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A critical review of definitions used to describe Pseudomonas aeruginosa microbiological status in patients with cystic fibrosis for application in clinical trials

Open ArchivePublished:September 13, 2019DOI:https://doi.org/10.1016/j.jcf.2019.08.014

      Highlights

      • Definitions of Pseudomonas aeruginosa initial, chronic infection and eradication.
      • Microbiological monitoring, sampling and standardization of methods is required.
      • Serology should be incorporated in definitions of initial infection and eradication.

      Abstract

      Background

      Definition of Pseudomonas aeruginosa (Pa) microbiological status is essential for patients' inclusion in clinical trials. The aim of this study was to agree on the definitions of Pa infection status for initial infection, eradication and chronic infection to be used in clinical trials and to propose additional future study areas.

      Methods

      An exhaustive literature search was performed. The clinimetric properties of different definitions of Pa microbiological status were evaluated.

      Results

      Historical studies have mostly used culture-based definitions, although some have also involved complementary anti-Pa antibodies. Clinimetric analysis showed great variability in the definitions used, leading to differences in reliability, validity, responsiveness to treatment and correlation with outcome measures.
      Use of serology for initial Pa infection and successful Pa eradication introduced a greater level of complexity as antibody tests are not standardised. Moreover, the chronology of the immune response to Pa antigenic determinants was not completely clear.
      Chronic Pa infection was characterized by high levels of antibodies and good concordance between culture results and serology.

      Conclusions

      Microbiological monitoring, regular sampling from the airways and standardization of culture methods remain essential requisites for microbiological definitions. Despite limitations, serology should be incorporated in the definitions of initial infection and eradication used in clinical trials to better classify patients at enrolment, mainly in non-expectorating children. This requires standardization of serological testing.

      Keywords

      Abbreviations:

      ABx (antibiotics), AET (antibiotic eradication therapy), AP (alkaline protease), AUROC (area under the ROC curve), Azithro (azithromycin), AZLI (aztreonam lysine), BAL (bronchoalevolar lavage), Ceft (ceftazidime), CF (cystic fibrosis), CFTR (cystic fibrosis transmembrane conductance regulator), CFU/ml (colony forming units/ml), CIE (Crossed immune electrophoresis), Cip (Ciprofloxacin), clav (clavulanate), Col (colistin), EAT (early antibiotic therapy), Ela (elastase), ELISA (Enzyme-linked immunosorbent assay), ECFS-CTN (European Cystic Fibrosis Society-Clinical Trials Network), EU (ELISA unit), Exo/ExoA/T/S (exotoxin A/T/S), FEV1% pred (forced expiratory volume % predicted), FVC (forced vital capacity), grp/s (group/s), inh (inhaled), IgG (immunoglobulin G), IQR (interquartile range), IV (intravenous), MAg (multiple antigenic blend), MC (monoclonal antibody), mg (milligrams), mo (month/s), n/no (number), NA (not applicable), neb (nebulized), NIH (National Institutes of Health Score), NPV (negative predictive value), NR (not reported), NS (not significant), OP (oropharyngeal), OR (odds ratio), p (p-value), Pa (Pseudomonas aeruginosa), popn (population), PopB (PopB protein), PopD (PopD protein), PPV (positive predictive value), PS/PI (pancreatic sufficient/pancreatic insufficient), pred (predicted), resp. (respiratory), rhDNase therapy (Recombinant human deoxyribonuclease therapy), RIA (radioimmunoassay), ROC (receiver operating curve), Rx (treatment), SD (standard deviation), SOP (standard operating procedure), St-Ag (standard antigen), Tic Clav (ticarcillin clavulanate), TIS/TSI (tobramycin inhalation solution), TIV (tobramycin by IV administration), tobi (tobramycin), WBC (white blood cell count), wk./s (week/s), VC (vital capacity), vs (versus), yr/s (year/s), −ve (negative), +ve (positive)

      1. Introduction

      Pseudomonas aeruginosa (Pa) is the most important pathogen in progressive lung disease in patients with cystic fibrosis (CF) [
      • De Boeck K.
      • Bulteel V.
      • Fajac I.
      Disease-specific clinical trials networks: the example of cystic fibrosis.
      ]. Following initial infection, in the absence of prompt antibiotic treatment, chronic infection is likely to develop, resulting in severe inflammation that leads to progressive lung damage and deterioration in the clinical condition of the patient. Determination of the stage of infection, e.g., initial or chronic Pa infection, as well as successful Pa eradication, is essential for standard of care as it dictates antibiotic strategy. Initial Pa infection usually occurs in young patients; it is typically asymptomatic and caused by strains which show susceptibility to anti-pseudomonal agents [
      • Van Devanter D.R.
      • Van Dalfsen J.M.
      • Burns J.L.
      • Mayer-Hamblett N.
      In vitro antibiotic susceptibility of initial Pseudomonas aeruginosa isolates from United States cystic fibrosis patients.
      ]. Recent studies have emphasized the importance of promptly initiating antibiotic eradication treatment (AET) as once chronic infection is established, it is rarely possible to eradicate Pa [
      • Döring G.
      Prevention of Pseudomonas aeruginosa infection on patients with cystic fibrosis.
      ,
      • European Medicines Agency (EMA) Report
      Report of the workshop on endpoints for cystic fibrosis clinical trials.
      ]. Chronic Pa infection is associated with a steeper deterioration in lung function, frequent pulmonary exacerbations and a shorter median survival [
      • Sansgiry S.S.
      • Joish V.N.
      • Boklage S.
      • Goyal R.K.
      • Chopra P.
      • Sethi S.
      Economic burden of Pseudomonas aeruginosa infection on patients with cystic fibrosis.
      ,
      • Baumann U.
      • Stocklossa C.
      • Greiner W.
      • von der Schulenberg J.-M.G.
      • von der Hardt H.
      Cost of care and clinical condition in paediatric cystic fibrosis patients.
      ,
      • Emerson J.
      • Rosenfeld M.
      • McNamara S.
      • Ramsey B.
      • Gibson R.L.
      Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis.
      ,
      • Kosorok M.R.
      • Zeng L.
      • West S.E.
      • Rock M.J.
      • Splaingard M.L.
      • Laxova A.
      • et al.
      Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition.
      ]. In clinically stable patients, chronic Pa infection is usually managed by administering long-term courses of inhaled antibiotics and/or azithromycin [
      • Saiman L.
      • Marshall B.C.
      • Mayer-Hamblett N.
      • Burns J.L.
      • Quittner A.
      • Cibene D.A.
      • et al.
      Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial.
      ]. Definition of Pa infection status relies on standardised procedures for respiratory sampling, specialised laboratory expertise in recognising CF pathogens and careful microbiological follow-up [
      • Kerem E.
      • Conway S.
      • Elborn J.S.
      • Heijerman H.
      Standards of care for patients with cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Høiby N.
      Consensus report: early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ]. As detection of Pa is very dependent on sputum sampling and culture techniques, other biomarkers of infection have been considered. Anti-Pa antibodies have long been used to support microbiological diagnosis both in the initial stage of infection, and in chronicity. Commercial kits are easily available in Europe and previous studies have measured antibody responses to individual Pa virulence factors or to multiple antigens from 17 of the most common serotypes (St-Ag:1–17) [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ]. However, the chronology of the immune response to Pa antigenic determinants is only partially known and there is no consensus regarding the best performing kit.
      There is great interest in the CF community in developing standardization for clinical trial outcome measures. The European Cystic Fibrosis Society Clinical Trials Network (ECFS-CTN) has established a Standardization Committee to undertake a rigorous evaluation of clinical trial endpoint [
      • De Boeck K.
      • Bulteel V.
      • Fajac I.
      Disease-specific clinical trials networks: the example of cystic fibrosis.
      ,
      • Kent L.
      • Reix P.
      • Innes J.A.
      • Zielen S.
      • Le Bourgeois M.
      • Braggion C.
      • et al.
      Lung clearance index:evidence for use in clinical trials in cystic fibrosis.
      ]. This article summarises the work of the Standardization Committee's Microbiology Group on definitions of Pa infection status for application in clinical trials. It is crucial for anti-infective clinical trials to confirm an individual patient's eligibility and assessment outcome criteria. Historically, trials of antimicrobials in CF have been based on different definitions of Pa infection status [
      • European Medicines Agency (EMA) Report
      Report of the workshop on endpoints for cystic fibrosis clinical trials.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn JS for the Consensus Study Group
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ]. The aim of this document is to offer guidance for investigators, pharmaceutical companies, and regulatory authorities when planning trials in CF patients including Pa microbiological status as an outcome. After critical review of the literature in support of definitions for Pa infection status, the group gained consensus on the definitions of initial Pa infection, eradication and chronic infection and proposed additional future study directions.

