CF Patients With Intermittent P. aeruginosa Infection Are at Higher Risk to Be Infected With Human Rhinovirus
To examine the biological significance of the above findings, we next analyzed a collection of P. aeruginosa isolates obtained from CF patients at various stages of P. aeruginosa infection. We measured total protease activity of P. aeruginosa isolates and their ability to degrade recombinant IFNλ. We observed a strong correlation (R = 0.62) and significant interdependency between protease activity and IFNλ degradation (Figure 6A). Moreover, when grouping the P. aeruginosa isolates according to their stage of infection (intermittent/early or chronic infection/late) we observed that especially P. aeruginosa of intermittent infected patients were able to degrade IFNλ (Figure 6B). This could indicate that especially intermittent infected CF patients are prone to acquire respiratory virus infections. Therefore, we checked which viruses are present in respiratory material (nose or throat swabs, sputum) of CF patients (Figure 6C). Using a multiplex diagnostic virus panel, we screened 818 samples of 526 visits. We could detect viruses in 162 samples (30.85%). Most of the viruses found in these patients were human rhinoviruses (hRV) (66%) whereas RSV was only found to a lower extent and at similar frequency to Influenza, Adenovirus or Parainfluenza Virus (3.7–7.4%). Since hRV was the most prevalent virus detected and P. aeruginosa was also able to suppress hRV induced antiviral response (Figure 1B), we decided to further analyze the presence of hRV in the sputum of these patients, since P. aeruginosa can be reliably detected in this material. In accordance with the literature we did not detect any difference between the three groups if all samples (nose, throat, and sputum) from one patient were considered (Figure 6D). However, restriction of the analysis to lower airways' sputum samples from CF patients (n = 499) revealed that the prevalence of hRV detection was significantly higher (p = 0.037) in intermittent P. aeruginosa infected CF patients (16.5%) than in chronic patients (8.5%) or in patients not infected with P. aeruginosa (11.0%) (Figure 6E). In line with this, the detectable virus load indicated by the hRV Ct value by RT-PCR was highest in samples of intermittent infected CF patients, which were culture-positive for P. aeruginosa at the time of sampling (Figure 6F). These observations in clinical samples indicate that P. aeruginosa may also be able to modulate the antiviral response in vivo in the clinical setting of CF.
Figure 6  CF patients with intermittent P. aeruginosa infection are at higher risk to be infected with hRV. (A) Capacity of CF P. aeruginosa isolates to degrade IFNλ was analyzed by western blot and protease activity was measured by skim milk agar. Correlation was assessed using Spearman analysis (n = 51). (B) P. aeruginosa isolates from (A) were classified according to infection status and plotted against relative IFNλ degradation after 1 h. Statistical analysis was done using non-parametric Mann-Whitney test. (C) Presence of respiratory viruses in sputum samples of CF patients were assessed using a multiplex diagnostic virus panel assay. Relative occurrence of respiratory virus positive samples and total samples are indicated (n = 340). (D) The prevalence of hRV in CF samples (nose, throat and sputum) was plotted according to the infection status (n = 340). Statistics was done by using Fisher's Exact Test. (E) The prevalence of hRV in CF sputum samples was plotted according to the infection status (n = 499). Statistics was done by using Fisher's Exact Test. (F) Presence of hRV in sputum from CF patients was assessed by qPCR. The Ct values of hRV was plotted according to the infection status (n = 24). Significant differences were considered at *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the control condition. n.s., not significant.