title: Still Searching for a Suitable Molecular Test to Detect Hidden Plasmodium Infection: A Proposal for Blood Donor Screening in Brazil.
abstract: BACKGROUND
  Efforts have been made to establish sensitive diagnostic tools for malaria screening in blood banks in order to detect malaria asymptomatic carriers. Microscopy, the malaria reference test in Brazil, is time consuming and its sensitivity depends on microscopist experience. Although molecular tools are available, some aspects need to be considered for large-scale screening: accuracy and robustness for detecting low parasitemia, affordability for application to large number of samples and flexibility to perform on individual or pooled samples.
  
  
  METHODS
  In this retrospective study, we evaluated four molecular assays for detection of malaria parasites in a set of 56 samples previously evaluated by expert microscopy. In addition, we evaluated the effect of pooling samples on the sensitivity and specificity of the molecular assays. A well-characterized cultured sample with 1 parasite/μL was included in all the tests evaluated. DNA was extracted with QIAamp DNA Blood Mini Kit and eluted in 50 μL to concentrate the DNA. Pools were assembled with 10 samples each. Molecular protocols targeting 18S rRNA, included one qPCR genus specific (Lima-genus), one duplex qPCR genus/Pf (PET-genus, PET-Pf) and one duplex qPCR specie-specific (Rougemont: Roug-Pf/Pv and Roug-Pm/Po). Additionally a nested PCR protocol specie-specific was used (Snou-Pf, Snou-Pv, Snou-Pm and Snou-Po).
  
  
  RESULTS
  The limit of detection was 3.5 p/μL and 0.35p/μl for the PET-genus and Lima-genus assays, respectively. Considering the positive (n = 13) and negative (n = 39) unpooled individual samples according to microscopy, the sensitivity of the two genus qPCR assays was 76.9% (Lima-genus) and 72.7% (PET-genus). The Lima-genus and PET-genus showed both sensitivity of 86.7% in the pooled samples. The genus protocols yielded similar results (Kappa value of 1.000) in both individual and pooled samples.
  
  
  CONCLUSIONS
  Efforts should be made to improve performance of molecular tests to enable the detection of low-density parasitemia if these tests are to be utilized for blood transfusion screening.
authors.affiliation: Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Giselle Fernandes Maciel de Castro
authors.initials: GF
authors.lastname: Lima
authors.affiliation: Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Naomi W
authors.initials: NW
authors.lastname: Lucchi
authors.affiliation: Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.|Atlanta Research and Education Foundation, Decatur-GA, Atlanta, Georgia, United States of America.
authors.forename: Luciana
authors.initials: L
authors.lastname: Silva-Flannery
authors.affiliation: Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Macedo-de-Oliveira
authors.affiliation: Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Angelica D
authors.initials: AD
authors.lastname: Hristov
authors.affiliation: Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Juliana
authors.initials: J
authors.lastname: Inoue
authors.affiliation: Núcleo de Estudos em Malária, Superintendência de Controle de Endemias, Secretaria de Estado da Saúde de São Paulo /Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Maria de Jesus
authors.initials: Mde J
authors.lastname: Costa-Nascimento
authors.affiliation: Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Venkatachalam
authors.initials: V
authors.lastname: Udhayakumar
authors.affiliation: Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.|Núcleo de Estudos em Malária, Superintendência de Controle de Endemias, Secretaria de Estado da Saúde de São Paulo /Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Di Santi
mesh_terms: D001938:Brazil; D016054:DNA, Protozoan / Q000235:genetics; D003955:Diagnostic Tests, Routine / Q000639:trends*; D046148:Donor Selection; D006801:Humans; D008288:Malaria / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan