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.

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

“…Measuring gene expression is increasingly important for a diverse range of clinical applications [23][24][25][26] . Purification of RNA, an essential prerequisite for qPCR, removes other cellular components and data must be normalised based on the stable expression of endogenous control genes.…”

A replication-competent late liver stage–attenuated human malaria parasite

“…Alternatively, parasite nucleic acid can be detected by a highly sensitive parasite 18S qRT-PCR assay (50,51). This has been used as a diagnostic tool to detect the presence of blood stage parasites in human blood samples following CHMI (52). The assay is capable of detecting as few as 1 ring stage parasite equivalent in 50 μL of blood sample, which comprises 3.86 log 10 copies of 18S rRNA/mL of blood (50).…”

“…studies to detect parasites in blood after challenge by 5-7 infectious mosquito bites (49)(50)(51)(52). The assay has a detection limit of 20 parasite equivalents/mL (52). However, qRT-PCR for 18S rRNA can only provide information regarding the presence or absence of parasite nucleic acids.…”

Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests

“…This result demonstrates that the ENAR clearly co-extracts DNA and RNA. The Pf 18S ribosomal RNA, detected by the Pspp18S RT-qPCR assay, is constantly and highly expressed during the life cycle of the parasite 27,28 , while the acidic terminal sequence of the var genes (PfEMP1), detected by the PfvarATS assay, is associated with lower RNA levels 29 . The ability of the ENAR protocol to co-extract DNA and RNA was also demonstrated with the following experiment: Five µL of an in vitro-generated stage V gametocyte culture was applied onto the RDTs and stored at RT for three weeks before NAs were extracted by ENAR.…”