      2. Methods

      An exhaustive literature search was conducted in PUBMED using the following search criteria: Initial/first lifetime/early/new Pa colonisation/infection, Pa eradication, anti-Pa antibodies, chronic Pa infection AND cystic fibrosis. A bibliography search was also conducted of all included articles and relevant reviews. The papers were checked in order to verify that definitions regarding Pa microbiological status were indeed reported.
      The clinimetric properties of Pa microbiological status definitions were assessed, including their reliability, validity, responsiveness to treatment, reference values, and correlation with other outcome measures. Definitions of each clinimetric property are presented in Supplementary Table S1 [
      • De Boeck K.
      • Kent L.
      • Davies J.
      • Derichs N.
      • Amaral M.
      • Rowe S.M.
      • et al.
      CFTR biomarkers: time for promotion to surrogate endpoint?.
      ].
      The expert panel searched for a consensus on definitions regarding Pa microbiological status and the use of anti-Pa antibodies, including the performance of tests available, the availability of standardised procedures, training requirements, their cost, the equipment/space required, and their applicability to different age groups.
      The levels of evidence that underlies recommendations in this document is based upon Scottish Intercollegiate Guidelines Network [
      • Scottish Intercollegiate Guidelines Network (SIGN)
      SIGN 50: A guideline developer's handbook.
      ].

      3. Results

      3.1 Definitions found in literature

      3.1.1 Definition of initial (first lifetime/early/new) Pa infection

      Table 1 describes the different definitions used for first lifetime or positive Pa culture after a stated time scale in studies aiming for Pa eradication. This includes first lifetime documented culture positive for Pa [
      • Schelstraete P.
      • Deschaght P.
      • Van Simaey L.
      • Van Daele S.
      • Haerynck F.
      • Vaneechoutte M.
      • et al.
      Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients.
      ,
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ,
      • Ratjen F.
      • Döring G.
      • Nikolaizik W.H.
      Effect of inhaled tobramycin on early Pseudomonas aeruginosa colonization in patients with cystic fibrosis.
      ,
      • Valerius N.
      • Koch C.
      • Høiby N.
      Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment.
      ] or positive Pa culture following a negative Pa timescale for: 6 months [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ,
      • Taccetti G.
      • Bianchini E.
      • Cariani L.
      • Buzzetti R.
      • Costantini D.
      • Trevisan F.
      • et al.
      Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomized multicentre study comparing two different protocols.
      ,
      • Frederiksen B.
      • Koch C.
      • Høiby N.
      Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis.
      ], 1 year [
      • Ratjen F.
      • Moeller A.
      • McKinney M.L.
      • Asherova I.
      • Alon N.
      • Maykut R.
      • et al.
      Eradication of early P. aeruginosa infection in children <7 years of age with cystic fibrosis: the early study.
      ,
      • Stanojevic S.
      • Waters V.
      • Mathew J.L.
      • Taylor L.
      • Ratjen F.
      Effectiveness of inhaled tobramycin in eradicating Pseudomonas aeruginosa in children with cystic fibrosis.
      ,
      • Amin R.
      • Lam M.
      • Dupuis A.
      • Ratjen F.
      The effect of early Pseudomonas aeruginosa treatment on lung function in pediatric cystic fibrosis.
      ,
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ,
      • Hansen C.R.
      • Pressler T.
      • Høiby N.
      Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonisation in CF patients: 15 years' experience.
      ,
      • Wiesemann H.G.
      • Steinkamp G.
      • Ratjen F.
      • Baurenfeind A.
      • Przyklenk B.
      • Döring G.
      • et al.
      Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis.
      ] and 2 years [
      • Mayer-Hamblett N.
      • Retsch-Bogart G.
      • Kloster M.
      • Accurso F.
      • Rosenfeld M.
      • Albers G.
      • et al.
      Azithromycin for early Pseudomonas infection in cystic fibrosis. The OPTIMIZE randomized trial.
      ,
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ,
      • Treggiarri M.M.
      • Retsch-Bogart G.Z.
      • Mayer-Hamblett N.
      • Khan U.
      • Kulich M.
      • Kronmal R.
      • et al.
      Comparative efficacy and safety of 4 randomised regimens to treat early Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ].
      Table 1Studies assessing first lifetime or positive Pseudomonas aeruginosa (Pa) culture after stated timescale in studies aiming for Pa eradication.
      Author yearPatients (n)Age (yrs)Mean (SD)Or (median) where statedSample origin (OP swab/sputum/BAL)TreatmentSerology used in definition of early infectionSerology used as study inclusion/exclusion criteria or endpoint?
      First lifetime documented culture positive for Pa
      Mayer-Hamblett [
      • Mayer-Hamblett N.
      • Retsch-Bogart G.
      • Kloster M.
      • Accurso F.
      • Rosenfeld M.
      • Albers G.
      • et al.
      Azithromycin for early Pseudomonas infection in cystic fibrosis. The OPTIMIZE randomized trial.
      ]
      2216 mo to 18 yrsOP swab or lower resp. tract cultureOral Azithromycin and TISNoNo
      Schelstraete [
      • Schelstraete P.
      • Deschaght P.
      • Van Simaey L.
      • Van Daele S.
      • Haerynck F.
      • Vaneechoutte M.
      • et al.
      Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients.
      ]
      416mo to 30 yrsNaso-pharyngeal aspirates or sputumOral Cip and TIS or ColYesNo
      Taccetti [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ]
      588.9 (±6.6)SputumInh Col and oral CipYesYes

      All patients included had −ve serum antibody titres against Pa and precipitating anti-Pa antibodies <2
      Ratjen [
      • Ratjen F.
      • Döring G.
      • Nikolaizik W.H.
      Effect of inhaled tobramycin on early Pseudomonas aeruginosa colonization in patients with cystic fibrosis.
      ]
      159 (+4.6)Swab or sputumTISNoYes

      Exclusion criteria: if patients had previous throat swabs or sputum positive for Pa or positive serum antibodies against Pa

      Endpoint: Absence of infection confirmed by −ve serum antibody titres against Pa
      Valerius [
      • Valerius N.
      • Koch C.
      • Høiby N.
      Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment.
      ]
      148.5 (range 36–228 mo)SputumInh Col and oral CipNoYes: study endpoint: when colonisation with Pa became chronic and/or precipitins ≥2
      2 year negative Pa timescale
      >2 negative cultures/year
      Mayer-Hamblett [
      • Mayer-Hamblett N.
      • Retsch-Bogart G.
      • Kloster M.
      • Accurso F.
      • Rosenfeld M.
      • Albers G.
      • et al.
      Azithromycin for early Pseudomonas infection in cystic fibrosis. The OPTIMIZE randomized trial.
      ]
      2216 mo to 18 yrsOP swab or lower resp. tract cultureOral Azithromycin and TISNoNo
      Tiddens [
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ]
      1056.2 (±4.7)Sputum or swabAZLI (inh)NoStated as other endpoint of interest: Pa specific serum antibodies (Exo A, Ela, AP)
      Minimum of 1 documented negative Pa culture per year
      Treggiarri [
      • Treggiarri M.M.
      • Retsch-Bogart G.Z.
      • Mayer-Hamblett N.
      • Khan U.
      • Kulich M.
      • Kronmal R.
      • et al.
      Comparative efficacy and safety of 4 randomised regimens to treat early Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ]
      3041 to 12Sputum or swabTIS with either oral Cip or placeboNoNo
      1 year negative Pa timescale
      >3 Pa negative cultures in previous 1 year (previously Pa positive)
      Stanojevic [
      • Stanojevic S.
      • Waters V.
      • Mathew J.L.
      • Taylor L.
      • Ratjen F.
      Effectiveness of inhaled tobramycin in eradicating Pseudomonas aeruginosa in children with cystic fibrosis.
      ]
      657.4 (median) range 3.2 to 10.3Swab, sputum or BALTISNoNo
      Hansen [
      • Hansen C.R.
      • Pressler T.
      • Høiby N.
      Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonisation in CF patients: 15 years' experience.
      ]
      994.8 (range 0.3 to 28.1)Sputum or endo-laryngeal suctionInh Col and oral CipNoYes

      Inclusion criteria: All patients included had precipitins <2.

