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Background  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. 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).    Methods  Standardized 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.    Results  On 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.    Conclusion  Smear 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.

malaria journal

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