Abstract
BackgroundIn the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. This hampers many aspects of P. vivax research. This study aimed to assess the safety of apheresis, a method for selective removal of specific components of blood as a means of extracting and concentrating P. vivax parasites.MethodsAn iterative approach was employed across four non-immune healthy human subjects in single subject cohorts. All four subjects were inoculated with ~ 564 blood stage P. vivax (HMP013-Pv) and subjected to apheresis 10 to 11 days later. Blood samples collected during apheresis (haematocrit layers 0.5% to 11%) were tested for the presence and concentration of P. vivax by microscopy, flow cytometry, 18S rDNA qPCR for total parasites, and pvs25 qRT-PCR for female gametocyte transcripts. Safety was determined by monitoring adverse events. Malaria transmission to mosquitoes was assessed by membrane feeding assays.ResultsThere were no serious adverse events and no significant safety concerns. Apheresis concentrated asexual parasites by up to 4.9-fold (range: 0.9–4.9-fold) and gametocytes by up to 1.45-fold (range: 0.38–1.45-fold) compared to pre-apheresis densities. No single haematocrit layer contained > 40% of all the recovered P. vivax asexual parasites. Ex vivo concentration of parasites by Percoll gradient centrifugation of whole blood achieved greater concentration of gametocytes than apheresis. Mosquito transmission was enhanced by up to fivefold in a single apheresis sample compared to pre-apheresis.ConclusionThe modest level of parasite concentration suggests that the use of apheresis may not be an ideal method for harvesting P. vivax.Trial Registration Australia New Zealand Clinical Trials Registry (ANZCTR) Trial ID: ACTRN12617001502325 registered on 19th October 2017. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373812.
Highlights
In the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo
The 1% HCT layer contained the greatest concentration of all parasites as determined by 18S Quantitative polymerase chain reaction (qPCR), with a 1.3-fold concentration of all parasites and a 3.7 and eightfold concentration for female and male gametocytes, compared to pre-apheresis (Additional file 6: Fig. S5 and Tables 1, 2 and 3)
The highest concentration of gametocytes detected by microscopy was seen in samples collected from the 1% HCT layer (76fold; Additional file 6: Fig. S6A and Table S4) with stage 1 gametocytes making up the largest proportion (37.5%)
Summary
In the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. In the absence of a method for continuous in vitro culture of P. vivax, parasites are usually sourced ex vivo from infected humans. This limits many aspects of the study of P. vivax research, including the development of interventions to control and eliminate P. vivax, such as diagnostics, drugs and vaccines. A reliable source of P. vivax sporozoites is required to test and develop new drugs targeting the dormant liver-stage parasites—the hypnozoites This entails an expensive, logistically complex and unreliable process of sourcing P. vivaxinfected mosquitoes from endemic areas. Experiments are subject to possible effects of strain variability
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