Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes.

Abstract

BackgroundThe malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is small membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of \"infectious\" gametocytes requires 10-12 days with considerable physico-chemical demands imposed on host RBCs and thus, \"fresh\" RBCs that are [≤]1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (cryo-preserved RBCs herein) is approved by the European and US FDAs as an alternative to refrigeration (4{degrees}C) for preserving RBC quality and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for >4 days.\n\nResultsUsing the standard laboratory strain, P. falciparum NF54, we first demonstrate that cryo-preserved RBCs preserved in the gaseous phase of liquid nitrogen and thawed after storage for 1, 4, 8 and 12 weeks, supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Using data from 11 SMFAs and RBCs from 4 separate donors with 3 donors re-tested following various periods of cryo-preservation, we show that overall levels of sporogony in the mosquito, as measured by oocyst prevalence and burdens in the midguts and sporozoites in salivary glands, were similar or better than using [≤]1-week old refrigerated RBCs. Additionally, the potential for cryo-preserved RBCs to serve as a universal substrate for SMFAs is shown for a Cambodian isolate of P. falciparum.\n\nConclusionsConsidering the suitability of cryo-preserved RBCs for P. falciparum SMFAs, we suggest guidelines for their use and how they can be integrated into an existing laboratory/insectary framework with the potential to significantly reduce running costs and provide greater reliability. Finally, we discuss scenarios where cryo-preserved RBCs may be especially useful in enhancing our understanding and/or providing novel insights into the patterns and process underlying human-to-mosquito transmission.