title: Molecular characterization of misidentified Plasmodium ovale imported cases in Singapore.
abstract: BACKGROUND
  Plasmodium ovale, considered the rarest of the malaria parasites of humans, consists of two morphologically identical but genetically distinct sympatric species, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. These parasites resemble morphologically to Plasmodium vivax with which they also share a tertian periodicity and the ability to cause relapses, making them easily misidentified as P. vivax. Plasmodium ovale infections are rarely reported, but given the likelihood of misidentification, their prevalence might be underestimated.
  
  
  METHODS
  Morphological and molecular analysis of confirmed malaria cases admitted in Singapore in 2012-2014 detected nine imported P. ovale cases that had been misidentified as P. vivax. Since P. ovale had not been previously officially reported in Singapore, a retrospective analysis of available, frozen, archival blood samples was performed and returned two additional misidentified P. ovale cases in 2003 and 2006. These eleven P. ovale samples were characterized with respect to seven molecular markers (ssrRNA, Potra, Porbp2, Pog3p, dhfr-ts, cytb, cox1) used in recent studies to distinguish between the two sympatric species, and to a further three genes (tufa, clpC and asl).
  
  
  RESULTS
  The morphological features of P. ovale and the differential diagnosis with P. vivax were reviewed and illustrated by microphotographs. The genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was assessed by ten molecular markers distributed across the three genomes of the parasite (Genbank KP050361-KP050470). The data obtained for seven of these markers were compared with those published and confirmed that both P. ovale species were present. This dimorphism was also confirmed for the first time on: (1) two genes from the apicoplast genome (tufA and clpC genes); and, (2) the asl gene that was used for phylogenetic analyses of other Plasmodium species, and that was found to harbour the highest number of dimorphic loci between the two P. ovale species.
  
  
  CONCLUSIONS
  Misidentified P. ovale infections are reported for the first time among imported malaria cases in Singapore. Genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was confirmed using markers from the parasites' three genomes. The apparent increase of imported P. ovale since 2012 (with yearly detection of cases) is puzzling. Given decrease in the overall number of malaria cases recorded in Singapore since 2010 the 'resurgence' of this neglected species raises public health concerns.
authors.affiliation: Malaria Reference Centre - National Public Health Laboratory, Ministry of Health, Singapore, 3 Biopolis Drive, Synapse #05-14/16, 138623, Singapore, Singapore. jean-marc_chavatte@moh.gov.sg.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chavatte
authors.affiliation: Malaria Reference Centre - National Public Health Laboratory, Ministry of Health, Singapore, 3 Biopolis Drive, Synapse #05-14/16, 138623, Singapore, Singapore. sarah_tan@moh.gov.sg.
authors.forename: Sarah Bee Hui
authors.initials: SB
authors.lastname: Tan
authors.affiliation: Sorbonne Universités, UPMC Université Paris 06, UPMC UMRS CR7, 75005, Paris, France. georges.snounou@upmc.fr.|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, 75013, Paris, France. georges.snounou@upmc.fr.
authors.forename: Georges
authors.initials: G
authors.lastname: Snounou
authors.affiliation: Malaria Reference Centre - National Public Health Laboratory, Ministry of Health, Singapore, 3 Biopolis Drive, Synapse #05-14/16, 138623, Singapore, Singapore. raymond_lin@moh.gov.sg.|Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore. raymond_lin@moh.gov.sg.|Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, 117545, Singapore, Singapore. raymond_lin@moh.gov.sg.
authors.forename: Raymond Tzer Pin Valentine
authors.initials: RT
authors.lastname: Lin
mesh_terms: D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D017125:Genes, Protozoan; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology*; D008969:Molecular Sequence Data; D041122:Plasmodium ovale / Q000166:cytology / Q000235:genetics* / Q000302:isolation & purification*; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D012846:Singapore
chemical_list: D016054:DNA, Protozoan