title: Diagnosis of bacteremia in pediatric oncologic patients by in-house real-time PCR.
abstract: BACKGROUND
  Infections are the major cause of morbidity and mortality in children with cancer. Gaining a favorable prognosis for these patients depends on selecting the appropriate therapy, which in turn depends on rapid and accurate microbiological diagnosis. This study employed real-time PCR (qPCR) to identify the main pathogens causing bloodstream infection (BSI) in patients treated at the Pediatric Oncology Institute IOP-GRAACC-UNIFESP-Brazil. Antimicrobial resistance genes were also investigated using this methodology.
  
  
  METHODS
  A total of 248 samples from BACTEC® blood culture bottles and 99 whole-blood samples collected in tubes containing EDTA K2 Gel were isolated from 137 patients. All samples were screened by specific Gram probes for multiplex qPCR. Seventeen sequences were evaluated using gender-specific TaqMan probes and the resistance genes bla SHV, bla TEM, bla CTX, bla KPC, bla IMP, bla SPM, bla VIM, vanA, vanB and mecA were detected using the SYBR Green method.
  
  
  RESULTS
  Positive qPCR results were obtained in 112 of the blood culture bottles (112/124), and 90 % agreement was observed between phenotypic and molecular microbial detection methods. For bacterial and fungal identification, the performance test showed: sensitivity 87 %; specificity 91 %; NPV 90 %; PPV 89 % and accuracy of 89 % when compared with the phenotypic method. The mecA gene was detected in 37 samples, extended-spectrum β-lactamases were detected in six samples and metallo-β-lactamase coding genes in four samples, with 60 % concordance between the two methods. The qPCR on whole blood detected eight samples possessing the mecA gene and one sample harboring the vanB gene. The bla KPC, bla VIM, bla IMP and bla SHV genes were not detected in this study.
  
  
  CONCLUSIONS
  Real-time PCR is a useful tool in the early identification of pathogens and antimicrobial resistance genes from bloodstream infections of pediatric oncologic patients.
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. milenequiles@yahoo.com.br.
authors.forename: Milene Gonçalves
authors.initials: MG
authors.lastname: Quiles
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. licarballo@ig.com.br.
authors.forename: Liana Carballo
authors.initials: LC
authors.lastname: Menezes
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. karenbauab@yahoo.com.br.
authors.forename: Karen de Castro
authors.initials: Kde C
authors.lastname: Bauab
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. elkekgumpl@hotmail.com.
authors.forename: Elke Kreuscher
authors.initials: EK
authors.lastname: Gumpl
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. talitaunesp@gmail.com.
authors.forename: Talita Trevizani
authors.initials: TT
authors.lastname: Rocchetti
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. fla_bio@hotmail.com.
authors.forename: Flavia Silva
authors.initials: FS
authors.lastname: Palomo
authors.affiliation: Institute of Pediatric Oncology IOP-GRAACC, Federal University of São Paulo, São Paulo, Brazil. fabiannecarlesse@graacc.org.br.
authors.forename: Fabianne
authors.initials: F
authors.lastname: Carlesse
authors.affiliation: Special Laboratory of Clinical Microbiology (LEMC), Federal University of São Paulo/UNIFESP, São Paulo, Brazil. pignatari@terra.com.br.
authors.forename: Antonio Carlos Campos
authors.initials: AC
authors.lastname: Pignatari
mesh_terms: D000293:Adolescent; D000328:Adult; D016470:Bacteremia / Q000175:diagnosis / Q000453:epidemiology* / Q000382:microbiology; D001938:Brazil; D002648:Child; D002675:Child, Preschool; D005260:Female; D006090:Gram-Negative Bacteria / Q000145:classification / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D016867:Immunocompromised Host; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D019095:Point-of-Care Systems; D011237:Predictive Value of Tests; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D055815:Young Adult; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D001618:beta-Lactamases