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

Susanne H. Hodgson, Alexander D. Douglas, Nick J. Edwards, Domtila Kimani, Sean C. Elias, Ming Chang, Glenda Daza, Annette M. Seilie, Charles Magiri, Alfred Muia, Elizabeth A. Juma, Andrew O. Cole, Thomas W. Rampling, Nicholas A. Anagnostou, Sarah C. Gilbert, Stephen L. Hoffman, Simon J. Draper, Philip Bejon, Bernhards Ogutu, Kevin MarshAdrian V.S. Hill, Sean C. Murphy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

Background: Controlled 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. Methods: Samples 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. Results: Large 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. Conclusions: Standard 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.

Original languageEnglish
Article number33
JournalMalaria Journal
Volume14
Issue number1
DOIs
Publication statusPublished - 2015
Externally publishedYes

Bibliographical note

Funding Information:
This work is published with the permission of the director of KEMRI. We thank the nursing, administrative and laboratory teams at the Centre for Clinical Research KEMRI and the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford; the laboratory teams at the KEMRI Centres for Clinical Research, Nairobi and Geographic Medicine Research, Kilifi and the Jenner Institute, University of Oxford, the Sanaria Manufacturing, Quality Systems, Clinical, Legal, and Operations Teams and all the study participants. This work was supported by the European and Developing Countries Clinical Trial Partnership (grant SP 2011.41304.062 to BO, KM, SHH and AVSH); the Wellcome Trust (grants 097940/Z/11/Z to SHH and 45488/Z/05 to AVSH); the U.S. National Institutes of Health (grant K08AI097238 to SCM) and the UK Medical Research Council (grant G1000527 to SJD). Funding for manufacture, quality control release and stability studies of the Sanaria PfSPZ Challenge product was provided by the National Institute of Allergy and Infectious Disease Business Innovation Research grant (R44AI058375). The authors thank the UW Center for AIDS Research (CFAR, AI27757) Clinical Research and Retrovirology Core (Robert Coombs). AVSH and SJD are Jenner Investigators. The funders had no role in the design, collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.

Publisher Copyright:
© 2015 Hodgson et al.; licensee Biomed Central.

Keywords

  • 18S rRNA
  • Calibrators
  • Controls
  • PCR
  • Plasmodium falciparum
  • Pre-erythrocytic
  • RT-PCR
  • Standards

Fingerprint

Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this