Beyond Blood Smears: Qualification of Plasmodium 18S rRNA as a Biomarker for Controlled Human Malaria Infections

Abstract

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

Figure 1.

Time to positivity for Plasmodium 18S rRNA/rDNA biomarker and blood smears in published controlled human malaria infection (CHMI) studies. Day 0 is the day of CHMI. Squares indicate mean onset of biomarker detection and circles indicate blood smear positivity, unless otherwise denoted as median values in Supplemental Table 1. Shadowed data points are PfSPZ direct venous injection CHMI, and non-shadowed points are mosquito bite CHMI. Error bars indicate minimum/maximum ranges, if available. In some studies, mean or range data could not be determined from the primary publications. Y-axis labels indicate references in the main text, with naive or vaccinated subgroups from each study denoted in parentheses.

Figure 2.

Differences in time to positivity (TTP) for Plasmodium 18S rRNA biomarker vs. blood smears or symptoms across all studies. (A) Time to positivity for quantitative reverse transcription PCR (qRT-PCR) biomarker-estimated parasite densities or to TBS positivity as shown. Differences in TTP for qRT-PCR biomarker compared with blood smears (B) using various biomarker-defined thresholds (any positive or quantitative positive ≥ 20 estimated parasites/mL to ≥ 10,000 estimated parasites/mL as shown). Differences in TTP for qRT-PCR biomarker compared with the first solicited malaria-related symptom of any grade (C) or first solicited grade 2 malaria-related symptom (D) using biomarker-defined thresholds as in A. Each data point corresponds to an individual participant. TBS = thick blood smear; p/mL = estimated parasites/mL. Error bars, 95% CI.

Figure 3.

Ability of biomarker-based measurements to predict delayed blood smear patency. (A) Comparison of blood smear patency (participants grouped based on patency on days 7–9, 10–11, 12–13, or ≥ 14 days post-controlled human malaria infection) vs. first positive quantitative reverse transcription PCR–defined biomarker result of any density including qualitative low positives. (B) Comparison of blood smear patency as in A vs. time to first biomarker result equivalent to ≥ 250 estimated parasites/mL. (C) Comparison of blood smear patency as in A vs. estimated parasite density based on the first positive biomarker result. Each data point corresponds to an individual participant. TBS = thick blood smear; p/mL = estimated parasites/mL. P-values shown for two-sided unpaired Student’s t-tests. Error bars, 95% CI.