WHO Standard Calibration for Malaria NAT Assays

The WHO international standard for P. falciparum DNA nucleic acid amplification technology (NAT) assays, obtained from the National Institute for Biological Standards and Control (NIBSC; Hertfordshire, United Kingdom) was used as the calibration reference reagent for of the Plasmodium spp. and P. falciparum assays. The standard consists of a freeze-dried preparation of whole blood collected by exchange transfusion from a patient infected with P. falciparum. Following NIBSC recommendations, this lyophilized material was suspended in 500 µl of sterile, nuclease-free water to a final concentration of 1×10⁹ IU/ml, which corresponds to a parasitemia of 9.79 parasites/100 red blood cells [11] . The parasite density of the NAT assays after reconstitution was estimated to be 469,920 parasites/µl, based on the average red blood cell count [from uninfected donor] of 4.8×10⁶ RBC/µl. Unless otherwise indicated, fresh uninfected whole blood was used as a diluent to prepare serial dilutions. The uninfected whole blood was obtained from donors from Washington DC metropolitan area under WRAIR approved protocol. After reconstitution, genomic DNA was extracted with the EZ1 DNA blood kit on the EZ1 Advanced XL automated sample purification system (Qiagen, Valencia, CA) as recommended by the manufacturer.