Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies

Abstract

BackgroundControlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates.MethodsSamples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood.ResultsLarge volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge.ConclusionsStandard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

Microscopic and molecular evidence of the presence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in an area with low, seasonal and unstable malaria transmission in Ethiopia

“…Moreover, PCR failed to detect two of the microscopy positive P. falciparum samples and three of the microscopy positive P. vivax samples. The lack of detection of infections by PCR in microscopy and RDT positive samples could be due to the small amount of DNA used in the PCR reaction as evidenced by Hodgson et al [ 42 ] where higher sample volumes will increase the detectability of infections.…”

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

“…The RNA-based methods include traditional and quantitative reverse transcriptase PCRs and nucleic acid sequence-based amplification(NASBA) methods, which rely on detection of the parasite 18S rRNA transcript in purified RNA samples [44, 45]. The field application of the above RNA amplification techniques, however, is limited by the requirement for highly trained personnel, electrified instrumentation, and large sample volumes (approximately 50 μL of paper filter-spotted blood or >200 μL of tube collected blood)[46, 47], as well as their susceptibility to cross contamination and to common PCR inhibitors[18]. …”

Workshop report: Malaria vaccine development in Europe–preparing for the future

“…For diagnosis, some centres therefore explore the possibility of using a quantitative polymerase chain reaction (qPCR) [54] along with the current gold standard, thick blood smear microscopy [55]. Preliminary data suggest that qPCR-guided treatment begins earlier but does not significantly affect the incidence of adverse events (personal communication R. Sauerwein).…”