title: Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene.
abstract: BACKGROUND
  Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis. However, they are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. Here the use of "direct PCR" was investigated with the aim of improving diagnosis in the malaria elimination era.
  
  
  METHODS
  The performance of the Plasmodium cytochrome oxidase III gene (COX-III) based novel malaria detection strategies (direct nested PCR and direct single PCR) were compared using a 18s-rRNA direct nested PCR as a reference tool. Evaluations were based on sensitivity, specificity and the ability to detect mixed infections using control blood spot samples and field collected blood samples with final species diagnosis confirmation by sequencing.
  
  
  RESULTS
  The COX-III direct PCR (limit of detection: 0.6-2 parasites/μL) was more sensitive than the 18s-rRNA direct nested PCR (limit of detection: 2-10 parasites/μL). The COX-III direct PCR identified all 21 positive controls (no mixed infections detected) while the 18s-rRNA direct nested PCR identified 18/21 (including four mixed infections). Different concentrations of simulated mixed infections (Plasmodium vivax and Plasmodium falciparum) suggest that the COX-III direct PCR detects only the predominant species. When the 18s-rRNA direct nested PCR was used to detect Plasmodium in field collected bloods spots (n = 3833), there was discrepancy in the results from the genus PCR (16 % positive) and the species-specific PCR (5 % positive). Further, a large portion of a subset of these positive samples (93 % for genus and 60 % for P. vivax), did not align with Plasmodium sequences. In contrast, the COX-III direct PCR clearly identified (single bands confirmed with sequencing) 2 % positive Plasmodium samples including P. vivax, P. falciparum, Plasmodium malariae and Plasmodium ovale wallikeri.
  
  
  CONCLUSIONS
  The COX-III single direct PCR is an alternative method for accurate detection of Plasmodium microscopic and submicroscopic infections in humans, especially when a large number of samples require screening. This PCR does not require DNA isolation, is sensitive, quick, produces confident/clear results, identifies all the Plasmodium species infecting humans, and is cost-effective.
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. decheve1@nd.edu.
authors.forename: Diego F
authors.initials: DF
authors.lastname: Echeverry
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. nicholas.deason@nih.gov.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Deason
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. jenna.r.davidson.60@nd.edu.
authors.forename: Jenna
authors.initials: J
authors.lastname: Davidson
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. victoria.makuru.1@nd.edu.
authors.forename: Victoria
authors.initials: V
authors.lastname: Makuru
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. hxiao@nd.edu.
authors.forename: Honglin
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. niedbalski.14@nd.edu.
authors.forename: Julie
authors.initials: J
authors.lastname: Niedbalski
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. marcia.k.kern.11@nd.edu.
authors.forename: Marcia
authors.initials: M
authors.lastname: Kern
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, 4870, Australia. tanya.russell@jcu.edu.au.
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Russell
authors.affiliation: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, 4870, Australia. tom.burkot@jcu.edu.au.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Burkot
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. frank@nd.edu.
authors.forename: Frank H
authors.initials: FH
authors.lastname: Collins
authors.affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA. neil.f.lobo.2@nd.edu.
authors.forename: Neil F
authors.initials: NF
authors.lastname: Lobo
mesh_terms: D001483:Base Sequence; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics; D059788:Dried Blood Spot Testing; D003576:Electron Transport Complex IV / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000175:diagnosis; D010961:Plasmodium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D003576:Electron Transport Complex IV