Abstract
The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities ≥102 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.
Highlights
Cystic fibrosis (CF) is a life-shortening, autosomal-recessive genetic condition
Discordant results consisted of 10 culture positive/ Quantitative real-time PCR (qPCR) negative and 8 qPCR positive/culture negative for P. aeruginosa, 43 culture positive/qPCR negative and 1 qPCR positive/culture negative for S. aureus, and 6 culture positive/ qPCR negative and 42 qPCR positive/culture negative for H. influenzae. (Table 3) For S. aureus, we examined the quantity detected by quantitative culture for sputa specimens with discordant results
Our results indicate that bacterial DNA, including all bacteria, typical CF pathogens, and anaerobic bacteria, can be reproducibly quantified from CF airway specimens using qPCR
Summary
Lung disease characterized by chronic airway infection and inflammation is the leading cause of morbidity and mortality in patients with CF [1]. Airway infection is primarily attributed to a narrow spectrum of bacteria, most commonly Pseudomonas aeruginosa and Staphylococcus aureus [2,3]. CF airway infections are frequently polymicrobial, and a broad range of bacteria including anaerobes likely contribute to CF lung disease [4,5,6]. Reliance on standard microbial culture methods limits detection of polymicrobial infections, especially anaerobic bacteria, and the routine use of anaerobic culture is labor-intensive and unreliable [8]. The ability to study polymicrobial and anaerobic infection in CF has been limited
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