(P-BC-21) Design and Validation of a Pan-Plasmodium Quantitative PCR for Whole Blood Lysates: A Supplemental Assay for Malaria Donor Screening

Background/Case Studies: Transfusion-transmitted malaria remains a concern in non-endemic regions due to persistent, asymptomatic parasitemia among blood donors previously exposed in endemic countries. In 2025, the Food and Drug Administration issued draft guidance recommending selective nucleic acid testing for malaria as an alternative to questionnaire-based deferral policies, aiming to enhance blood safety while promoting an inclusive, genetically diverse donor pool. To address this regulatory shift, we developed a highly sensitive pan-Plasmodium reverse transcription quantitative PCR (RT-qPCR) assay targeting the conserved 18S rRNA gene of all five Plasmodium species infecting human. This target was selected due to its high expression of several thousand copies per parasite, which enhances analytical sensitivity. The assay was designed as a supplemental test for Grifols’ Procleix Plasmodium Assay for donor screening and served as the primary comparator in Grifols’ clinical trial involving high-risk donor populations. Here, we present results from the development and analytical validation phases.

Study

Design/Methods: Initial optimization focused on selecting RNA extraction reagents compatible with blood lysates stabilized in Grifols’ Parasite Transport Medium (PTM) to maximize RNA recovery. The assay was designed to have 100% primer-probe identity across all five Plasmodium species infecting human, but it harbors sequence mismatches with Babesia. Amplification efficiency was evaluated using serial dilutions of P. falciparum in vitro transcripts (IVTs). Analytical sensitivity was determined by Probit analysis of dilutions of Plasmodium-infected cultured red blood cells (iRBCs) in PTM blood lysates. Analytical specificity was assessed through in silico analysis and experimental testing of Babesia-infected blood samples; clinical specificity was evaluated with lysates from 300 healthy donors.

Results/Findings: Amplification efficiency was 96.8%. The 95% limit of detection (LoD) of this assay was 4.3 iRBCs/mL (95% CI: 3.1–7.6 iRBCs/mL), slightly higher than Grifols’ assay (95% LoD = 2.1 iRBCs/mL, 95% CI: 1.4–4.7 iRBCs/mL) tested using the same analytical sensitivity panel. The assay detected IVTs of all five Plasmodium species across multiple concentrations. Clinical sensitivity was 100% based on testing 25 naturally infected clinical specimens. No cross-reactivity with Babesia-infected blood was detected; clinical specificity was 100% (95% CI: 98.8–100%).

Conclusions: This pan-Plasmodium RT-qPCR serves as a robust supplemental assay for malaria donor screening, with future development aimed at enabling species classification and quantitative applications to support longitudinal studies of low-level parasitemia in semi-immune donors from endemic regions.