title: Verification of the ProPneumo-1 assay for the simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in clinical respiratory specimens.
abstract: BACKGROUND
  Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples.
  
  
  OBJECTIVE
  The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay.
  
  
  METHODS
  A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea).
  
  
  RESULTS
  Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool.
  
  
  CONCLUSIONS
  The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.
authors.affiliation: Ontario Public Health Laboratories, Ministry of Health and Long-Term Care, Toronto, Ontario, Canada. rachel.higgins@ontario.ca
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Higgins
authors.forename: Ernesto
authors.initials: E
authors.lastname: Lombos
authors.forename: Patrick
authors.initials: P
authors.lastname: Tang
authors.forename: Karl
authors.initials: K
authors.lastname: Rohoman
authors.forename: Anne
authors.initials: A
authors.lastname: Maki
authors.forename: Shirley
authors.initials: S
authors.lastname: Brown
authors.forename: Frances
authors.initials: F
authors.lastname: Jamieson
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Drews
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D023521:Chlamydophila Infections / Q000175:diagnosis*; D016993:Chlamydophila pneumoniae / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D009177:Mycoplasma pneumoniae / Q000302:isolation & purification*; D011019:Pneumonia, Mycoplasma / Q000175:diagnosis*; D012141:Respiratory Tract Infections / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D055815:Young Adult