      Study endpoint: 10/12 patients developing chronic infection (Copenhagen definition)
      Wieseman [
      • Wiesemann H.G.
      • Steinkamp G.
      • Ratjen F.
      • Baurenfeind A.
      • Przyklenk B.
      • Döring G.
      • et al.
      Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis.
      ]
      119.8 (+8.3)Sputum or swabTIS or placeboNoYes

      Inclusion criteria: Pa + ve respiratory cultures in at least 2 of 3 specimens and seronegative for Pa antibodies(Exo A, Ela, AP)
      At least four documented negative cultures (or up to 2 years with 4 negative cultures in this time period in the absence of anti-Pa treatment):
      Ratjen [
      • Ratjen F.
      • Moeller A.
      • McKinney M.L.
      • Asherova I.
      • Alon N.
      • Maykut R.
      • et al.
      Eradication of early P. aeruginosa infection in children <7 years of age with cystic fibrosis: the early study.
      ]
      5139 mo (median) (range 6–82)Sputum or swabTISNoNo
      Ratjen [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ]
      886mo to >18 yrSputum or swabTISNoNo
      Amin [
      • Amin R.
      • Lam M.
      • Dupuis A.
      • Ratjen F.
      The effect of early Pseudomonas aeruginosa treatment on lung function in pediatric cystic fibrosis.
      ]
      1167.5 (±1.9)SputumTISNoNo
      6 month negative Pa timescale
      Proesmans [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ]
      589 (median) range 4.7–13.1Swab, sputum or BALTIS or Col plus oral CipNoYes

      Study endpoint: Pa antibodies determined at 1 year after study entry (ELISA St Ag 1–17)
      Taccetti [
      • Taccetti G.
      • Bianchini E.
      • Cariani L.
      • Buzzetti R.
      • Costantini D.
      • Trevisan F.
      • et al.
      Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomized multicentre study comparing two different protocols.
      ]
      105

      118
      7.45 (IQR:1 to 25.5)

      7.64 (IOR:1 to 35.2)
      Sputum or swabTIS and oral Cip

      vs Inh Col and oral Cip
      NoNo
      Frederiksen [
      • Frederiksen B.
      • Koch C.
      • Høiby N.
      Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis.
      ]
      487.4 (range 0.9–18.4)Sputum or swab or nasopharyngeal aspirate or BALInh Col and oral CipYes

      Intermittent colonisation: presence of Pa in sputum at least once in association with normal values of Pa precipitins (0–1).
      Yes

      Endpoint was chronic Pa infection defined as persistent presence of Pa in sputum for at least 6 months or less when combined with presence of ≥2 precipitins.
      Presence of Pa in BAL culture: 1st lifetime Pa isolation:
      Douglas [
      • Douglas T.A.
      • Brennan S.
      • Gard S.
      • Berry L.
      • Gangell C.
      • Stick S.M.
      • et al.
      Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.
      ]
      332.5 (range 3–71 months)BAL at diagnosis (6 to 12 wks) and annually until able to expectorate sputumIV Tobi and tic clav OR Ceft, followed by TIS and oral CipNoNo
      Nixon [
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]
      243.7(±2.1) (range 0.3–7)BAL at diagnosis (6 to 12 wks) and annually until able to expectorate sputumTic clav and Tobi, followed by oral Cip and/or neb TobiNoNo
      Other definitions
      First/New Pa isolation (not stated if first lifetime isolation):
      Blanchard [
      • Blanchard A.C.
      • Horton E.
      • Stanojevic S.
      • Taylor L.
      • Waters V.
      • Ratjen F.
      Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis.
      ]
      1289 (median) IQR 6.8–12.3Sputum or swab or BALStep 1: TIS

      Step 2: TIS

      Step 3: IV Ceft and Tobi plus TIS
      NoNo
      Kenny [
      • Kenny S.L.
      • Shaw T.D.
      • Downey D.G.
      • Moore J.E.
      • Rendall J.C.
      • Elborn J.S.
      Eradication of Pseudomonas aeruginosa in adults with cystic fibrosis.
      ]
      2027 (median) range 18–81 yrsSputumOral Cip/neb Col

      IV ABx therapy

      Other: TIS and oral Cip; Oral Cip

      Neb Col
      NoNo
      Isolation of mucoid Pa: at least 1 positive Pa sample in 9-year retrospective study period
      McPherson [
      • McPherson H.
      • Rosenthal M.
      • Bush A.
      Can mucoid Pseudomonas aeruginosa be eradicated in children with cystic fibrosis?.
      ]
      116<16 yrsSputum or swab or nasopharyngeal aspirate or BALInh Col and gentamicinNoNo
      First Pa isolation: OP culture positive for Pa within 2 weeks to 12 months before screening (not stated if first lifetime isolation)
      Gibson [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ]
      28>6mo–<6 yrs.OP culture positive for Pa then eligible for BALTISNoYes (ExoA serology assessed at baseline)
      Initial isolation: presence of Pa in sputum and fewer than 2 precipitin antibodies against Pa (not stated if first lifetime isolation)
      Munck [
      • Munck A.
      • Bonacorsi S.
      • Mariani-Kurkdjian P.
      • Lebourgeois M.
      • Gérardin M.
      • Brahimi N.
      • et al.
      Genotypic characterization of Pseudomonas aeruginosa strains recovered from patients with cystic fibrosis after initial and subsequent colonization.
      ]
      195.0 (+4.0) (range 3mo–14 yrs)Sputum or laryngeal suctionIV Tobi and neb ColYes

      Colonisation: presence of Pain sputum and <2 precipitins against Pa
      No
      ABx: antibiotics; AP: alkaline protease; AZLI: aztreonam lysine; BAL: bronchoalveolar lavage; ceft: ceftazidime; Cip: Ciprofloxacin; clav: clavulanate; Col: colistin; Ela: elastase, ELISA/St-Ag1–17: Enzyme-linked immunosorbent assay/standard antigens 1–17; Exo/ExoA: exotoxin A; FEV1% pred: forced expiratory volume % predicted; grp/s: group/s; inh: inhaled; IQR: interquartile range; IV: intravenous; mg: milligrams; mo: month/s; n: number; neb: nebulized; NS: not significant; OP: oropharyngeal; p: p-value; Pa: Pseudomonas aeruginosa; resp.: respiratory; Rx: treatment; SD: standard deviation; Tic Clav: ticarcillin Clavulanate; TIS: inhaled tobramycin solution; Tobi: tobramycin; wk./s: week/s; vs: versus; yr/s: year/s.
      Other differences among the studies included the type/combination of samples collected sputum versus oropharyngeal (OP) swabs versus BAL [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ,
      • Frederiksen B.
      • Koch C.
      • Høiby N.
      Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis.
      ,
      • Stanojevic S.
      • Waters V.
      • Mathew J.L.
      • Taylor L.
      • Ratjen F.
      Effectiveness of inhaled tobramycin in eradicating Pseudomonas aeruginosa in children with cystic fibrosis.
      ,
      • Blanchard A.C.
      • Horton E.
      • Stanojevic S.
      • Taylor L.
      • Waters V.
      • Ratjen F.
      Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis.
      ,
      • McPherson H.
      • Rosenthal M.
      • Bush A.
      Can mucoid Pseudomonas aeruginosa be eradicated in children with cystic fibrosis?.
      ,
      • Douglas T.A.
      • Brennan S.
      • Gard S.
      • Berry L.
      • Gangell C.
      • Stick S.M.
      • et al.
      Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.
      ,
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ,
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ] versus laryngeal suction [
      • Hansen C.R.
      • Pressler T.
      • Høiby N.
      Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonisation in CF patients: 15 years' experience.
      ,
      • Munck A.
      • Bonacorsi S.
      • Mariani-Kurkdjian P.
      • Lebourgeois M.
      • Gérardin M.
      • Brahimi N.
      • et al.
      Genotypic characterization of Pseudomonas aeruginosa strains recovered from patients with cystic fibrosis after initial and subsequent colonization.
      ]. Most of the studies combined different measurements which further complicates interpretation of the data. The studies with bronchoalveolar lavage (BAL) focused on infants until able to expectorate sputum and were performed at diagnosis of CF (6 to 12 wks) and annually thereafter [
      • Douglas T.A.
      • Brennan S.
      • Gard S.
      • Berry L.
      • Gangell C.
      • Stick S.M.
      • et al.
      Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.
      ,
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]; or on patients with OP culture positive for Pa [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ].
      The most sophisticated studies included verification of positive results from previous clinical cultures with further cultures during a screening period in combination with anti-Pa antibodies [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ,
      • Wiesemann H.G.
      • Steinkamp G.
      • Ratjen F.
      • Baurenfeind A.
      • Przyklenk B.
      • Döring G.
      • et al.
      Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis.
      ,
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ]. Ratjen [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ] also used positive antibody titres for any of the three Pa exoproducts as an exclusion criterion [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ].

