Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: A Gambian field study

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.