A pilot randomised trial of induced blood-stage Plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs.

BACKGROUND: Critical to the development of new drugs for treatment of malaria is the capacity to safely evaluate their activity in human subjects. The approach that has been most commonly used is testing in subjects with natural malaria infection, a methodology that may expose symptomatic subjects to the risk of ineffective treatment. Here we describe the development and pilot testing of a system to undertake experimental infection using blood stage Plasmodium falciparum parasites (BSP). The objectives of the study were to assess the feasibility and safety of induced BSP infection as a method for assessment of efficacy of new drug candidates for the treatment of P. falciparum infection. METHODS AND FINDINGS: A prospective, unblinded, Phase IIa trial was undertaken in 19 healthy, malaria-naïve, male adult volunteers who were infected with BSP and followed with careful clinical and laboratory observation, including a sensitive, quantitative malaria PCR assay. Volunteers were randomly allocated to treatment with either of two licensed antimalarial drug combinations, artemether-lumefantrine (A/L) or atovaquone-proguanil (A/P). In the first cohort (n = 6) where volunteers received ∼360 BSP, none reached the target parasitemia of 1,000 before the day designated for antimalarial treatment (day 6). In the second and third cohorts, 13 volunteers received 1,800 BSP, with all reaching the target parasitemia before receiving treatment (A/L, n = 6; A/P, n = 7) The study demonstrated safety in the 19 volunteers tested, and a significant difference in the clearance kinetics of parasitemia between the drugs in the 13 evaluable subjects, with mean parasite reduction ratios of 759 for A/L and 17 for A/P (95% CI 120-4786 and 7-40 respectively; p<0.01). CONCLUSIONS: This system offers a flexible and safe approach to testing the in vivo activity of novel antimalarials. TRIAL REGISTRATION: ClinicalTrials.gov NCT01055002.

A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers.

BACKGROUND: The accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents. METHODS: A real-time quantitative PCR (rt-qPCR) method using primers and a hydrolysis probe that targets the 18S rRNA gene was adapted and optimized to estimate parasite load in blood samples. Samples included laboratory prepared blood samples of varying parasite concentrations (6.4 × 10(5) to 6.4 parasites per 500 µl of packed red blood cells (500pRBC)) and blood samples collected from an experimentally infected human subject collected at 19 time points over 10 days. Sample preparation and extraction, detection chemistry, assay reproducibility, and limit of detection were compared to a previously published SYBR Green rt-qPCR used in a malaria vaccine clinical trial. RESULTS: Both the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 10(1) parasites per 500pRBC. However non-specific amplification in the SYBR Green rt-qPCR assay led to either inaccurate estimation of parasite load at levels below 6.4 × 10(2) parasites per 500pRBC and to false-positive detection of parasites in negative samples. The rt-qPCR hydrolysis probe assay was specific and provided reliable quantification of parasitaemia down to 6.4 × 10(1) parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 10(2) copies per 500pRBC. CONCLUSIONS: These results show that the hydrolysis probe rt-qPCR assay is superior to the SYBR Green rt-qPCR for the quantification of P. falciparum in human blood samples. The hydrolysis probe rt-qPCR is now in use in the Queensland paediatric infectious diseases laboratory (QPID) to monitor parasitaemia in experimentally-infected clinical trial subjects.