Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in patients with cystic fibrosis (CF) 1 2 3 4 5 . Its presence in the lungs is associated with an increased mortality and morbidity of CF patients 6 . Early detection of this bacterium from respiratory tract is determinant because it ensures effective patient management 5 7 8 . Indeed, after intermittent colonization by different strains, once acquired, chronic P. aeruginosa colonization by mucoid and biofilm-growing isolates is difficult to eradicate 2 4 9 10 . Thus, the earlier the treatment toward P. aeruginosa onset, the higher the chance to efficiently control P. aeruginosa 5 7 8 .

However, accurate identification of this bacterium in CF sputum by conventional microbiology techniques is known to be limited. This can be explained by a large phenotypic diversity of P. aeruginosa isolates recovered from CF patients such as loss of pigment production or exopolysaccharide production. Moreover, Singh et al. demonstrated that P. aeruginosa can form biofilms in the airways of CF patients 11 . Biofilms contain bacterial cells that are in a wide range of physiological states. One of the mechanisms contributing to this physiological heterogeneity includes the adaptation to the local environmental conditions.

For instance, bacterial cells from the deep layers of biofilm depleted of oxygen 12 can grow in anaerobic conditions. Therefore, the CF patients isolates obtained from biofilms, i.e. in anaerobic conditions, grow hardly in aerobic conditions on a conventional culture medium 13 . Another limitation of conventional culture is that P. aeruginosa can be easily misidentified with closely related Gram-negative bacilli in CF sputum 14 15 16 17 18 19 .

The use of molecular techniques such as PCR could improve accurate identification of P. aeruginosa 14 15 16 17 18 19 , and consequently, its early detection in CF sputum patients 20 21 22 23 24 . To date, there is no consensus for a universal protocol for the molecular detection of P. aeruginosa. Indeed, its genome is known to be highly polymorphic. Changes that can occur at the genetic level could compromise the reliability of molecular identification techniques. In particular in CF patients lungs, most of the recovered isolates are hypermutable 2 4 25 26 , and show a high genetic plasticity by acquisition or loss of genes 27 28 . Since mutations or gene deletions occur on PCR target sequences, they could decrease the sensitivity of the method 29 . Moreover, horizontal genetic transfer with other bacterial species present in the CF lung niche can impact upon the specificity of the PCR 14 .

In a prospective multicenter study, we aimed to assess the role of PCR for the early detection of P. aeruginosa in CF patients; we evaluated two qPCRs in detection of P. aeruginosa: a simplex qPCR targeting oprL gene 30 , and a multiplex qPCR, targeting gyrB and ecfX genes 14 . The sensitivity and the specificity of both qPCRs were initially evaluated testing a large panel of P. aeruginosa isolates and closely related non-P. aeruginosa gram-negative bacilli isolates from CF patients. Then, the two different qPCRs ability in detection of P. aeruginosa were tested ex vivo, i.e in CF sputum samples. Finally, we were able to propose a promising reference protocol combining these two qPCRs for an optimal detection of P. aeruginosa in clinical setting.

Several studies have suggested that qPCR is superior to culture for detecting early colonization of P. aeruginosa in CF sputum 20 22 23 24 . Today, the main goal is to have an optimal protocol as the gold standard for the molecular detection of P. aeruginosa. Therefore, we performed in vitro and ex vivo evaluation of two qPCRs, one targeting the oprL gene and the other targeting simultaneously gyrB and ecfX genes 14 30 . Numerous DNA targets have been described for the amplification of P. aeruginosa 15 17 19 34 35 36 , of these three housekeeping genes, oprL, gyrB and ecfX have been reported to be reliable targets in the detection of P. aeruginosa 14 19 30 35 .

The first criterion for an optimal technique in early detection of P. aeruginosa in CF sputum is related to the choice of the PCR format and its optimization. Today, the DNA molecules counting of a particular sequence in a complex sample can be achieved with exceptional accuracy and sensitivity sufficient to detect a single molecule 36 . As underlined by Deschagt et al. 35 , the choice of PCR format may influence the performance of the molecular detection. We chose a probe-based assay, which is known to be more sensitive and specific than the SYBR Green-based qPCR 35 .

The second criterion is a good sensitivity to prevent false negative results. Despite wide genetic variability of P. aeruginosa isolates recovered from CF patients 2 4 25 26 27 28 , results of previous studies aiming at detecting P. aeruginosa by PCR have been encouraging. In our study, both evaluated qPCRs showed an excellent sensitivity covering all the tested panel of P. aeruginosa isolates. Focusing on the lower detection threshold, the difference was significant between the two qPCR assays with a detection threshold of 10 CFU/mL for the oprL qPCR versus 730 CFU/mL for the multiplex PCR. The sensitivity of the in vitro oprL qPCR in our study was higher than that recommended by the French guidelines, i.e. a detection threshold of 10² CFU/mL for CF sputum sample 37 .

