Measurement of parasitological data by quantitative real-time PCR from controlled human malaria infection trials at the Walter Reed Army Institute of Research.

Edwin Kamau,Saba Alemayehu,Jessica Cowden,Jason Regules,Christian F Ockenhouse,Karla C Feghali,Jack Komisar

doi:10.1186/1475-2875-13-288

Edwin Kamau, Saba Alemayehu + Show 5 more

Open Access

https://doi.org/10.1186/1475-2875-13-288

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Journal: Malaria journal	Publication Date: Jul 28, 2014
Citations: 38	License type: cc-by

Affiliation: Walter Reed Army Institute of Research

Abstract

BackgroundThe use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Parasitological data presented here are from non-immunized, control-challenged subjects who participated in two CHMI trials conducted at the Walter Reed Army Institute of Research (WRAIR).MethodsStandardized sporozoite challenge was achieved through the bite of five Anopheles stephensi mosquitoes infected with the 3D7clone of the NF54 strain of Plasmodium falciparum. Blood smears were scored positive when two unambiguous parasites were found. Analysis of parasitological PCR data was performed on log-transformed data using an independent sample t-test when comparing the two studies. The multiplication rate of blood-stage parasites was estimated using the linear model.ResultsOn average, parasites were detected 4.91 days (95% CI = 4.190 to 5.627) before smears. The earliest parasites were detected within 120 hours (5.01 days) after challenge. Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers. The parasite multiplication rates for both studies was 8.18 (95% CI = 6.162 to 10.20). Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit.ConclusionSmear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint. This would allow for early treatment to reduce clinical illness and risk for study participants. To use qPCR as the primary endpoint in CHMI trials, an algorithm of two positives by qPCR where one of the positives must have parasite density of at least 2 parasites/μL is proposed.

Highlights

The use of quantitative real-time PCR has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials
Time to parasitaemia Data from 16 unvaccinated infectivity control subjects enrolled in two different malaria vaccine trials at the Walter Reed Army Institute of Research (WRAIR) were analysed by quantitative real-time PCR (qPCR)
This study show detailed estimations of critical parameters in the parasite life cycle from CHMI studies conducted at WRAIR using qPCR measurements of parasitaemia in infectivity control, malaria-naive volunteers

Summary

Introduction

The use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Controlled human malaria infection (CHMI) is increasingly being used to assess the efficacy of malaria vaccines as well as to evaluate antimalarial drug candidates [1,2]. CHMI trials allow for detailed evaluation of parasite growth kinetics and provide an opportunity to characterize immunological responses [1,2], which can be informative for further optimization of a vaccine or drug candidate. The comparative prepatent period of treated subjects and control subjects is used to assess efficacy of the vaccine or drug being tested [3]

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