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

Malaria Journal

Research

Andrew O Cole¹  Alfred Muia¹  Charles Magiri¹  Elizabeth A Juma²  Bernhards Ogutu²  Glenda Daza³  Sean C Murphy³  Ming Chang³  Annette M Seilie³  Philip Bejon⁴  Domtila Kimani⁴  Kevin Marsh⁴  Stephen L Hoffman⁵  Alexander D Douglas⁶  Thomas W Rampling⁶  Nick J Edwards⁶  Sarah C Gilbert⁶  Susanne H Hodgson⁶  Sean C Elias⁶  Simon J Draper⁶  Nicholas A Anagnostou⁶  Adrian VS Hill⁶ 

[1] Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya;Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya;Centre for Research in Therapeutic Sciences, Strathmore University, Nairobi, Kenya;Department of Laboratory Medicine and Center for Emerging and Re-Emerging Infectious Diseases, University of Washington (UW), 750 Republican St., E633, 98109, Seattle, WA, USA;Kenya Medical Research Institute - Wellcome Trust, Centre for Geographical Medical Research (Coast), Kilifi, Kenya;Sanaria, Inc, Rockville, MD, USA;The Jenner Institute, University of Oxford, Oxford, UK;

关键词: Plasmodium falciparum;    18S rRNA;    PCR;    RT-PCR;    Standards;    Controls;    Calibrators;    Pre-erythrocytic;   

DOI  :  10.1186/s12936-015-0541-6

 received in 2014-09-18, accepted in 2015-01-05,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Hodgson et al.; licensee Biomed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.