1017. Real-Time PCR Detection of áMycoplasma pneumoniae, Legionella spp. and Chlamydia pneumoniae in Oropharyngeal Swabs and Broncho-aveolar Lavage Specimens Using the BD MAX« Instrument
Session: Poster Abstract Session: Diagnosing Pneumonia and Meningitis
Saturday, October 22, 2011
Room: Poster Hall B1
  • Summersgill.pdf (255.8 kB)
  • Background: We developed and evaluated an RT-PCR assay for the detection of Mycoplasma pneumoniae, Legionella species and Chlamydia pneumoniae in oropharyngeal swabs (OS) and brocho-alveolar lavage (BAL) specimens on the BD Max« instrument (BD-Ann Arbor, Ann Arbor, MI). This is a fully integrated, walk-away instrument for RT-PCR. Methods: OS and BAL specimens were spiked with 10-fold dilutions of M. pneumoniae, L. pneumophila and C. pneumoniae. BAL specimens were diluted 1:4 in M4 prior to processing. 750 ul of each specimen was then added to an SP-3 specimen collection tube. Primers and probes for M. pneumoniae were those targeting the P1 attachment gene; for Legionella spp. those targeting the mip gene; and for C. pneumoniae those targeting the 16S rRNA gene. Results: Level of detection (LOD) was determined for both sample types and was defined as the lowest concentration of organism in the sample which produces a Ct value of <40. Thus, the LODs for OS was 101 cfu/ml (avg. CT = 33.50 ▒ 1.04) for each agent and the LOD for BAL was 102 cfu/ml (avg. CT = 30.98 ▒ 0.94) for each agent. A total of 2,336 specimens have been processed in our reference laboratory, with 6 positive M. pneumoniae, 5 positive Legionella spp. and 1 positive C. pneumoniae specimen. The average Ct value for these positive specimens was 29.09 ▒ 3.11. The lower LOD seen for BAL specimens was presumed to be due to specimen quality and viscosity. Conclusion: The availability of a RT-PCR assay greatly enhances the ability to rapidly diagnose infection caused by these agents. The BD Max« system involves minimal technologist time in sample preparation. The DNA Unitized Strips« contain all necessary reagents for DNA extraction and purification, and the microfluidic cartridge facilitates two-channel RT-PCR amplification. This instrument is also easily adapted to additional target sequences.

    Subject Category: D. Diagnostic microbiology

    James Summersgill, PhD1, Laura Schindler, MT(SACP)2, Karen Campbell, MT(SACP)2, Sabrena Garr, MT(SACP)2 and Julio Ramirez, MD1, (1)Division of Infectious Diseases, University of Louisville, Louisville, KY, (2)Medicine, University of Louisville, Louisville, KY


    J. Summersgill, None

    L. Schindler, None

    K. Campbell, None

    S. Garr, None

    J. Ramirez, None

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