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. The bacterial load assay and specific organism assays had high intra-assay repeatability for specimens with rRNA gene copy numbers ≥102. In addition, qPCR results for split samples had excellent agreement for bacterial DNA detection and quantification indicating that little variability was introduced by our DNA extraction technique. Results from between-plate and within-plate analyses also indicate that variability in sample measurements was primarily attributable to subject and specimen differences and that processing variability was negligible. Anaerobes were detected frequently by qPCR from all airway specimen types.
Our results also indicate that the qPCR assays for P. aeruginosa, S. aureus and H. influenzae have variable sensitivity compared to culture for bacterial detection from CF airway specimens. Quantitative PCR and culture had similar sensitivity for detection of P. aeruginosa from sputa, but qPCR was less sensitive for detection of P. aeruginosa from oropharyngeal specimens. Staphylococcus aureus was detected less frequently by qPCR, particularly from oropharyngeal swabs and from sputum specimens with low quantities of S. aureus by quantitative culture (<105 cfu/ml). The increased sensitivity from sputum, especially for specimens with higher bacterial counts, and the high agreement between split-samples despite overall low sensitivity suggests that the limitation of qPCR is related to specimen characteristics, particularly low quantities of bacterial DNA, rather than inconsistency in assay performance. Unlike P. aeruginosa and S. aureus, H. influenzae was detected more often by qPCR than by culture from all specimen types.
The ability to reproducibly quantify anaerobic bacteria from CF airway samples is an important advance in studying the complex CF airway microbiome. Two important developments sparked recent interest in anaerobic infection in CF. First, Worlitzsch and co-workers demonstrated that P. aeruginosa
was found in hypoxic mucus plugs within the CF airway lumen in patients with chronically infected with P. aeruginosa 
. Second was the development of molecular techniques that allow detection of fastidious bacteria including anaerobic bacteria without the need for culture 
. Multiple studies using molecular techniques or strict anoxic culture have demonstrated anaerobic bacteria in CF airway samples, including during pulmonary exacerbations 
. Studies using molecular detection have relied primarily on PCR amplification with universal bacterial primers followed by terminal restriction fragment length polymorphism (t-RFLP) analysis or sequencing. These methods provide an important overview of the microbial community present in each sample. Unlike microbial community analysis, qPCR allows quantification of specific bacteria of interest. Potential applications of qPCR to CF include rapid detection and quantification of potentially pathogenic anaerobic bacteria, assessment of response to antimicrobial therapy, and tracking longitudinal changes in airway microbiology.
There are several potential limitations to our study and the use of qPCR in CF. As oropharyngeal swabs are the primary means of non-invasive bacterial surveillance in young children with CF, the low sensitivity of these qPCR assays for P. aeruginosa
and S. aureus
may limit the utility of qPCR in this population 
. Unlike our findings, Matsuda and co-workers reported excellent sensitivity of qPCR compared to culture for detection of these bacteria from blood specimens using these primers. There are several possible explanations for our lower sensitivity. CF sputum is highly tenacious and typically contains a large amount of inflammatory cells, primarily neutrophils resulting in release of large amounts of human DNA which may interfere with qPCR detection of relatively small quantities of bacterial DNA
. Second, S. aureus
bacterium may not have been sufficiently lysed by our processing methods. However, specimens were processed twice with bead-beating techniques prior to qPCR so help ensure adequate lysis. Also, there was excellent agreement between parallel DNA extractions suggesting that lysis was at least consistent between samples. Third, bacterial DNA may have been present below the level of detection for qPCR. To investigate this further, we determined the sensitivity of qPCR relative to quantitative culture results and found significantly higher sensitivity in specimens with ≥105
cfu/ml. As oropharyngeal swabs may have lower quantities of bacterial DNA than sputa samples, the finding of lower sensitivity for oropharyngeal specimens for both P. aeruginosa
and S. aureus
supports this explanation. Finally, the growth phase of bacteria or the presence of biofilms also may impact the quantity and detection of rRNA genes 
Another limitation to our study was the lack of anaerobic culture with which to compare our qPCR assays for anaerobic bacteria. Anaerobic cultures of airway samples are problematic in that they are labor intensive, outside standard practice for most clinical microbiologic laboratories and may have variable performance; thus, their utility as a gold-standard comparison is limited. To address this concern, we analyzed our qPCR products using gel electrophoresis to identify side products that may have interfered with our results and performed cross-reactivity studies for the Prevotella
assays. We found minimal cross-reactivity among Prevotella
species except at extreme low and high levels of bacterial DNA. The qPCR assays used for P. micros
are published assays that have been shown to have good agreement with anaerobic culture from periodontal samples with minimal cross-reactivity with other bacterial species 
Unlike our findings with S. aureus
and P. aeruginosa
, qPCR appears more sensitive than culture for detection of H. influenzae.
Dithiothreitol, used for sputum homogenization as recommended in CF airway culture guidelines, has been shown to inhibit the growth of H. influenzae
in culture 
. Although the use of dithiothreitol may in part explain the low sensitivity of sputum culture compared with qPCR, oropharyngeal specimens not processed with dithiothreitol also had a low detection rate of H. influenzae
by culture. In findings similar to our results, Van Belkum and colleagues detected H. influenzae
from 4 of 6 CF untreated sputa samples by PCR but not by culture using PCR amplification of bacterial small subunit rDNA followed by probe mediated bacterial identification 
. Thus, PCR may be more sensitive than culture for H. influenzae
. To examine the specificity of the qPCR assay for H. influenzae
, sequencing was performed on a sub-group of assays. The amplified PCR product was confirmed as H. influenzae
with ≥99% sequence identity. We found some cross-reactivity between H. influenzae
and H. parainfluenzae
in mixed reactions, but in the absence of H. influenzae
, there was no amplification of H. parainfluenzae
within the Tm range determined by standards.
We also explored the use of a quality control matrix designed to manage missing values and to identify unreliable results for repeat analysis. Overall, less than 1% of sample measurements had improved agreement between split-sample measurements with the application of our QC matrix. For the large majority of our samples, simple averaging of triplicate measurements (with missing values set to not-detected) resulted in equivalent values and agreement between split-samples as our results with the application of QC.
In conclusion, we found that qPCR is a reproducible method for detection of bacteria including anaerobic bacteria from CF airway samples. Using qPCR, anaerobes were detected frequently from CF airway specimens, in quantities similar to that of typical CF associated pathogens. The sensitivity of our qPCR assays for detection of P. aeruginosa and S. aureus, especially from oropharyngeal specimens may limit its usefulness for detection of these pathogens. Refinement of these qPCR assays, including the use of alternative primers, may help address this limitation. The application of qPCR to airway specimens may improve our understanding of the clinical importance of polymicrobial infection and anaerobic bacteria, progression of lung disease and ultimately improve antimicrobial treatments in CF.