0.22-14.04 nmol/L). Susceptibility in vitro to CQ and the Pfmdr1 genotype were determined for 31 fresh isolates. The allele was present in 12 of 22 isolates found to be resistant to CQ in vitro, but in none of the 9 isolates which were susceptible (Fisher's exact test, P = 0.005). All P. falciparum isolates with the tyr86 allele were CQ resistant in vitro, but since only half of all resistant isolates contained the allele, additional explanations for CQ resistance are required.