Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

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

Search for other papers by Sean C. Murphy in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Jennifer L. Prentice in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Kathryn Williamson in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Carolyn K. Wallis in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Ferric C. Fang in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Michal Fried in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Cris Pinzon in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Ruobing Wang in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Angela K. Talley in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Stefan H. I. Kappe in
Current site
Google Scholar
PubMed
Close
,
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

Search for other papers by Patrick E. Duffy in
Current site
Google Scholar
PubMed
Close
, and
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

Search for other papers by Brad T. Cookson in
Current site
Google Scholar
PubMed
Close
Restricted access

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

*Address correspondence to Sean C. Murphy, Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific St., Box 357110, Seattle, WA 98195-7110. E-mail: murphysc@u.washington.edu

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.

  • 1.

    Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92: 419433.

  • 2.

    Bejon P, Andrews L, Hunt-Cooke A, Sanderson F, Gilbert SC, Hill AV, 2006. Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density. Malar J 5: 104.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3.

    Stauffer WM, Cartwright CP, Olson DA, Juni BA, Taylor CM, Bowers SH, Hanson KL, Rosenblatt JE, Boulware DR, 2009. Diagnostic performance of rapid diagnostic tests versus blood smears for malaria in US clinical practice. Clin Infect Dis 49: 908913.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Chinkhumba J, Skarbinski J, Chilima B, Campbell C, Ewing V, San Joaquin M, Sande J, Ali D, Mathanga D, 2010. Comparative field performance and adherence to test results of four malaria rapid diagnostic tests among febrile patients more than five years of age in Blantyre, Malawi. Malar J 9: 209.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K, 2004. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 42: 56365643.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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: 850854.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Tangin A, Komichi Y, Wagatsuma Y, Rashidul H, Wataya Y, Kim HS, 2008. Detection of malaria parasites in mosquitoes from the malaria-endemic area of Chakaria, Bangladesh. Biol Pharm Bull 31: 703708.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Das A, Holloway B, Collins WE, Shama VP, Ghosh SK, Sinha S, Hasnain SE, Talwar GP, Lal AA, 1995. Species-specific 18S rRNA gene amplification for the detection of P. falciparum and P. vivax malaria parasites. Mol Cell Probes 9: 161165.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Ciceron L, Jaureguiberry G, Gay F, Danis M, 1999. Development of a Plasmodium PCR for monitoring efficacy of antimalarial treatment. J Clin Microbiol 37: 3538.

  • 12.

    Kho WG, Chung JY, Sim EJ, Kim MY, Kim DW, Jongwutiwes S, Tanabe K, 2003. A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax. Parasitol Int 52: 229236.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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: 422428.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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: 687692.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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: 10171024.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Snounou G, Singh B, 2002. Nested PCR analysis of Plasmodium parasites. Methods Mol Med 72: 189203.

  • 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: 12141219.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18.

    Coleman RE, Sattabongkot J, Promstaporm S, Maneechai N, Tippayachai B, Kengluecha A, Rachapaew N, Zollner G, Miller RS, Vaughan JA, Thimasarn K, Khuntirat B, 2006. Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand. Malar J 5: 121.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19.

    Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T, 2007. Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol 45: 25212528.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20.

    Mangold KA, Manson RU, Koay ES, Stephens L, Regner M, Thomson RB Jr, Peterson LR, Kaul KL, 2005. Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43: 24352440.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 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: 402405.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    Schoone GJ, Oskam L, Kroon NC, Schallig HD, Omar SA, 2000. Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification. J Clin Microbiol 38: 40724075.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Kamau E, Tolbert LS, Kortepeter L, Pratt M, Nyakoe N, Muringo L, Ogutu B, Waitumbi JN, Ockenhouse CF, 2011. Development of a highly sensitive genus-specific qRT-PCR assay for detection and quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J Clin Microbiol 49: 29462953.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24.

    Mixson-Hayden T, Lucchi NW, Udhayakumar V, 2010. Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection. BMC Res Notes 3: 88.

  • 25.

    Snounou G, 2002. Genotyping of Plasmodium spp. Nested PCR. Methods Mol Med 72: 103116.

  • 26.

    Epstein JE, Rao S, Williams F, Freilich D, Luke T, Sedegah M, de la Vega P, Sacci J, Richie TL, Hoffman SL, 2007. Safety and clinical outcome of experimental challenge of human volunteers with Plasmodium falciparum-infected mosquitoes: an update. J Infect Dis 196: 145154.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27.

    Webster DP, Dunachie S, Vuola JM, Berthoud T, Keating S, Laidlaw SM, McConkey SJ, Poulton I, Andrews L, Andersen RF, Bejon P, Butcher G, Sinden R, Skinner MA, Gilbert SC, Hill AV, 2005. Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara. Proc Natl Acad Sci USA 102: 48364841.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28.

    Walther M, Thompson FM, Dunachie S, Keating S, Todryk S, Berthoud T, Andrews L, Andersen RF, Moore A, Gilbert SC, Poulton I, Dubovsky F, Tierney E, Correa S, Huntcooke A, Butcher G, Williams J, Sinden RE, Hill AV, 2006. Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect Immun 74: 27062716.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29.

    Walther M, Dunachie S, Keating S, Vuola JM, Berthoud T, Schmidt A, Maier C, Andrews L, Andersen RF, Gilbert S, Poulton I, Webster D, Dubovsky F, Tierney E, Sarpotdar P, Correa S, Huntcooke A, Butcher G, Williams J, Sinden RE, Thornton GB, Hill AV, 2005. Safety, immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, ICC-1132 formulated in Seppic ISA 720. Vaccine 23: 857864.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30.

    Hermsen CC, Telgt DS, Linders EH, van de Locht LA, Eling WM, Mensink EJ, Sauerwein RW, 2001. Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCR. Mol Biochem Parasitol 118: 247251.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31.

    Schneider P, Schoone G, Schallig H, Verhage D, Telgt D, Eling W, Sauerwein R, 2004. Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification. Mol Biochem Parasitol 137: 3541.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32.

    Andrews L, Andersen RF, Webster D, Dunachie S, Walther RM, Bejon P, Hunt-Cooke A, Bergson G, Sanderson F, Hill AV, Gilbert SC, 2005. Quantitative real-time polymerase chain reaction for malaria diagnosis and its use in malaria vaccine clinical trials. Am J Trop Med Hyg 73: 191198.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33.

    McCutchan TF, de la Cruz VF, Lal AA, Gunderson JH, Elwood HJ, Sogin ML, 1988. Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum. Mol Biochem Parasitol 28: 6368.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34.

    McCutchan TF, Li J, McConkey GA, Rogers MJ, Waters AP, 1995. The cytoplasmic ribosomal RNAs of Plasmodium spp. Parasitol Today 11: 134138.

  • 35.

    Kuypers J, Wright N, Morrow R, 2004. Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children. J Clin Virol 31: 123129.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 36.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 37.

    Ribaut C, Berry A, Chevalley S, Reybier K, Morlais I, Parzy D, Nepveu F, Benoit-Vical F, Valentin A, 2008. Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J 7: 45.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38.

    Lambros C, Vanderberg JP, Ribaut C, Berry A, Chevalley S, Reybier K, Morlais I, Parzy D, Nepveu F, Benoit-Vical F, Valentin A, 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. J Parasitol 65: 418420.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39.

    Chulay JD, Schneider I, Cosgriff TM, Hoffman SL, Ballou WR, Quakyi IA, Carter R, Trosper JH, Hockmeyer WT, 1986. Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum. Am J Trop Med Hyg 35: 6668.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 40.

    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611622.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 41.

    Chesher D, 2008. Evaluating assay precision. Clin Biochem Rev 29 (Suppl 1):S23S26.

  • 42.

    Bejon P, Andrews L, Andersen RF, Dunachie S, Webster D, Walther M, Gilbert SC, Peto T, Hill AV, 2005. Calculation of liver-to-blood inocula, parasite growth rates, and preerythrocytic vaccine efficacy, from serial quantitative polymerase chain reaction studies of volunteers challenged with malaria sporozoites. J Infect Dis 191: 619626.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 43.

    Bachmann LH, Desmond RA, Stephens J, Hughes A, Hook EWI, 2002. Duration of persistence of gonococcal DNA detected by ligase chain reaction in men and women following recommended therapy for uncomplicated gonorrhea. J Clin Microbiol 40: 35963601.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 44.

    Johnson RE, Newhall WJ, Papp JR, Knapp JS, Black CM, Gift TL, Steece R, Markowitz LE, Devine OJ, Walsh CM, Wang S, Gunter DC, Irwin KL, DeLisle S, Berman SM, 2002. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections. MMWR Recomm Rep 18: 138.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 45.

    Hermsen CC, de Vlas SJ, van Gemert GJ, Telgt DS, Verhage DF, Sauerwein RW, 2004. Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reaction. Am J Trop Med Hyg 71: 196201.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 46.

    McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, Butcher G, Vuola JM, Blanchard TJ, Gothard P, Watkins K, Hannan CM, Everaere S, Brown K, Kester KE, Cummings J, Williams J, Heppner DG, Pathan A, Flanagan K, Arulanantham N, Roberts MT, Roy M, Smith GL, Schneider J, Peto T, Sinden RE, Gilbert SC, Hill AV, 2003. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med 9: 729735.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47.

    Waters AP, van Spaendonk RM, Ramesar J, Vervenne RA, Dirks RW, Thompson J, Janse CJ, 1997. Species-specific regulation and switching of transcription between stage-specific ribosomal RNA genes in Plasmodium berghei. J Biol Chem 272: 35833589.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48.

    Miller LH, Baruch DI, Marsh K, Doumbo OK, 2002. The pathogenic basis of malaria. Nature 415: 673679.

  • 49.

    Waters AP, McCutchan TF, 1989. Rapid, sensitive diagnosis of malaria based on ribosomal RNA. Lancet 333: 13431346.

  • 50.

    Gauffin F, Nordgren A, Barbany G, Gustafsson B, Karlsson H, 2009. Quantitation of RNA decay in dried blood spots during 20 years of storage. Clin Chem Lab Med 47: 14671469.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 51.

    Karlsson H, Guthenberg C, von Dobeln U, Kristenssson K, 2003. Extraction of RNA from dried blood on filter papers after long-term storage. Clin Chem 46: 979981.

Past two years Past Year Past 30 Days
Abstract Views 387 387 63
Full Text Views 755 55 4
PDF Downloads 380 54 5
 
Membership Banner
 
 
 
Affiliate Membership Banner
 
 
Research for Health Information Banner
 
 
CLOCKSS
 
 
 
Society Publishers Coalition Banner
Save