      3.1.2 Definition of Pa eradication

      Definitions of successful eradication varied according to the studies in terms of time-point for sampling (end of treatment versus a time point after treatment) (Table 2). The Pa free interval varied from (i) 3 months [
      • Schelstraete P.
      • Deschaght P.
      • Van Simaey L.
      • Van Daele S.
      • Haerynck F.
      • Vaneechoutte M.
      • et al.
      Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients.
      ,
      • Douglas T.A.
      • Brennan S.
      • Gard S.
      • Berry L.
      • Gangell C.
      • Stick S.M.
      • et al.
      Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.
      ] (ii) 6 months with 3 successive negative cultures [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ,
      • Taccetti G.
      • Bianchini E.
      • Cariani L.
      • Buzzetti R.
      • Costantini D.
      • Trevisan F.
      • et al.
      Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomized multicentre study comparing two different protocols.
      ]; (iii) 12 months [
      • McPherson H.
      • Rosenthal M.
      • Bush A.
      Can mucoid Pseudomonas aeruginosa be eradicated in children with cystic fibrosis?.
      ,
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ,
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ,
      • Kenny S.L.
      • Shaw T.D.
      • Downey D.G.
      • Moore J.E.
      • Rendall J.C.
      • Elborn J.S.
      Eradication of Pseudomonas aeruginosa in adults with cystic fibrosis.
      ] with 2 negative consecutive respiratory cultures [
      • Amin R.
      • Lam M.
      • Dupuis A.
      • Ratjen F.
      The effect of early Pseudomonas aeruginosa treatment on lung function in pediatric cystic fibrosis.
      ]; (iv) a variable follow-up timescale e.g. free of Pa at 28, 56 and 84 days [
      • Blanchard A.C.
      • Horton E.
      • Stanojevic S.
      • Taylor L.
      • Waters V.
      • Ratjen F.
      Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis.
      ] or weeks 4,8,16 and 28 [
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ]. Other definitions included Pa free interval 1 month after end of treatment [
      • Ratjen F.
      • Moeller A.
      • McKinney M.L.
      • Asherova I.
      • Alon N.
      • Maykut R.
      • et al.
      Eradication of early P. aeruginosa infection in children <7 years of age with cystic fibrosis: the early study.
      ,
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ] or negative BAL Pa culture at day 56, 84 or 112 after treatment [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ].
      Table 2Definitions of Pa eradication in studies assessing the efficacy of early eradication treatment.
      Author yearPatients (n)Age (yrs) mean (SD) or median where statedSample Origin (OP swab/sputum/BAL)TreatmentCulture time-pointsFollow up durationEfficacy of treatment (% eradication)Eradicated popn vs non-eradicated popnSerology used in definition of eradication?
      12 Months
      Free of Pa for 12 months at quarterly assessment (all quarterly cultures free of Pa)
      Mayer Hamblett [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ]
      3047.6(4.1)Sputum or swabTIS

      Oral Cip

      Oral placebo
      Quarterly for 1 to 2 yr study period5 yrs69Eradicated cohort reduced risk of developing chronic Pa by 74% (p < 0.001)

      Eradicated cohort 50 to 60% less ABx and macrolide usage (p < 0.001)
      No
      Kenny [
      • Kenny S.L.
      • Shaw T.D.
      • Downey D.G.
      • Moore J.E.
      • Rendall J.C.
      • Elborn J.S.
      Eradication of Pseudomonas aeruginosa in adults with cystic fibrosis.
      ]
      2027(median) 18–81SputumOral Cip/neb ColBimonthly intervals pre-Pa isolation then median interval of 24 days post isolation until eradication1 yr23.9NS between grpsNo
      McPherson [
      • McPherson H.
      • Rosenthal M.
      • Bush A.
      Can mucoid Pseudomonas aeruginosa be eradicated in children with cystic fibrosis?.
      ]
      11610.3Sputum or swab or nasopharyngeal aspirate or BALReported as earliest culture available until last available result over 9-year period9 yrs81Eradicated cohort mean age at clearance lower (p = 0.049)No
      Nixon [
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]
      243.7 ± 2.1 (0.3–7)BAL at diagnosis (6 to 12 wks) annually thereafter until able to expectorateIV Tic/Clav & Tobi followed by oral Cip and/or TISInitial then annual for 2 years7 yrs25Non-eradicated cohort: Lower NIH score (p < 0.01)

      Higher no. days hospitalized (p < 0.01)
      No
      Free of Pa for 2 negative consecutive respiratory cultures, measured quarterly for 1 year of follow up
      Amin [
      • Amin R.
      • Lam M.
      • Dupuis A.
      • Ratjen F.
      The effect of early Pseudomonas aeruginosa treatment on lung function in pediatric cystic fibrosis.
      ]
      1167.1 ± 1.9SputumTISQuarterly follow-up5 to 7 yrs52NS between grpsNo
      6 Months
      Pa eradication: 3 successive negative cultures in 6mo
      Taccetti [
      • Taccetti G.
      • Bianchini E.
      • Cariani L.
      • Buzzetti R.
      • Costantini D.
      • Trevisan F.
      • et al.
      Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomized multicentre study comparing two different protocols.
      ]
      105

      118
      7.45 (IQR:1 to 25.5)

      7.64 (IOR:1 to 35.2)
      Sputum or swabInh Col plus oral Cip OR TIS plus oral CipMonth 2,4,616 mo63 (Cip/Col)

      65 (Cip/Tobi)
      FEV1% mean relative change from baseline 2.15% (+8.50) in grp A and 4.55% (+11.54) in grp B (p = 0.18)No
      Taccetti [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ]
      588.9 ± 6.6SputumInh Col plus oral CipQuarterlyNot stated80

      Pa isolates from Rx grp more sensitive to Pa ABx (p < 0.05)
      Average FEV1% decline sig lower in early treated-patients vs chronically-infected (p < 0.05)Yes

      Serology used in definition of eradication: −ve titres ExoA and precipitins <2 within 6-month period post Rx
      3 Months
      Schelstraete [
      • Schelstraete P.
      • Deschaght P.
      • Van Simaey L.
      • Van Daele S.
      • Haerynck F.
      • Vaneechoutte M.
      • et al.
      Genotype based evaluation of Pseudomonas aeruginosa eradication treatment success in cystic fibrosis patients.
      ]
      416mo to 30 yrsNaso-pharyngeal aspirates or sputumOral Cip plus TIS or Oral Cip plus ColMonthly during eradication period then every 3 months post eradication treatment period50 mo median83

      47 at median follow up at 39 mo

      Genotypes of 1st and subsequent Pa isolates: chronic colonised patients identical genotypes 1st and 2nd isolates (p < 0.0001)
      Pa free period between 1st ever and 2nd Pa isolation: chronic colonised patients shorter time to 2nd isolate (p < 0.05)No
      Douglas [
      • Douglas T.A.
      • Brennan S.
      • Gard S.
      • Berry L.
      • Gangell C.
      • Stick S.M.
      • et al.
      Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.
      ]
      332.5 (3–71) moBAL at diagnosis (6 to 12 wks) and annually thereafter until able to expectorate sputumIV Tobi plus Tic/Clav OR

      IV Tobi plus Ceft followed by TIS and oral Cip
      Annually, 12 months prior, at Pa detection and 12 months post eradication19 mo median88.5Decrease pulmonary inflammation p < 0.05 (neutrophils % total cell count; IL-1B); p < 0.001 (neutrophils log, NE, IL-6)No
      Variable timescale:
      Free of Pa at 28, 56, 84 days
      Blanchard [
      • Blanchard A.C.
      • Horton E.
      • Stanojevic S.
      • Taylor L.
      • Waters V.
      • Ratjen F.
      Effectiveness of a stepwise Pseudomonas aeruginosa eradication protocol in children with cystic fibrosis.
      ]
      1289

      median IQR 6.8–12.3
      Sputum or swabTIS (step 1)

      TIS (step2)

      IV Ceft and Tobi and TIS (step 3)
      Day 28,56,845 yrs76.9 at end step 1 (28 days TIS)

      33.3 at end step 2 (additional 28 days TIS)

      87.1 at all time points
      PS increased risk of failure to eradicate

      BMI z score lower in PI
      No
      Free of Pa at weeks 4,8,16 and 28
      Tiddens [
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ]
      1056.2 ± 4.7Sputum or swabAZLIWeek 4,8,16,2824 wks89end of Rx

      75 4 weeks post Rx

      58 at all time points
      Patients –ve for Pa maintained mean baseline FEV1% pred throughout 24-week follow-up (n = 25)Yes antibodies as endpoint of interest: AP, Ela, ExoA

      Titres: –ve: <500

      Borderline: >500 to <1250

      +ve: >1250 to <10,000
      Free of Pa upon completion of treatment
      Stanojevic [
      • Stanojevic S.
      • Waters V.
      • Mathew J.L.
      • Taylor L.
      • Ratjen F.
      Effectiveness of inhaled tobramycin in eradicating Pseudomonas aeruginosa in children with cystic fibrosis.
      ]
      657.4Swab, sputum or BALTIS83 days median (TIS 80 mg grp)

      21.5 days median (TIS 300 mg grp)
      1 yr89 (80 mg grp)

      89 (300 mg grp)
      Pa recurrence in 12 months post Rx: female, age, PS patients, lower lung function, lower BMI p < 0.001No
      Free of Pa upon completion of treatment as primary outcome at 28 days (TIS) or 3 months (CC), and as secondary outcome at 6 months
      Proesmans [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ]
      584.6–14.6

      9 median (IQR 4.7–13.1)
      Swab, sputum or BALCip-TIS or Inh Col plus oral Cipconsecutive months2 yrs79.3 Cip-TIS

      89.7 Cip-Col
      NS between grpsYes

      St-Ag 1–17 antibodies determined at 1 year
      1 month after the end of treatment
      Ratjen [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ]
      886mths to >18 yrsOP swabs/sputumTISMonth 3,12,2727 mo93 TIS/28-day 1mo post Rx

