Christopher L. Peatey

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

Effect of Antimalarial Drugs on Plasmodium falciparum Gametocytes

Gametocytes are the sexual stage of the malaria parasite and are essential for transmission to the mosquito. Antimalarial drugs have been reported to affect gametocyte production in vivo, which leads to a potential increase in transmission. We used transgenic Plasmodium falciparum parasites expressing a green fluorescent protein tag in a fluorescence-activated cell sorting-based assay to measure the effect of 8 antimalarial drugs on gametocyte production in vitro. Exposure to antimalarial drugs resulted in an increase in the number of gametocytes in test cultures. Although a dose-dependent reduction in late-stage gametocyte viability was observed, none of the drugs tested statistically significantly reduced gametocyte numbers.

A high-throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes

Recent success in the global reduction campaign against malaria has resulted in the possibility that it may be feasible to drastically reduce or even eradicate malaria even without the introduction of a vaccine. However, while there has been significant effort to design the next generation of antimalarial drugs, one area that is underrepresented in the current antimalarial pharmacopeia is that of transmission blocking drugs directed at late-stage gametocytes. Here we describe the development of a robust and simple assay that is amenable to a high throughput format for the discovery of new antigametocyte drugs.

Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes?

BackgroundRecent renewed emphasis on the eradication of malaria has highlighted the need for more tools with which to achieve this ambitious goal. One high priority area is the need to determine the gametocytocidal activity of both currently used anti-malarial drugs and those in the development pipeline. However, testing the activity of compounds against Plasmodium falciparum gametocytes is technically challenging both in vivo and in vitro.MethodsHere the use of a simple robust assay to screen a panel of currently used and experimental anti-malarial drugs against mature P. falciparum gametocytes is described.ResultsEight of 44 compounds tested reduced gametocyte viability by at least 50% and three showed IC50 values in nM range.ConclusionsThere is a need to identify new compounds with activity against late stage gametocytes and the information provided by this in vitro assay is a valuable first step, which can guide future clinical studies.

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

BackgroundThe 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.MethodsA 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 × 105 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.ResultsBoth the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 101 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 × 102 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 × 101 parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 102 copies per 500pRBC.ConclusionsThese 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.

A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum.

Study of the formation of the sexual blood stages of the malaria parasite has been significantly hampered by the absence of a reliable, reproducible assay devoid of operator bias and error. Here we report on the development of an assay utilizing a green fluorescent protein chimera of the early sexual blood stage protein Pfs16 as a marker for commitment to gametocytogenesis. Analysis of parasites via fluorescence activated cell sorting allows for the accurate assessment of gametocyte production well before morphological changes are apparent.

Piperaquine Monotherapy of Drug-Susceptible Plasmodium falciparum Infection Results in Rapid Clearance of Parasitemia but Is Followed by the Appearance of Gametocytemia

Background. Piperaquine, coformulated with dihydroartemisinin, is a component of a widely used artemisinin combination therapy. There is a paucity of data on its antimalarial activity as a single agent. Such data, if available, would inform selection of new coformulations. Methods. We undertook a study in healthy subjects, using the induced blood stage malaria (IBSM) model to test the antimalarial activity of single doses of piperaquine (960, 640, and 480 mg) in 3 cohorts. In a pilot study in the third cohort, gametocyte clearance following administration of 15 mg, or 45 mg or no primaquine was investigated. Results. Parasite clearance over the 48-hour period after piperaquine administration was more rapid in the 960 mg cohort, compared with the 640 mg cohort (parasite reduction ratio, 2951 [95% confidence interval {CI}, 1520–5728] vs 586 [95% CI, 351–978]; P < .001). All 24 subjects developed gametocytemia as determined by pfs25 transcripts. Clearance of pfs25 was significantly faster in those receiving primaquine than in those not receiving primaquine (P < .001). Conclusions. Piperaquine possesses rapid parasite-clearing activity, but monotherapy is followed by the appearance of gametocytemia, which could facilitate the spread of malaria. This new information should be taken into account when developing future antimalarial coformulations. Clinical Trials Registration ACTRN12613000565741.

Antimalarial Asexual Stage-Specific and Gametocytocidal Activities of HIV Protease Inhibitors

ABSTRACT The stage-specific antimalarial activities of a panel of antiretroviral protease inhibitors (PIs), including two nonpeptidic PIs (tipranavir and darunavir), were tested in vitro against Plasmodium falciparum. While darunavir demonstrated limited antimalarial activity (effective concentration [EC50], >50 μM), tipranavir was active at clinically relevant concentrations (EC50, 12 to 21 μM). Saquinavir, lopinavir, and tipranavir preferentially inhibited the growth of mature asexual-stage parasites (24 h postinvasion). While all of the PIs tested inhibited gametocytogenesis, tipranavir was the only one to exhibit gametocytocidal activity.

Mitochondrial Membrane Potential in a Small Subset of Artemisinin-Induced Dormant Plasmodium falciparum Parasites In Vitro

Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage.

Enhanced Gametocyte Formation in Erythrocyte Progenitor Cells: A Site-Specific Adaptation by Plasmodium falciparum

Gametocytogenesis by Plasmodium falciparum is essential for transmission of the parasite from human to mosquito, yet developing gametocytes lack expression of surface proteins required for cytoadherence. Therefore, elimination from the circulation should occur unless they are sequestered in regions of low blood flow such as the extracellular spaces of the bone marrow. Our data indicate that gametocytogenesis is enhanced in the presence of erythroid progenitors found within the bone marrow. Furthermore, atomic force microscopy indicates that developing gametocytes undergo remarkable shifts in their erythrocyte membrane elasticity, which may allow them to be retained within the bone marrow until maturation.