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Reference Manager
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Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials
Sean C. Murphy
Sean C. Murphy
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Jennifer L. Prentice
Jennifer L. Prentice
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Kathryn Williamson
Kathryn Williamson
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Carolyn K. Wallis
Carolyn K. Wallis
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Ferric C. Fang
Ferric C. Fang
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Michal Fried
Michal Fried
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Cris Pinzon
Cris Pinzon
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Ruobing Wang
Ruobing Wang
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Angela K. Talley
Angela K. Talley
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Stefan H. I. Kappe
Stefan H. I. Kappe
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Patrick E. Duffy
Patrick E. Duffy
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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Brad T. Cookson
Brad T. Cookson
Departments of Laboratory Medicine, Microbiology and Medicine (Division of Allergy and Infectious Diseases), University of Washington Medical Center, Seattle, Washington; Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, Washington; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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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 log10 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 1×104 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.
Author Notes
Financial support: Funding in support of the human challenge trial was provided by the PATH Malaria Vaccine Initiative and the U.S. Department of Defense (award no. W81XWH-09-1-0531; the U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering acquisition office.)
Authors' addresses: Sean C. Murphy and Jennifer L. Prentice, Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA, E-mails: murphysc@u.washington.edu and jprentic@u.washington.edu. Kathryn Williamson and Michal Fried, Seattle Biomedical Research Institute, Seattle, WA, E-mails: kathryn.williamson@seattlebiomed.org and michal.fried@seattlebiomed.org. Carolyn K. Wallis, Department of Laboratory Medicine, Harborview Medical Center, Seattle, WA, E-mail: ckw@u.washington.edu. Ferric C. Fang and Brad T. Cookson, Departments of Laboratory Medicine and Microbiology, University of Washington Medical Center, Seattle, WA, E-mails: fcfang@u.washington.edu and cookson@u.washington.edu. Cris Pinzon and Ruobing Wang, Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, WA, E-mails: cris.pinzon@seattlebiomed.org and ruobing.wang@seattlebiomed.org. Angela K. Talley, Malaria Clinical Trials Center, Seattle Biomedical Research Institute, Seattle, WA, E-mail: angela.talley@seattlebiomed.org (also affiliated with the Department of Medicine [Allergy and Infectious Diseases], University of Washington Medical Center]. Stefan H. I. Kappe, Seattle Biomedical Research Institute, Seattle, WA, E-mail: stefan.kappe@seattlebiomed.org (also affiliated with the Department of Global Health, University of Washington, Seattle, WA). Patrick E. Duffy, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, E-mail: duffype@niaid.nih.gov (also affiliated with Seattle Biomedical Research Institute, Seattle, WA).
Reprints requests: Sean C. Murphy, Department of Laboratory Medicine, University of Washington, 1959 NE Pacific St., Box 357110, Seattle, WA 98195-7110; Tel: 206-598-6131; Fax: 206-598-6189; E-mail: murphysc@u.washington.edu.
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
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-
2.
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-
3.
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-
4.
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-
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-
6.
Swan H, Sloan L, Muyombwe A, Chavalitshewinkoon-Petmitr P, Krudsood S, Leowattana W, Wilairatana P, Looareesuwan S, Rosenblatt J, 2005. Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand. Am J Trop Med Hyg 73: 850–854.
-
7.
Mens PF, Schoone GJ, Kager PA, Schallig HD, 2006. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. Malar J 5: 80.
-
8.
Safeukui I, Millet P, Boucher S, Melinard L, Fregeville F, Receveur MC, Pistone T, Fialon P, Vincendeau P, Fleury H, Malvy D, 2008. Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travelers and migrants. Malar J 7: 70.
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9.
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-
10.
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-
13.
Vo TK, Bigot P, Gazin P, Sinou V, De Pina JJ, Huynh DC, Fumoux F, Parzy D, 2007. Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travelers. Trans R Soc Trop Med Hyg 101: 422–428.
-
14.
Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA, 1999. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687–692.
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15.
Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363: 1017–1024.
-
16.
Snounou G, Singh B, 2002. Nested PCR analysis of Plasmodium parasites. Methods Mol Med 72: 189–203.
-
17.
Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici MC, Arcangeletti MC, Snounou G, Dettori G, Chezzi C, 2004. Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42: 1214–1219.
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-
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-
21.
Schneider P, Wolters L, Schoone G, Schallig H, Sillekens P, Hermsen R, Sauerwein R, 2005. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol 43: 402–405.
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