title: A rapid, real-time quantitative polymerase chain reaction test for the identification of pathogens in bronchoalveolar lavage samples.
abstract: BACKGROUND
  Standard bacteriologic culture techniques offer results within 2 days to 3 days, precluding a focused and timely antibiotic therapy in ventilated trauma patients. Our laboratory developed a real-time quantitative polymerase chain reaction (qPCR) test that can detect 25 different bacteria and fungi and methicillin resistance and offers results within 3 hours. The objective of this study was to compare the qPCR method to standard culture techniques.
  
  
  METHODS
  This was a prospective observational cohort study at a Level I trauma center from 2009 to 2012. Adult trauma patients on ventilation, receiving at least one bronchoalveolar lavage (BAL) with culture results were eligible for inclusion. DNA was isolated from the BAL samples and analyzed in 96-well plates using qPCR. Student's t tests were used to examine differences in mean qPCR cycle counts. Sensitivities, specificities, negative predictive values, and positive predictive values were calculated for the qPCR primer sets.
  
  
  RESULTS
  There were 28 BALs in the study. The qPCR method detected a total of 165 organisms, and culture methods found 54. The qPCR test had an overall sensitivity of 85%, specificity of 74%, negative predictive value of 98%, and positive predictive value of 27%. Those organisms that were only identified through qPCR had significantly less DNA than those identified through both qPCR and quantitative culture (28.8 vs. 23.3, p < 0.001). Concurrent antibiotic therapy was found to decrease the qPCR specificity in some primer sets, and methicillin resistance was only found in BAL samples that were concurrent with antibiotics.
  
  
  CONCLUSIONS
  The qPCR method shows promising initial diagnostic value. Many of the organisms not identified by quantitative culture had late cycle calls, suggesting that they might have been in quantities too low to result in culture identification. Once refined, our qPCR method has the potential to identify pathogens faster and earlier than standard quantitative culture methods, allowing for targeted antibiotic therapy within 3 hours.
  
  
  METHODS
  Diagnostic test, level II.
authors.affiliation: From the Trauma Research Department (A.O., G.T., C.W.M., D.B.-O.), and Trauma Services Department (C.W.M.), St. Anthony Hospital, Lakewood; Trauma Research Department (A.O., G.T., D.B.-O.), and Trauma Services Department (D.S.S.), Swedish Medical Center, Englewood; and College of Osteopathic Medicine (C.W.M., D.B.-O.), Rocky Vista University, Parker, Colorado.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Orlando
authors.forename: Gregory
authors.initials: G
authors.lastname: Thoma
authors.forename: Denetta S
authors.initials: DS
authors.lastname: Slone
authors.forename: Charles W
authors.initials: CW
authors.lastname: Mains
authors.forename: David
authors.initials: D
authors.lastname: Bar-Or
mesh_terms: D000328:Adult; D000368:Aged; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D016106:Methicillin Resistance / Q000235:genetics; D008875:Middle Aged; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004271:DNA, Fungal