title: A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots.
abstract: BACKGROUND
  As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material.
  
  
  METHODS
  The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared.
  
  
  RESULTS
  Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P<0.001) and symptomatic (Pearson's r=0.70, P<0.0001) P. falciparum infections.
  
  
  CONCLUSIONS
  Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook 2, Room 125, 12441 Parklawn Drive, Rockville, Maryland 20852, USA. tuan.tran@nih.gov.
authors.forename: Tuan M
authors.initials: TM
authors.lastname: Tran
authors.forename: Amirali
authors.initials: A
authors.lastname: Aghili
authors.forename: Shanping
authors.initials: S
authors.lastname: Li
authors.forename: Aissata
authors.initials: A
authors.lastname: Ongoiba
authors.forename: Kassoum
authors.initials: K
authors.lastname: Kayentao
authors.forename: Safiatou
authors.initials: S
authors.lastname: Doumbo
authors.forename: Boubacar
authors.initials: B
authors.lastname: Traore
authors.forename: Peter D
authors.initials: PD
authors.lastname: Crompton
mesh_terms: D016054:DNA, Protozoan / Q000097:blood*; D059788:Dried Blood Spot Testing / Q000379:methods*; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000469:parasitology; D008853:Microscopy; D018512:Parasitemia; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D016054:DNA, Protozoan