Amplification reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and 45 ng of template cDNA prepared from each sample. Samples were set up in duplicate and experiments were repeated independently twice. PCR products were amplified and detected on an ABI Prism 7700 (Applied Biosystems). Cycling parameters for PCR were an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. To analyse the relative transcript levels, the threshold cycle value (Ct) of each sample was used to calculate and compare the ΔCt of each sample to that of the P. vivax housekeeping gene Sal I β-tubulin; the ΔΔCt was also calculated as in [17] to compare the transcript levels of pvcrt-o and pvmdr 1 in the patient with severe symptoms and in the patient with mild symptoms.