      92 TIS/56-day 1mo post Rx

      66 TIS 28 day at mo 27

      69 TIS 56 day at mo 27
      NSYes

      Patients excluded if AP, Ela, ExoA titres were >1000
      Ratjen 20185139 mo median (range 6–82)OP swabs/sputumTISDay 29, 63, 91 for persistent eradication273 days (median)

      Range 65–379 days
      84.6 at day 29NSNo
      At time of follow up BAL (day 56, 84 or 112)
      Gibson [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ]
      282.4 + 2.0BALDay 30,56,84,112,168168-day BAL follow up74-ve serology at baseline higher proportion of eradication p < 0.04

      Inflammatory markers: TSI Rx associated with reduced neutrophilic inflammation
      Yes

      ExoA +ve >1:200 or higher Non-mucoid Pa at baseline and/or ExoA seronegativity associated with higher rates of eradication
      Negative sequential respiratory cultures
      Ratjen [
      • Ratjen F.
      • Döring G.
      • Nikolaizik W.H.
      Effect of inhaled tobramycin on early Pseudomonas aeruginosa colonization in patients with cystic fibrosis.
      ]
      159 + 4.6SwabTISMonth 3,6,9,122 yrs.14/15 at 1 yearNSYes

      AP, Ela, ExoA Titres not stated. Serology

      -ve titres for Pa
      Pa eradication: absence of Pa or presumed presence at a level below the detection limit any time point
      Munck [
      • Munck A.
      • Bonacorsi S.
      • Mariani-Kurkdjian P.
      • Lebourgeois M.
      • Gérardin M.
      • Brahimi N.
      • et al.
      Genotypic characterization of Pseudomonas aeruginosa strains recovered from patients with cystic fibrosis after initial and subsequent colonization.
      ]
      195.0 + 4.0 (3 m–14 yrs)Sputum or laryngeal suctionIV ceft or imipenem with Tobi, inh ColBimonthly or more often if clinically indicated3 yrs100

      74
      NSYes

      All patients had <2 precipitins after first and second Pa isolation
      ABx: antibiotic/s; AP: alkaline protease; AZLI: aztreonam for inhalation solution; BAL: bronchoalveolar lavage; BMI z score: body mass index z score; Ceft: ceftazidime; CIE: Crossed immune electrophoresis; Cip: Ciprofloxacin; Col: Colistin; Ela: elastase; ELISA: Enzyme-linked immunosorbent assay; Exo A: exotoxin A; EAT: early antibiotic therapy; FEV1% pred: forced expiratory volume predicted; grp/s: group/s; inh: inhaled; IL-1B: Interleukin 1beta; IL-6: Interleukin 6; IQR: interquartile range; IV: intravenous; mg: milligrams; mo: month/s; Pa: Pseudomonas aeruginosa; n/no: number; NE: neutrophil elastase; NIH: National Institutes of Health Score; NR: not reported; NS: not significant; OP: oropharyngeal; p: p-value; Pa: Pseudomonas aeruginosa; popn: population; pred: predicted; PS/PI: pancreatic sufficient/pancreatic insufficient; resp.: respiratory; Rx: treatment; SD: standard deviation; sig: significant; St-Ag: standard antigen; Tic Clav: ticarcillin clavulanate; Cip: oral ciprofloxacin; Tic Clav: ticarcillin Clavulanate; TIS: tobramycin inhalation solution; tobi: tobramycin; −ve: negative; +ve: positive; vs: versus; wk./s: week/s; yr/s: year/s.
      As stated above, some definitions of eradication varied according to the number of follow-up samples collected at various time-points (single culture versus several cultures over 6 months or more). In the case of several samples the strategy was very variable: e.g. quarterly follow up samples for the study duration of up to 2 years [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ]. Studies also varied by type of samples collected (sputum versus OP swab versus BAL versus laryngeal suction), the use of anti-Pa antibody testing and the time to Pa recurrence or chronic Pa. As for initial infection, within individual papers there is often no clear discrimination between sample types which renders studies difficult to analyze.
      Several studies incorporated the use of serology in the definition of eradication and/or study outcomes. Tiddens [
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ] determined baseline anti-Pa antibodies (negative/positive) [
      • Tiddens H.A.W.M.
      • De Boeck K.
      • Clancy J.P.
      • Fayon M.
      • Arets H.G.M.
      • Bresnik M.
      • et al.
      Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: the ALPINE study.
      ]; whilst Proesmans [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ] assessed Pa antibodies (ELISA St-Ag 1–17) at one year follow up [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ] and Gibson [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ] assessed serology at end of treatment [
      • Gibson R.L.
      • Emerson J.
      • Mayer-Hamblett N.
      • Burns J.L.
      • McNamara S.
      • Accurso F.J.
      • et al.
      Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis.
      ], follow-up BAL and day 168 visits. Ratjen [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ] used positive antibody titres as an exclusion criterion [
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ] and assessed serology at baseline and 1 year follow up [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]; Taccetti [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ] employed negative titres within a 6-month period post treatment [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ].

