title: Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.
abstract: To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified > 98.7% of parasite-containing samples to ±0.5 log(10) parasites/mL of the nominal value without false positives. The analytical sensitivity was ≥ 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1 × 10(4) copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 μL) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.
authors.affiliation: Department of Laboratory Medicine, Division of Allergy and Infectious Diseases, University of Washington Medical Center, Seattle, 98195-7110, USA. murphysc@u.washington.edu
authors.forename: Sean C
authors.initials: SC
authors.lastname: Murphy
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Prentice
authors.forename: Kathryn
authors.initials: K
authors.lastname: Williamson
authors.forename: Carolyn K
authors.initials: CK
authors.lastname: Wallis
authors.forename: Ferric C
authors.initials: FC
authors.lastname: Fang
authors.forename: Michal
authors.initials: M
authors.lastname: Fried
authors.forename: Cris
authors.initials: C
authors.lastname: Pinzon
authors.forename: Ruobing
authors.initials: R
authors.lastname: Wang
authors.forename: Angela K
authors.initials: AK
authors.lastname: Talley
authors.forename: Stefan H I
authors.initials: SH
authors.lastname: Kappe
authors.forename: Patrick E
authors.initials: PE
authors.lastname: Duffy
authors.forename: Brad T
authors.initials: BT
authors.lastname: Cookson
mesh_terms: D000328:Adult; D001483:Base Sequence; D002986:Clinical Trials as Topic; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000188:drug therapy / Q000469:parasitology; D025202:Molecular Diagnostic Techniques / Q000379:methods; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics*; D016053:RNA, Protozoan / Q000032:analysis* / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA; D013045:Species Specificity
chemical_list: D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S