Abstract
BackgroundStaphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.MethodsWe examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion.Results87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs.ConclusionSequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.
Highlights
Airway infection is a significant contributor to morbidity and mortality in patients with cystic fibrosis (CF) [1]
Previous studies have demonstrated poor sensitivity of this quantitative PCR (qPCR) assay in the identification of S. aureus in airway samples from CF subjects when compared to culture [6]. 16S rRNA qPCR assays specific for other CF pathogens were more sensitive for the identification of other important species in CF including P. aeruginosa and H. influenzae, suggesting that different approaches may be needed for detection of S. aureus [6]
Sensitivity and specificity of qPCR assays and sequencing compared to culture For both the 16S rRNA assay and the femA assay without enzymatic digestion, OP samples were less sensitive compared to expectorated sputum (ES) samples [16S rRNA: 7.1% versus 58.1% femA: 21.4% versus 80.6%, p < 0.01 for both] (Fig 3)
Summary
Airway infection is a significant contributor to morbidity and mortality in patients with cystic fibrosis (CF) [1]. Previous studies have demonstrated poor sensitivity of this qPCR assay in the identification of S. aureus in airway samples from CF subjects when compared to culture [6]. 16S rRNA qPCR assays specific for other CF pathogens were more sensitive for the identification of other important species in CF including P. aeruginosa and H. influenzae, suggesting that different approaches may be needed for detection of S. aureus [6]. Modified extraction methods using lysostaphin and lysozyme may improve staphylococcal DNA extraction [7] Another potential reason for poor sensitivity is the gene target used, alternate targets such as the femA gene may offer improved sensitivity. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections
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