      3.1.3 Definition of chronic Pa infection

      Definitions of chronic Pa infection previously used in trials of maintenance therapy were assessed by culture time point and sample origin. Data is outlined in Table 3. Similar to initial Pa acquisition and eradication, studies differed widely in the criteria used to define chronic Pa infection. This included a wide range of timeframes required for sputum specimen positive for Pa: from 3 months [
      • Assael B.M.
      • Pressler T.
      • Bilton D.
      • Fayon M.
      • Fischer R.
      • Chiron R.
      • et al.
      Inhaled aztreonam lysine vs. inhaled tobramycin in cystic fibrosis: a comparative efficacy trial.
      ]; six months prior to screening [
      • Konstan M.W.
      • Flume P.A.
      • Kappler M.
      • Chiron R.
      • Higgins M.
      • Brockhaus F.
      • et al.
      Safety, efficacy and convenience of tobramycin inhalation powder in cystic fibrosis patients: the EAGER trial.
      ,
      • Konstan M.W.
      • Geller D.A.
      • Minić P.
      • Brockhaus F.
      • Zhang J.
      • Angyalosi G.
      Tobramycin inhalation powder for P. aeruginosa infection in cystic fibrosis: the EVOLVE trial.
      ,
      • Herrmann G.
      • Yang L.
      • Wu H.
      • Song Z.
      • Wang H.
      • Høiby N.
      • et al.
      Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa.
      ,
      • Nasr S.Z.
      • Sakmar E.
      • Christodoulou E.
      • Eckhardt B.P.
      • Streetmen D.S.
      • Strouse P.J.
      The use of high-resolution computerized tomography (HRCT) of the chest in evaluating the effect of tobramycin solution for inhalation in cystic fibrosis in lung disease.
      ,
      • Chuchalin A.
      • Csiszér E.
      • Gyurkovics K.
      • Bartnicka M.T.
      • Sands D.
      • Kapranov N.
      • et al.
      A formulation of aerosolized tobramycin (Bramitob) in the treatment of patients with cystic fibrosis and Pseudomonas aeruginosa infection: a double-blind, placebo-controlled, multicenter study.
      ,
      • Lenoir G.
      • Antypkin Y.G.
      • Miano A.
      • Moretti P.
      • Zanda M.
      • Varoli G.
      • et al.
      Efficacy, safety, and local pharmacokinetics of highly concentrated nebulized tobramycin in patients with cystic fibrosis colonized with Pseudomonas aeruginosa.
      ,
      • Hodson M.E.
      • Gallagher C.G.
      • JRW Govan
      A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.
      ], 12 months [
      • Saiman L.
      • Marshall B.C.
      • Mayer-Hamblett N.
      • Burns J.L.
      • Quittner A.
      • Cibene D.A.
      • et al.
      Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial.
      ,
      • Hodson M.E.
      • Gallagher C.G.
      • JRW Govan
      A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.
      ,
      • Flume P.A.
      • Clancy J.P.
      • Retsch-Bogart G.Z.
      • Tullis D.E.
      • Bresnik M.
      • Derchak P.A.
      • et al.
      Continuous alternating inhaled antibiotics for chronic Pseudomonal infection in cystic fibrosis.
      ,
      • Elborn J.S.
      • Geller D.E.
      • Conrad D.
      • Aaron S.D.
      • Smyth A.R.
      • Fischer R.
      • et al.
      A phase-3, open-label randomized trial to evaluate the safety and efficacy of levofloxacin inhalation solution (APT-1026) versus tobramycin inhalation solution in stable cystic fibrosis patients.
      ,
      • Trapnell B.C.
      • McColley S.A.
      • Kissner D.G.
      • Rolfe M.W.
      • Rosen J.M.
      • McKevitt, et al.
      Fosfomycin/tobramycin for inhalation in patients with cystic fibrosis with Pseudomonas airway infection.
      ,
      • Wainwright C.E.
      • Quittner A.L.
      • Geller D.E.
      • Nakamura C.
      • Wooldridge J.L.
      • Gibson R.L.
      • et al.
      Aztreonam for inhalation solution (AZLI) in patients with cystic fibrosis, mild lung impairment and P aeruginosa.
      ,
      • Okusanya O.O.
      • Bhavani S.M.
      • Hammel J.
      • Minic P.
      • Dupont L.J.
      • Forrest A.
      • et al.
      Pharmacokinetic and pharmacodynamic evaluation of liposomal amikacin for inhalation in cystic fibrosis patients with chronic pseudomonal infection.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.
      • Gibson R.L.
      • Oermann C.M.
      • McCoy K.S.
      • Montgomery B.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis.
      ]; 18 months [
      • Geller D.E.
      • Flume P.A.
      • Staab D.
      • Fischer R.
      • Loutit J.S.
      • Conrad D.J.
      Levofloxacin inhalation solution (MP-376) in patients with cystic fibrosis with Pseudomonas aeruginosa.
      ] and finally 24 months prior to screening [
      • Okusanya O.O.
      • Bhavnani S.M.
      • Hammel J.P.
      • Forrest A.
      • Bulik C.C.
      • Ambrose P.G.
      • et al.
      Evaluation of the pharmacokinetics and pharmacodynamics of liposomal amikacin for inhalation in cystic fibrosis patients with chronic Pseudomonal infections using data from two phase 2 clinical studies.
      ]. Five other studies did not specify a timeframe. The majority of studies did not specify the total number or type of samples required apart from the following specific definitions: 3 positive specimens within 2 years prior to screening and 1 within 3 months prior to screening [
      • Okusanya O.O.
      • Bhavnani S.M.
      • Hammel J.P.
      • Forrest A.
      • Bulik C.C.
      • Ambrose P.G.
      • et al.
      Evaluation of the pharmacokinetics and pharmacodynamics of liposomal amikacin for inhalation in cystic fibrosis patients with chronic Pseudomonal infections using data from two phase 2 clinical studies.
      ]; 2 positive cultures within 12 months prior to screening (1 within the previous 3 months) or positive at screening [
      • Hodson M.E.
      • Gallagher C.G.
      • JRW Govan
      A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.
      ,
      • Trapnell B.C.
      • McColley S.A.
      • Kissner D.G.
      • Rolfe M.W.
      • Rosen J.M.
      • McKevitt, et al.
      Fosfomycin/tobramycin for inhalation in patients with cystic fibrosis with Pseudomonas airway infection.
      ,
      • Wainwright C.E.
      • Quittner A.L.
      • Geller D.E.
      • Nakamura C.
      • Wooldridge J.L.
      • Gibson R.L.
      • et al.
      Aztreonam for inhalation solution (AZLI) in patients with cystic fibrosis, mild lung impairment and P aeruginosa.
      ,
      • Retsch-Bogart G.Z.
      • Quittner A.
      • Gibson R.L.
      • Oermann C.M.
      • McCoy K.S.
      • Montgomery B.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis.
      ]. Pa was confirmed using repeated culture as part of the screening or baseline measurements in most of the studies [
      • Saiman L.
      • Marshall B.C.
      • Mayer-Hamblett N.
      • Burns J.L.
      • Quittner A.
      • Cibene D.A.
      • et al.
      Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial.
      ,
      • Konstan M.W.
      • Flume P.A.
      • Kappler M.
      • Chiron R.
      • Higgins M.
      • Brockhaus F.
      • et al.
      Safety, efficacy and convenience of tobramycin inhalation powder in cystic fibrosis patients: the EAGER trial.
      ,
      • Konstan M.W.
      • Geller D.A.
      • Minić P.
      • Brockhaus F.
      • Zhang J.
      • Angyalosi G.
      Tobramycin inhalation powder for P. aeruginosa infection in cystic fibrosis: the EVOLVE trial.
      ,
      • Herrmann G.
      • Yang L.
      • Wu H.
      • Song Z.
      • Wang H.
      • Høiby N.
      • et al.
      Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa.
      ,
      • Nasr S.Z.
      • Sakmar E.
      • Christodoulou E.
      • Eckhardt B.P.
      • Streetmen D.S.
      • Strouse P.J.
      The use of high-resolution computerized tomography (HRCT) of the chest in evaluating the effect of tobramycin solution for inhalation in cystic fibrosis in lung disease.
      ,
      • Chuchalin A.
      • Csiszér E.
      • Gyurkovics K.
      • Bartnicka M.T.
      • Sands D.
      • Kapranov N.
      • et al.
      A formulation of aerosolized tobramycin (Bramitob) in the treatment of patients with cystic fibrosis and Pseudomonas aeruginosa infection: a double-blind, placebo-controlled, multicenter study.
      ,
      • Lenoir G.
      • Antypkin Y.G.
      • Miano A.
      • Moretti P.
      • Zanda M.
      • Varoli G.
      • et al.
      Efficacy, safety, and local pharmacokinetics of highly concentrated nebulized tobramycin in patients with cystic fibrosis colonized with Pseudomonas aeruginosa.
      ,
      • Hodson M.E.
      • Gallagher C.G.
      • JRW Govan
      A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.
      ,
      • Flume P.A.
      • Clancy J.P.
      • Retsch-Bogart G.Z.
      • Tullis D.E.
      • Bresnik M.
      • Derchak P.A.
      • et al.
      Continuous alternating inhaled antibiotics for chronic Pseudomonal infection in cystic fibrosis.
      ,
      • Elborn J.S.
      • Geller D.E.
      • Conrad D.
      • Aaron S.D.
      • Smyth A.R.
      • Fischer R.
      • et al.
      A phase-3, open-label randomized trial to evaluate the safety and efficacy of levofloxacin inhalation solution (APT-1026) versus tobramycin inhalation solution in stable cystic fibrosis patients.
      ,
      • Okusanya O.O.
      • Bhavani S.M.
      • Hammel J.
      • Minic P.
      • Dupont L.J.
      • Forrest A.
      • et al.
      Pharmacokinetic and pharmacodynamic evaluation of liposomal amikacin for inhalation in cystic fibrosis patients with chronic pseudomonal infection.
      ,
      • Gibson R.L.
      • Emerson J.
      • McNamara S.
      • Burns J.L.
      • Rosenfeld M.
      • Yunker A.
      • et al.
      Significant microbiological effect of inhaled tobramycin in young children with cystic fibrosis.
      ,
      • Okusanya O.O.
      • Bhavnani S.M.
      • Hammel J.P.
      • Forrest A.
      • Bulik C.C.
      • Ambrose P.G.
      • et al.
      Evaluation of the pharmacokinetics and pharmacodynamics of liposomal amikacin for inhalation in cystic fibrosis patients with chronic Pseudomonal infections using data from two phase 2 clinical studies.
      ,
      • Hodson M.E.
      • Penketh A.R.L.
      • Batten J.C.
      Aerosol carbenicillin and gentamicin treatment of Pseudomonas aeruginosa infection in patients with cystic fibrosis.
      ]. Only one study incorporated anti-Pa antibodies, defining chronic Pa infection as a continuous presence of Pa in the lungs for more than 6 months and/or the presence of more than 2 precipitating Pa antibodies [
      • Chuchalin A.
      • Csiszér E.
      • Gyurkovics K.
      • Bartnicka M.T.
      • Sands D.
      • Kapranov N.
      • et al.
      A formulation of aerosolized tobramycin (Bramitob) in the treatment of patients with cystic fibrosis and Pseudomonas aeruginosa infection: a double-blind, placebo-controlled, multicenter study.
      ].
      Table 3Studies defining chronic Pa infection: sample origin and culture time-point.
      First author/yearPatients (n)Age (yrs) mean (SD)Sample origin (OP swab/sputum/BAL)Culture time-points (days) [unless otherwise stated]
      24 Months
      Sputum specimen positive for Pa within 24 months prior to screening (3 positive cultures) and 1 positive culture within 3 months prior to screening
      Okusanya [
      • Okusanya O.O.
      • Bhavnani S.M.
      • Hammel J.P.
      • Forrest A.
      • Bulik C.C.
      • Ambrose P.G.
      • et al.
      Evaluation of the pharmacokinetics and pharmacodynamics of liposomal amikacin for inhalation in cystic fibrosis patients with chronic Pseudomonal infections using data from two phase 2 clinical studies.
      ]
      10520(6.68)SputumBaseline,14,28
      18 Months
      Sputum specimen positive for Pa within 18 months prior to screening and positive at screening
      Geller [
      • Geller D.E.
      • Flume P.A.
      • Staab D.
      • Fischer R.
      • Loutit J.S.
      • Conrad D.J.
      Levofloxacin inhalation solution (MP-376) in patients with cystic fibrosis with Pseudomonas aeruginosa.
      ]
      15128.7SputumBaseline,28
      12 Months
      Sputum specimen positive for Pa within 12 months prior to screening and positive at screening
      Flume [
      • Flume P.A.
      • Clancy J.P.
      • Retsch-Bogart G.Z.
      • Tullis D.E.
      • Bresnik M.
      • Derchak P.A.
      • et al.
      Continuous alternating inhaled antibiotics for chronic Pseudomonal infection in cystic fibrosis.
      ]
      33028.8 (10.9)