The third criterion needed for early P. aeruginosa detection technique, in particular, for molecular one, is to have a high specificity to prevent false positive amplification. When looking at a large panel of genes described in the literature e.g. oprI, oprL, rrl, ecfX, gyrB, or rrs, specificity varied from 74% to 100% 14 17 34 35 36 38 . In our study, specificity of the oprL qPCR was evaluated at 73% versus 90% for the multiplex PCR. Four previous studies have tested the specificity of the oprL primer pairs and found different values ranging from 87% to 100% 22 34 35 38 . Again, previous studies looking at gyrB and ecfX genes found a better specificity (100%) than in our study 14 35 . Different reasons could explain these discrepancies. Firstly, our specificity could have been influenced by a larger panel of closely related non P. aeruginosa gram-negative bacilli (41 isolates including 16 different species). Secondly, all the bacterial isolates (except one reference strain) were recovered from clinical samples (CF or non CF) or from environmental samples. These isolates, which were recovered from CF could have undergone genetic exchange with other species in the natural CF microenvironment, especially P. aeruginosa, influencing the specificity of the molecular method 38 . Thus, specificity in previous studies could have been overestimated 14 34 35 38 . As highlighted by Anuj et al. 14 35 , the higher specificity of our results for the multiplex PCR may be explained by the fact that we amplified at least 2 DNA targets. The use of two probes simultaneously seems to improve the specificity, providing at the same time the detection and the confirmation of the presence of P. aeruginosa 14 19 . Interestingly, our bacterial species that cross-reacted with the oprL qPCR did not do so when oprL qPCR was combined with the multiplex PCR thus allowing 100% specificity.

These results were successfully validated by the sputum samples of CF patients from the never or free categories according to the definition of Leeds 32 . The ex vivo experiments put forward a significant difference between the culture-based quantification and the qPCR-based quantification. In average, the qPCR detected 100 times more CFU of P. aeruginosa than the culture did.

This could be explained by different hypotheses. First, the difference in utilized sputum volumes contributes to this discrepancy. Indeed, only 10 μl were cultured whereas 1 ml was extracted for the qPCR. The lowest concentration that theoretically can be detected by qPCR equals the presence of one genome (i.e. equivalent to one CFU) per qPCR reaction mixture. Using 1 ml of 10-fold concentrated sputum by centrifugation and extraction (elution volume of 100 μl) and 4.5 μl for the PCR reaction (final volume of 25 μl), the detection limit of our molecular diagnosis is ≈22 CFU/mL. In comparison, the lowest concentration that theoretically can be detected by culture is 100 CFU/mL.

Second, given the phenotypic diversity of P. aeruginosa isolates and the large diversity of species found in pulmonary microbiota, the detection of P. aeruginosa by culture in CF sputum is a hard task 14 15 16 17 18 19 . Moreover, culture in aerobic conditions can fail in the detection of some isolates adapted to anaerobic conditions of the CF lung niche 13 , or of non-cultivable isolates present in the bacterial biofilm 39 .

Another explanation could be that qPCR detects P. aeruginosa DNA, i.e. not only live bacteria but also dead cells 40 . As CF patients are chronically treated with antibiotics, one can suppose that dead bacteria are significantly present in the pulmonary tract. In a study lead by Deschaght et al. in 2009, no difference in sensitivity between culture and oprL qPCR was found 41 . Their study was conducted on eight artificial P. aeruginosa-positive sputum pre-liquefied samples thus skipping the sample homogenization step, one of the cornerstones in amplification-based technique. Our ex vivo application of the two qPCR assays with real samples took into account the sample homogenization. It also put forward the importance of having a controlled amplification assay in particular to avoid false negatives due to inhibitors or a bad extraction. Indeed, the DNA-extraction method has been shown to be a critical step in the PCR performances 41 . In our study, we chose the DICO Extra r-gene kit, a totally artificial DNA, as internal control, which prevents from contamination during procedure handling, and allows to test extraction and amplification at the same time.

Altogether, our study showed that the oprL qPCR offers a good sensitivity whereas the multiplex PCR offers a good specificity. Based on these data, we decided to combine these two qPCR assays and proposed a molecular protocol for an optimal detection of P. aeruginosa by qPCR in CF sputum as follows (Figure 1). The oprL qPCR can be applied in screening because of its good sensitivity. In case of a doubtful or a positive result, we can proceed to the multiplex PCR. Interpretation of the multiplex PCR takes into account the quantification found with oprL PCR. Below the detection threshold of 730 CFU/mL, the oprL qPCR prevails over the multiplex PCR. Conversely, beyond this threshold, the multiplex PCR prevails over the oprL qPCR. Overall, this combined molecular protocol offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%.

This qPCR-based combined protocol can be adapted for instance in a subgroup of non-sputum producing patients and used for other future prospective studies. Indeed, the initial colonization of P. aeruginosa often occurs in CF patients who do not produce sputum (e.g. mainly children). This qPCR format should therefore be tested on the sample secretions routinely obtained from, e.g. deep throat swabs or endolaryngeal suction.

The authors declare that they have no competing interests.

GHA, FLG, and RLB conceived the study and designed the experiments. FLG, GHA and RLB wrote the manuscript. FLG, SR, JH, SG and GHA performed the experiments. SBG, SV, CP and GR helped with the manuscript discussion. All authors have read and approved the final manuscript.