title: A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients.
abstract: BACKGROUND
  Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients.
  
  
  METHODS
  DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures.
  
  
  RESULTS
  The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients.
  
  
  CONCLUSIONS
  Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.
authors.forename: Cleison Ledesma
authors.initials: CL
authors.lastname: Taira
authors.forename: Thelma Suely
authors.initials: TS
authors.lastname: Okay
authors.forename: Artur Figueiredo
authors.initials: AF
authors.lastname: Delgado
authors.forename: Maria Esther Jurfest Rivero
authors.initials: ME
authors.lastname: Ceccon
authors.forename: Margarete Teresa Gottardo
authors.initials: MT
authors.lastname: de Almeida
authors.affiliation: Laboratory of Medical Mycology (LIM-53), Clinical Dermartology Division, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) and Instituto de Medicina Tropical da Universidade de São Paulo (IMT-USP), Av, Dr, Enéas Carvalho de Aguiar, 500, Andar térreo, Predio 2, CEP, 05403-900 São Paulo, SP, Brazil. gildamdn@usp.br.
authors.forename: Gilda Maria Barbaro
authors.initials: GM
authors.lastname: Del Negro
mesh_terms: D000293:Adolescent; D002175:Candida / Q000235:genetics / Q000302:isolation & purification*; D058387:Candidemia / Q000097:blood / Q000175:diagnosis*; D002675:Child, Preschool; D016638:Critical Illness; D003428:Cross Infection / Q000097:blood / Q000175:diagnosis*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers