title: High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias.
abstract: The epidemiology of malaria in "low-transmission" areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (∼5 μl) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive "high-volume" quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ≥250 μl and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand mallika.imw@mahidol.ac.th.
authors.forename: Mallika
authors.initials: M
authors.lastname: Imwong
authors.affiliation: Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Sarun
authors.initials: S
authors.lastname: Hanchana
authors.affiliation: Laboratory of Malaria Immunobiology, Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR), Biopolis, Singapore Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore.
authors.forename: Benoit
authors.initials: B
authors.lastname: Malleret
authors.affiliation: Laboratory of Malaria Immunobiology, Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR), Biopolis, Singapore.
authors.forename: Laurent
authors.initials: L
authors.lastname: Rénia
authors.affiliation: Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Nicholas P J
authors.initials: NP
authors.lastname: Day
authors.affiliation: Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Arjen
authors.initials: A
authors.lastname: Dondorp
authors.affiliation: Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, Oxford, United Kingdom Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.
authors.forename: Francois
authors.initials: F
authors.lastname: Nosten
authors.affiliation: Sorbonne Universités, UPMC University Paris 06, UPMC UMRS CR7, Paris, France Centre d'Immunologie et de Maladies Infectieuses (CIMI)-Paris, Institut National de la Santé et de la Recherche Médicale (INSERM) U1135-Centre National de la Recherche Scientifique (CNRS) ERL 8255, Paris, France.
authors.forename: Georges
authors.initials: G
authors.lastname: Snounou
authors.affiliation: Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, Oxford, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: White
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D002353:Carrier State / Q000175:diagnosis / Q000469:parasitology; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology*; D059208:Parasite Load / Q000379:methods*; D018512:Parasitemia / Q000175:diagnosis* / Q000469:parasitology*; D010961:Plasmodium; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S