title: 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: 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.
authors: The Jenner Institute, University of Oxford, Oxford, UK. susanne.hodgson@ndm.ox.ac.uk.
authors: Susanne H
authors: SH
authors: Hodgson
authors: The Jenner Institute, University of Oxford, Oxford, UK. sandy.douglas@ndm.ox.ac.uk.
authors: Alexander D
authors: AD
authors: Douglas
authors: The Jenner Institute, University of Oxford, Oxford, UK. nick.edwards@ndm.ox.ac.uk.
authors: Nick J
authors: NJ
authors: Edwards
authors: Kenya Medical Research Institute - Wellcome Trust, Centre for Geographical Medical Research (Coast), Kilifi, Kenya. dkimani@kilifi.kemri-wellcome.org.
authors: Domtila
authors: D
authors: Kimani
authors: The Jenner Institute, University of Oxford, Oxford, UK. sean.elias@ndm.ox.ac.uk.
authors: Sean C
authors: SC
authors: Elias
authors: Department of Laboratory Medicine and Center for Emerging and Re-Emerging Infectious Diseases, University of Washington (UW), 750 Republican St., E633, Seattle, WA, 98109, USA. minchang@u.washington.edu.
authors: Ming
authors: M
authors: Chang
authors: Department of Laboratory Medicine and Center for Emerging and Re-Emerging Infectious Diseases, University of Washington (UW), 750 Republican St., E633, Seattle, WA, 98109, USA. gdaza@uw.edu.
authors: Glenda
authors: G
authors: Daza
authors: Department of Laboratory Medicine and Center for Emerging and Re-Emerging Infectious Diseases, University of Washington (UW), 750 Republican St., E633, Seattle, WA, 98109, USA. amseilie@uw.edu.
authors: Annette M
authors: AM
authors: Seilie
authors: Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. charles.magiri@usamru-k.org.
authors: Charles
authors: C
authors: Magiri
authors: Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. dealfremmdoh@yahoo.com.
authors: Alfred
authors: A
authors: Muia
authors: Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. jumaelizabeth@yahoo.com.|Centre for Research in Therapeutic Sciences, Strathmore University, Nairobi, Kenya. jumaelizabeth@yahoo.com.
authors: Elizabeth A
authors: EA
authors: Juma
authors: Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. omandi.cole@gmail.com.
authors: Andrew O
authors: AO
authors: Cole
authors: The Jenner Institute, University of Oxford, Oxford, UK. thomas.rampling@ndm.ox.ac.uk.
authors: Thomas W
authors: TW
authors: Rampling
authors: The Jenner Institute, University of Oxford, Oxford, UK. Nickanag@gmail.com.
authors: Nicholas A
authors: NA
authors: Anagnostou
authors: The Jenner Institute, University of Oxford, Oxford, UK. sarah.gilbert@ndm.ox.ac.uk.
authors: Sarah C
authors: SC
authors: Gilbert
authors: Sanaria, Inc, Rockville, MD, USA. slhoffman@sanaria.com.
authors: Stephen L
authors: SL
authors: Hoffman
authors: The Jenner Institute, University of Oxford, Oxford, UK. simon.draper@merton.ox.ac.uk.
authors: Simon J
authors: SJ
authors: Draper
authors: Kenya Medical Research Institute - Wellcome Trust, Centre for Geographical Medical Research (Coast), Kilifi, Kenya. pbejon@well.ox.ac.uk.
authors: Philip
authors: P
authors: Bejon
authors: Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. bernhards.ogutu@indepth-network.org.|Centre for Research in Therapeutic Sciences, Strathmore University, Nairobi, Kenya. bernhards.ogutu@indepth-network.org.
authors: Bernhards
authors: B
authors: Ogutu
authors: Kenya Medical Research Institute - Wellcome Trust, Centre for Geographical Medical Research (Coast), Kilifi, Kenya. kevin.marsh@ndm.ox.ac.uk.
authors: Kevin
authors: K
authors: Marsh
authors: The Jenner Institute, University of Oxford, Oxford, UK. adrian.hill@ndm.ox.ac.uk.
authors: Adrian V S
authors: AV
authors: Hill
authors: Department of Laboratory Medicine and Center for Emerging and Re-Emerging Infectious Diseases, University of Washington (UW), 750 Republican St., E633, Seattle, WA, 98109, USA. murphysc@uw.edu.
authors: Sean C
authors: SC
authors: Murphy
mesh_terms: D000328:Adult; D016054:DNA, Protozoan / Q000097:blood; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008288:Malaria / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D018512:Parasitemia / Q000175:diagnosis / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D055815:Young Adult
chemical_list: D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S
references: Vaccine. 2005 Jan 4;23(7):857-64
references: 15603885
references: Am J Trop Med Hyg. 2012 Mar;86(3):383-94
references: 22403305
references: PLoS One. 2014;9(5):e97398
references: 24838112
references: Am J Trop Med Hyg. 2013 Jan;88(1):3-4
references: 23303797
references: PLoS One. 2013 Jun 18;8(6):e65960
references: 23823332
references: PLoS One. 2012;7(6):e38434
references: 22701640
references: Am J Trop Med Hyg. 2014 Sep;91(3):471-80
references: 25070995
references: Nat Med. 2003 Jun;9(6):729-35
references: 12766765
references: J Infect Dis. 2013 Jul 15;208(2):340-5
references: 23570846
references: Nat Rev Immunol. 2011 Jan;11(1):57-64
references: 21179119
references: Am J Trop Med Hyg. 2005 Jul;73(1):191-8
references: 16014857
references: Malar J. 2014;13:288
references: 25066459
references: J Infect Dis. 2012 Aug 1;206(3):319-23
references: 22615322
references: Front Microbiol. 2014 Dec 12;5:686
references: 25566206
references: Trans R Soc Trop Med Hyg. 1947 May;40(5):621-76
references: 20243883
references: J Infect Dis. 2005 Feb 15;191(4):619-26
references: 15655787
references: J Clin Microbiol. 2012 Dec;50(12):4128-30
references: 23052309
references: Hum Vaccin Immunother. 2013 Sep;9(9):1831-40
references: 23778312
references: Curr HIV/AIDS Rep. 2012 Mar;9(1):91-100
references: 22215419
references: Am J Trop Med Hyg. 2013 Jan;88(1):5-13
references: 23149582
references: J Clin Microbiol. 1989 Jul;27(7):1434-7
references: 2671011
references: J Clin Microbiol. 2003 Oct;41(10):4531-6
references: 14532178