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 standard 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”) is accepted by European and US FDAs as an alternative to refrigeration (4 °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.ResultsUsing the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (− 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.ConclusionsBasic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.
Highlights
The malaria Eradication Research Agenda has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication
Overall trends were independent of storage method (Fig. 1a and Table 1, Odds ratios (OR) = 1.31, standard error = 0.41, p = 0.4) or duration (Fig. 1b and Table 1, odds ratios (OR) = 1.01, se = 0.04, p = 0.72) with most of the random variation explained by differences between experimental block (Table 1 and Additional file 5a) and negligible contribution by RBC donor (Fig. 1b, Table 1 and Additional file 5a)
The mean prevalence of mosquitoes infected with P. falciparum oocysts (NF54) was 53 ± 6.4% (mean ± se, n = 11 standard-membrane feeding assays (SMFAs), mean mosquito midguts sampled/SMFA = 26, total number of mosquito midguts sampled = 283) with 59 ± 4.6% (n = 2) and 52 ± 7.9% (n = 9) in mosquitoes infected with parasites cultured in refrigerated and cryo-preserved RBCs, respectively (Fig. 2a, left panel)
Summary
The malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The experimental model for evaluating basic parasite life history or the efficacy of various interventions typically involve standard-membrane feeding assays (SMFAs), where mature male and female gametocytes of P. falciparum cultured in vitro are supplemented with naïve RBCs and offered as part of a blood-meal to female Anopheles spp. mosquitoes. These assays are followed by the collection of various measures of parasite fitness in the mosquito vector [7,8,9,10,11]. Available methodology is a synthesis of several tools and techniques that have been elegantly summarized in two recent publications [13, 15]
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