Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies.

Susanne H Hodgson ,Bernhards Ogutu,Glenda Daza,Charles Magiri,Kevin Marsh,Philip Bejon,Domtila Kimani,Alfred Muia,Ming Chang,Nicholas Anagnostou ,Simon J Draper ,Nick J Edwards ,Sarah C Gilbert ,Tommy Rampling ,Adrian V S Hill ,Stephen L Hoffman ,Annette M Seilie ,Andrew O Cole ,Elizabeth Juma ,Alexander D Douglas ,Sean C Elias ,Sean C Murphy

doi:10.1186/s12936-015-0541-6

Abstract

BackgroundControlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates.MethodsSamples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood.ResultsLarge volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge.ConclusionsStandard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

Highlights

Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants
VAC052 was a CHMI trial conducted at the Centre for Clinical Vaccinology & Tropical Medicine, Oxford, UK to assess the efficacy of candidate pre-erythrocytic viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors containing multiple epitopes thrombospondin related adhesion protein (ME-TRAP), circumsporozoite protein (CS) and apical membrane antigen 1 (AMA1) administered in combination (Clinicaltrials.gov NCT01739036) (NAA et al, in preparation)
The study consisted of three groups: Group 1 immunized with ChAd63MVA ME-TRAP + CSP (n = 13), Group 2 immunized with ChAd63-MVA ME-TRAP + CSP + AMA1 (n = 13) and Group 3 unimmunized infectivity controls (n = 6)

Summary

Introduction

Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The controlled human malaria infection (CHMI) model is a well-established system to determine the efficacy of drug and vaccine candidates early in their clinical development process. In this model, healthy human subjects are infected with Plasmodium spp. either through the bite of infectious mosquitoes or by needle-based administration of cryopreserved sporozoites or infected red blood cells [1,2,3,4]. When applied in the post-CHMI setting, blood-stage infections can often be detected by NATs two to five days earlier than by TBS, with highly sensitive NAT assays becoming positive around Day 7 post-CHMI (D7.0) when standard CHMI doses of five infectious mosquito bites are used [6,7,8,9,10]

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