      29.4 (10.3) placebo
      OP or sputum14,28,42,56
      Elborn [
      • Elborn J.S.
      • Geller D.E.
      • Conrad D.
      • Aaron S.D.
      • Smyth A.R.
      • Fischer R.
      • et al.
      A phase-3, open-label randomized trial to evaluate the safety and efficacy of levofloxacin inhalation solution (APT-1026) versus tobramycin inhalation solution in stable cystic fibrosis patients.
      ]
      28228.8(10.94)

      28.1(8.96)
      OP or sputumBaseline, 28,56,84,112,140,168
      Okusanya [
      • Okusanya O.O.
      • Bhavani S.M.
      • Hammel J.
      • Minic P.
      • Dupont L.J.
      • Forrest A.
      • et al.
      Pharmacokinetic and pharmacodynamic evaluation of liposomal amikacin for inhalation in cystic fibrosis patients with chronic pseudomonal infection.
      ]
      2423.7(6.96)SputumScreening −14,0,1,7,14
      Saiman [
      • Saiman L.
      • Marshall B.C.
      • Mayer-Hamblett N.
      • Burns J.L.
      • Quittner A.
      • Cibene D.A.
      • et al.
      Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial.
      ]
      18520.2(7.9)OP or sputumScreening,168, 252,336
      Sputum specimen positive for Pa in 2 cultures within 12 months prior to screening (1 within previous 3 months) or positive at screening
      Trapnell [
      • Trapnell B.C.
      • McColley S.A.
      • Kissner D.G.
      • Rolfe M.W.
      • Rosen J.M.
      • McKevitt, et al.
      Fosfomycin/tobramycin for inhalation in patients with cystic fibrosis with Pseudomonas airway infection.
      ]
      11935(10.9)

      31(8.8) placebo
      OP or sputumBaseline,14,28,42,56
      Wainwright [
      • Wainwright C.E.
      • Quittner A.L.
      • Geller D.E.
      • Nakamura C.
      • Wooldridge J.L.
      • Gibson R.L.
      • et al.
      Aztreonam for inhalation solution (AZLI) in patients with cystic fibrosis, mild lung impairment and P aeruginosa.
      ]
      15731(10.2)

      18.9(9.1) placebo
      OP or sputumBaseline,28,42
      Retsch-Bogart [
      • Retsch-Bogart G.Z.
      • Quittner A.
      • Gibson R.L.
      • Oermann C.M.
      • McCoy K.S.
      • Montgomery B.
      • et al.
      Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis.
      ]
      13819.5(9.1) AZLI

      31.7(11.74) placebo

      27.4(7.54) AZLI
      SputumBaseline,14,28
      Hodson [
      • Hodson M.E.
      • Gallagher C.G.
      • JRW Govan
      A randomised clinical trial of nebulised tobramycin or colistin in cystic fibrosis.
      ]
      11521.3 ± 9.6 TNS

      20.1 ± 9.4 Colistin
      SputumScreening (week −2), baseline (week 0), week 4
      Sputum specimen positive for Pa (1 historical OP sample) within 12 months prior to screening (2 weeks to 12 months prior) and positive baseline BAL culture at screening
      Gibson [
      • Gibson R.L.
      • Emerson J.
      • McNamara S.
      • Burns J.L.
      • Rosenfeld M.
      • Yunker A.
      • et al.
      Significant microbiological effect of inhaled tobramycin in young children with cystic fibrosis.
      ]
      214.0(1.5) TIS

      3.7(1.6) placebo
      BALBaseline,28
      6 Months
      Sputum specimen positive for Pa within 6 months prior to screening and positive at screening
      Konstan et al., [
      • Konstan M.W.
      • Flume P.A.
      • Kappler M.
      • Chiron R.
      • Higgins M.
      • Brockhaus F.
      • et al.
      Safety, efficacy and convenience of tobramycin inhalation powder in cystic fibrosis patients: the EAGER trial.
      ] (JCF)
      51726(11.4) TIP

      25(10.2) TIS
      OP or sputumBaseline,28,56,84,112,140,168
      Konstan et al., [
      • Konstan M.W.
      • Geller D.A.
      • Minić P.
      • Brockhaus F.
      • Zhang J.
      • Angyalosi G.
      Tobramycin inhalation powder for P. aeruginosa infection in cystic fibrosis: the EVOLVE trial.
      ] (Ped Pulm)
      9513.4(4.42) TIP

      13.2(3.91) placebo
      OP or sputumBaseline,28,56,84,112,140
      Herrmann [
      • Herrmann G.
      • Yang L.
      • Wu H.
      • Song Z.
      • Wang H.
      • Høiby N.
      • et al.
      Colistin-tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa.
      ]
      527(9)SputumBaseline,30
      Nasr [
      • Nasr S.Z.
      • Sakmar E.
      • Christodoulou E.
      • Eckhardt B.P.
      • Streetmen D.S.
      • Strouse P.J.
      The use of high-resolution computerized tomography (HRCT) of the chest in evaluating the effect of tobramycin solution for inhalation in cystic fibrosis in lung disease.
      ]
      3211.81(7.46) TSI

      15.86(7.25) placebo
      OPBaseline,28 (PFTs end of study, not cultures)
      Chuchalin [
      • Chuchalin A.
      • Csiszér E.
      • Gyurkovics K.
      • Bartnicka M.T.
      • Sands D.
      • Kapranov N.
      • et al.
      A formulation of aerosolized tobramycin (Bramitob) in the treatment of patients with cystic fibrosis and Pseudomonas aeruginosa infection: a double-blind, placebo-controlled, multicenter study.
      ]
      24714.8(5.7) TSI

      14.7(6.6) placebo
      Sputum or IS/sputumBaseline,14,28,56,84,112,140,168
      Lenoir [
      • Lenoir G.
      • Antypkin Y.G.
      • Miano A.
      • Moretti P.
      • Zanda M.
      • Varoli G.
      • et al.
      Efficacy, safety, and local pharmacokinetics of highly concentrated nebulized tobramycin in patients with cystic fibrosis colonized with Pseudomonas aeruginosa.
      ]
      5911.0(5.0) TSI

      14.2(5.5) placebo
      IS/sputumBaseline,28,56
      Hodson [
      • Hodson M.E.
      • Penketh A.R.L.
      • Batten J.C.
      Aerosol carbenicillin and gentamicin treatment of Pseudomonas aeruginosa infection in patients with cystic fibrosis.
      ]
      2015–42 rangeSputumBaseline, monthly for 12 mo
      6 consecutive monthly sputum samples with growth of Pa or in case of shorter colonisation, rise in specific, precipitating antibodies against P. aeruginosa to at least 2
      Hansen [
      • Hansen C.R.
      • Pressler T.
      • Høiby N.
      Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonisation in CF patients: 15 years' experience.
      ]
      994.8(range 0.3 to 28.1)OP or sputumScreening, monthly until establishment of chronic infection
      3 Months
      Sputum specimen positive for Pa within 3 months prior to screening
      Assael [
      • Assael B.M.
      • Pressler T.
      • Bilton D.
      • Fayon M.
      • Fischer R.
      • Chiron R.
      • et al.
      Inhaled aztreonam lysine vs. inhaled tobramycin in cystic fibrosis: a comparative efficacy trial.
      ]
      26825.8(9.1) AZLI

      25.1(9.0) TNS
      SputumBaseline,28,84,140
      Variable sample/time-point parameters
      Sputum specimen positive for Pa in 2 prior samples
      Murphy [
      • Murphy T.D.
      • Anbar R.A.
      • Lester L.A.
      • Nasr S.Z.
      • Nickerson B.
      • VanDevanter D.R.
      • et al.
      Treatment with tobramycin solution for inhalation reduces hospitalizations in young CF subjects with mild lung disease.
      ]
      1819.9(2.4) control

      10.2(2.7) TIS
      OP or sputumBaseline,56,140,224,308,492
      Sputum specimen positive for Pa in 1 prior sample
      Burns [
      • Burns J.L.
      • Van Dalfsen J.M.
      • Shawar R.M.
      • Otto K.L.
      • Garber R.L.
      • Quan J.M.
      • et al.
      Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis.
      ]
      520>6 yearsOP or sputumBaseline,140 (168 for TSI)
      Sputum specimen positive for Pa at screening visit
      McCoy [
      • McCoy K.S.
      • Quittner A.L.
      • Oermann C.M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Montgomery A.B.
      Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis.
      ]
      21127.9(10.65) placebo

      26.5(10.50) AZLI

      24.1(7.5) AZLI
      OP or sputumBaseline,14,28,42
      Sputum specimen positive for Pa
      Ramsey [
      • Ramsey B.W.
      • Pepe M.S.
      • Quan J.M.
      • Otto K.L.
      • Montgomery A.B.
      • Williams-Warren J.
      • et al.
      For the cystic fibrosis inhaled tobramycin study group. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis.
      ]
      46420.8(9.5) TIS

