We will recruit 20 individuals affected by cystic fibrosis (≥12 years of age), chronically
infected with P. aeruginosa (defined as having at least 50% or more sputum cultures positive
for P. aeruginosa in the previous year), homozygous for the F508del mutation
(F508del/F508del) or heterozygous for the F508del mutation and with a minimal function
mutation in the other allele (F508del/minimal function), candidates for treatment with
Trikafta/Kaftrio according to clinical practice. P. aeruginosa strains will be collected from
the sputum before the start of therapy (T0) and after 12 (T12m) and 18 (T18m) months of
treatment with Trikafta/Kaftrio. Currently, some patients have already started treatment with
Trikafta/Kaftrio. The strains at T0 and for some of them also at T12m and T18m have already
been recovered from these patients for other studies. Of these strains it is not yet known
whether they are clonal at different times (T0, T12m and T18m). Of these patients, we will
include in the expected 20 (20 total between prospective and retrospective) only patients for
whom strains clonal between T0, T12m and T18m will be identified, asking for their
authorization to use the strains and their data through informed consent. If the number of
retrospective patients is sufficient to reach the expected number (20 total patients), no
other patients will be recruited prospectively.
Patients who will be prescribed Trikafta/Kaftrio therapy according to clinical practice will
be recruited at routine visits. The decision to prescribe treatment with Trikafta/Kaftrio,
according to current clinical practice, is completely independent of the decision to include
the patient in this study. The diagnostic and evaluation procedures and follow-up visits
foreseen by the study will coincide with those programmed for each patient according to the
standard operating procedures in force at the participating centres.
For retrospective patients, informed consent will be collected during the routine check-up
and, once the eligibility criteria have been verified, the data relating to the visit
performed before the start of therapy (T0) and, if already available, the data relating to
the visit performed at 12 and 18 months from the beginning of the therapy (T12m and T18m),
will be collected. P. aeruginosa recovered from the respiratory samples during the three
visits will be analysed. In the event that data are not available, data and samples will be
collected at the respective routine follow-ups (at 12 and/or 18 months).
For prospective patients, the enrollment visit will be performed during the routine visit
performed before the start of therapy (T0) which will be followed by visits scheduled for
clinical practice at 12 and 18 months from the start of therapy (T12m and T18m).
At T0, T12m and T18m the sputum samples, collected from the patient's airways, will be
treated according to standard procedures for the evaluation of the bacterial species present.
P. aeruginosa strains will be subjected to minimal inhibitory concentration tests to assay
their susceptibility to a panel of standard antibiotics, and analyzed by Multi-locus Sequence
Typing to detect their clonality.
The strains of each patient that will be clonal between T0, T12m and T18m, immediately stored
at -80°C, will then be shipped to Ospedale San Raffaele , where they will be analyzed for the
purposes of the project using methods validated in the literature. We will analyze i) the
phenotypes (in particular motility by pilus, motility by flagellum, secretion of proteases
and pyocyanin, and mucoidity) of P. aeruginosa strains using microbiological methods
validated in the literature, ii) the expression profiles of the strains by RNA sequencing,
iii) the presence of gene variants by whole genome sequencing of the strains, iv) and their
virulence by measuring their impact on the expression of the cystic fibrosis transmembrane
conductance regulator protein in cystic fibrosis bronchial epithelial cell lines.
The final aim will be to i) evaluate the differences in the phenotypes of the P. aeruginosa
strains isolated at T0 and those isolated at T12m and T18m post treatment with Kaftrio; ii)
to evaluate differences in the susceptibility to antibiotics of the P. aeruginosa strains
isolated at T0 and those isolated at T12m and T18m; iii) evaluate differences in the
expression profiles of the P. aeruginosa strains isolated at T0 and those isolated at T12m
and T18m and correlate them to the difference in respiratory function measured as FEV1 (%);
iv) evaluate the presence of gene variants between the strains isolated at T0 and those
isolated at T12m and T18m and correlate them to the difference in respiratory function
measured as FEV1 (%); v) to determine whether changes in bacterial phenotypes induced by
Kaftrio treatment may be associated with lower anti-CFTR activity of P. aeruginosa.
