Methods: A qualitative, in vitro,PCR-based assay for the detection of PA was developed for use with the GeneXpert platform. PCR primer sets to various target genes were empirically screened across multiple PA strains. The clinical assay requires ~1 minute of hands-on time and 52 minutes to complete. The specimen source was a swab dipped into an ETA sample (fresh or previously frozen) and placed in 5mL of sample reagent. The sample reagent was vortexed for 10 seconds and 1.7mL was placed into the Xpert cartridge for testing.
Results: The target amplicon region was conserved across 295 hospital PA strains collected globally from 2004-2012. The assay detected ~600 CFU of PA per mL of ETA. The assay was highly specific (0/291 false positives) for PA when tested against common respiratory flora. Respiratory samples were cultured on blood and MacConkey agar plates and organisms were identified by MALDI-TOF or using automated systems. A clinical trial was conducted in 3 European and 1 US laboratories in which a total of 291 (189 fresh and 102 frozen) samples were tested. The GeneXpert assay demonstrated overall sensitivity and specificity of 100% (95% CI: 92.1-100) and 97.6% (95% CI: 94.8-99.1), respectively. Of the 45 true positive specimens, 20 were fresh and 25 were frozen. Discrepant samples underwent bidirectional DNA sequencing using alternate primers for PA. After discrepant resolution, the specificity of the PA assay increased to 99.2%.
Conclusion: These data suggest that the GeneXpert PA assay is both a sensitive and specific method for determining PA colonization in ETA samples. This assay will enable more rapid detection of PA for immuno-prophylaxis of MEDI3902.
A. C. Nyborg,
D. E. Tabor, MedImmune LLC: Employee , Salary
R. Yu, None
A. Digiandomenico, MedImmune LLC: Employee , Salary
M. Patel, None
N. Shire, None
H. Jafri, MedImmune LLC: Employee , Salary
M. T. Esser, MedImmune LLC: Employee , Salary
H. Goossens, None
F. Tenover, None
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