Inactivation of Plasmodium falciparum in whole blood using the amustaline and glutathione pathogen reduction technology

Abstract

Background: Risk of transfusion-transmitted (TT) malaria is mainly associated with whole blood (WB) or red blood cell (RBC) transfusion. Risk mitigation relies mostly on donor deferral while a limited number of countries perform blood testing, both negatively impacting blood availability. This study investigated the efficacy of the pathogen reduction system using amustaline and glutathione (GSH) to inactivate Plasmodium falciparum in WB.

Study design and methods: WB units were spiked with ring stage P. falciparum infected RBCs. Parasite loads were measured in samples at time of infection, after 24 hours at room temperature (RT), and after a 24-hour incubation at RT post-treatment with 0.2 mM amustaline and 2 mM GSH. Serial 10-fold dilutions of the samples were inoculated to RBC cultures and maintained up to 4 weeks. Parasitemia was quantified by cytometry.

Results: The P. falciparum viability assay has a limit of detection of a single live parasite per sample. Input parasite titer was >5.7 log₁₀ TCID₅₀ per mL. A 24-hour incubation at RT paused parasite development in controls, but they retained viability and infectivity when tested in culture. In contrast, no infectious parasites were detected in the amustaline/GSH-treated sample after 4 weeks of culture.

Conclusion: A robust level of P. falciparum inactivation was achieved in WB using amustaline/GSH treatment. Parasite log reduction was >5.7 log₁₀ TCID₅₀ per mL. Development of such a pathogen reduction system may provide an opportunity to reduce the risk of TT malaria and improve blood availability.

Figure 1

Process and time points of the evaluation of the P. falciparum inactivation by amustaline/GSH in WB. WB unit (465 mL) was infected with 1.46 × 10⁹ ring‐infected RBC. Controls UT0h and UT24h were removed before the 24‐hour treatment with amustaline and GSH from the INTERCEPT Blood System for RBC. Controls and treated (T24h) samples were processed for P. falciparum viability assay to determine parasite load and reduction. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Example of a P. falciparum viability assay from untreated control UT0h. A WB unit (467 mL, 40% hematocrit) was infected with 1.46 × 10⁹ ring‐iRBCs. After mixing, a 5 mL sample was collected and processed for P. falciparum viability assay as described in Materials and Methods. Parasitemia was followed in assays of dilution 10⁻⁴ to 10⁻⁸. Good correlation was measured between parasitemia development for the different dilutions and time of culture (R² = 0.975 for the time to reach 1% parasitemia). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

P. falciparum death after amustaline/GSH treatment. Parasite morphology and development were followed on Diff‐Quik stained smears in cultures of 10⁻¹ dilutions of the control sample maintained 24 hours at RT (UT24h) and the amustaline/GSH treated sample (T24). Smears were prepared at the time of the culture of 10⁻¹ dilutions (t0) and after 36 hours of culture (t0 + 36 hr). In control UT24h, ring parasites (A) develop into trophozoite (B) and schizont stages with differentiation of infective merozoites (C), which will reproduce a new intraerythrocytic cycle. In contrast, in treated parasites T24h, rings (D) rapidly degenerate after maintaining in culture (E, F) and parasites were expelled from the RBCs. Images were produced on smears of one replicate and are representative of the two other study replicates. Bar: 5 μm. [Color figure can be viewed at http://wileyonlinelibrary.com]