      20.6(10) placebo
      SputumBaseline,12,28,42,56,84,112,140,168
      Ramsey [
      • Ramsey B.W.
      • Dorkin H.L.
      • Eisenberg J.D.
      • Gibson R.L.
      • Harwood I.R.
      • Kravitz R.M.
      • et al.
      Efficacy of aerosolized tobramycin in patients with cystic fibrosis.
      ]
      7117.7(1.25) TIS

      16.6(1.24) placebo
      SputumBaseline,14,28,56,84 (TIS)

      Baseline,14,21,28,35 (placebo)
      AZLI: aztreonam lysine; BAL: bronchoalveolar lavage; n: number; mo: month/s; OP: oropharyngeal; Pa: Pseudomonas aeruginosa; Rx: treatment; SD: standard deviation; TIS/TSI/TIP: tobramycin inhaled solution/powder; TNS: tobramycin nebulised solution; wk./s: week/s; yr/s: year/s.
      Due to the difficulties in defining chronic Pa infection and hence the identification of patients most at risk of progressive lung disease, recent research aims to redefine chronic infection in CF either by considering the emergence of mucoidity or by using molecular methods. Heltshe [
      • Heltshe S.L.
      • Khan U.
      • Beckett V.
      • Baines A.
      • Emerson J.
      • Sanders D.B.
      • et al.
      Longitudinal development of initial, chronic and mucoid Pseudomonas aeruginosa infection in young children with cystic fibrosis.
      ] demonstrated that the majority of children developed mucoid infection prior to meeting their accepted definition of chronic infection (at least 3 yearly quarters Pa positive in the preceding year) [
      • Heltshe S.L.
      • Khan U.
      • Beckett V.
      • Baines A.
      • Emerson J.
      • Sanders D.B.
      • et al.
      Longitudinal development of initial, chronic and mucoid Pseudomonas aeruginosa infection in young children with cystic fibrosis.
      ]. Boutin [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ] showed that Pa abundance, measured by quantitative polymerase chain reaction (qPCR), was more discriminatory than mucoidity to distinguish chronic from intermittent Pa strains [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ].

      3.2 Assessment of the clinimetric properties of microbiological definitions of Pa status based solely on cultures

      3.2.1 Feasibility

      The microbiological definitions of Pa status require a review of historical culture results. This requires all ECFS-CTN centres to adhere to agreed sampling frequencies, sample types and laboratory processes prior to trial enrolment. Therefore, these centres must agree to adhere to a set of minimum standards during their routine clinical practice. Culture of respiratory samples is easy to perform and is now supported by the use of ECFS-CTN Standard Operating Procedures (ECFS-CTN SOP Optimised Isolation and Identification of Pa; ECFS-CTN SOP Frequency and Methods of Respiratory Sampling; ECFS CTN SOP Sputum Induction; ECFS-CTN SOP Inflammatory Mediators in CF Patients Bronchoalveolar Lavage Through a Flexible Bronchoscope) which recommend sampling on a quarterly basis of sputum, or induced sputum in non-expectorating patients [
      • ECFS-CTNSOP 2.5/002
      ,
      • ECFS-CTNSOP 2.4/002
      ,
      • ECFS-CTNSOP 2.4/001
      ,

      ECFS-CTNSOP 2.5/001 Inflammatory mediators in cf patients bronchoalveolar lavage through a flexible bronchoscope; Fayon M and Dupont L (ECFS-CTN) and Konstan M, Hilliard J, Davis S (CFF-TDN) and Wainwright C (Aus.). 2015.

      ]. The feasibility of sampling will be variable depending on patient factors. Older children and adults who expectorate sputum will be easy to sample. It may be difficult to collect sputum from younger children unable to expectorate. As OP swabs or laryngeal suction lack optimal sensitivity, patients with early infection may be missed in clinical trials. Recent studies utilizing molecular methods such as qPCR may prove to be beneficial in the early detection of lower airway Pa infection using OP swabs in a non-expectorating paediatric population [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ]. Importantly, recent studies highlight the utility of sputum induction as a surrogate for BAL for paediatric lower airway pathogen assessment in symptomatic children [
      • Ronchetti K.
      • Tame J.D.
      • Paisey C.
      • Thia L.P.
      • Doull I.
      • Howe R.
      • et al.
      The CF-sputum induction trial (CF-SpIT) to assess lower airway bacterial sampling in young children with cystic fibrosis: a prospective internally controlled interventional trial.
      ].

      3.2.2 Risk

      There will be additional clinical and enrolment risks should invasive sampling be required (e.g. via two-lobe BAL).

      3.2.3 Responsiveness

      The outcome measure of ‘successful Pa eradication’ is required and conversion from positive to negative Pa culture results is optimal to monitor the efficacy of the intervention (Table 3).
      Eradication is associated with less antibiotic therapy [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ], reduced lung function decline [
      • Taccetti G.
      • Campana S.
      • Festini F.
      • Mascherini M.
      • Döring G.
      Early eradication therapy against Pseudomonas aeruginosa in CF patients.
      ], and reduced risk of worse lung function [
      • Stanojevic S.
      • Waters V.
      • Mathew J.L.
      • Taylor L.
      • Ratjen F.
      Effectiveness of inhaled tobramycin in eradicating Pseudomonas aeruginosa in children with cystic fibrosis.
      ]. In a 7-year prospective study by Nixon [
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ] Pa infection was associated with increased time of hospitalization, and higher rates of rhDNase therapy in survivors [
      • Nixon G.M.
      • Armstrong D.S.
      • Carziano R.
      • Carlin J.B.
      • Olinsky A.
      • Robertson C.F.
      • et al.
      Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]. Effect on nutritional status was controversial, with no change [
      • Proesmans M.
      • Vermeulen F.
      • Boulanger L.
      • Verhagen J.
      • De Boeck K.
      Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ,
      • Ratjen F.
      • Munck A.
      • Kho P.
      • Angyalosi G.
      Treatment of early Pseudomonas aeruginosa infection in patients with cystic fibrosis: the ELITE trial.
      ]; whereas 1 study demonstrated a significant increase in mean ideal weight for height of 3.5% (p < 0.001) [
      • Steinkamp G.
      • Tümmler B.
      • Malkottke R.
      • Von der Hardt H.
      Treatment of Pseudomonas aeruginosa colonisation in cystic fibrosis.
      ]. A possible side effect of EAT could be the emergence of new pathogens in the airways as reported in 2 studies [
      • Taccetti G.
      • Bianchini E.
      • Cariani L.
      • Buzzetti R.
      • Costantini D.
      • Trevisan F.
      • et al.
      Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: a randomized multicentre study comparing two different protocols.
      ,
      • Treggiarri M.M.
      • Retsch-Bogart G.Z.
      • Mayer-Hamblett N.
      • Khan U.
      • Kulich M.
      • Kronmal R.
      • et al.
      Comparative efficacy and safety of 4 randomised regimens to treat early Pseudomonas aeruginosa infection in children with cystic fibrosis.
      ].

      3.2.4 Cost

      The costs of non-invasive sampling are minimal. These will increase if either induced sputum or bronchoscopy is required. Culture costs should be minimal, although these would be increased if Pa quantitative counts are needed, if the trial protocol requires repeated cultures over a prolonged period of time or if additional transport costs were incurred (e.g. to a central laboratory).

      3.3 Assessment of the clinimetric properties of microbiological definitions of Pa status based on sputum culture and anti-Pa antibodies

      3.3.1 Validity

      3.3.1.1 Concurrent validity

      In the early stages of Pa infection, the use of anti-Pa antibodies can be justified because of the low sensitivity of OP swabs obtained from non-expectorating patients. Anti-Pa serology, in the initial phase of Pa infection (Tables 1 and 2), could be considered an adjunct to, rather than a replacement for cultures. At present, no clinical decisions regarding patient treatment are based on anti-Pa serology alone.
      Chronic Pa infection is characterized by high levels of anti-Pa antibodies and good concordance between culture results and serology (Table 4). Clear cut-offs have only been well defined for chronic Pa infection [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ,
      • Hayes D.
      • Farrell P.M.
      • Li Z.
      • West S.E.
      Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.
      ,
      • Douglas T.A.
      • Brennan S.
      • Berry L.
      • Winfield Wainwright C.E.
      • Grimwood K.
      • et al.
      Value of serology in predicting P.aeruginosa infection in young children with CF.
      ,
      • West S.E.
      • Zeng L.
      • Lee B.L.
      • Kosorok M.R.
      • Laxova A.
      • Rock M.J.
      • et al.
      Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors.
      ]. Cut-offs for ‘significant’ rises in Pa antibodies in the chronic phase of Pa infection would need to be defined.
      Table 4Anti-Pa antibodies at chronic Pa infection.
      First author yearPatients (n)Age range (yrs)Pa time samplingMethodTitre cutoffAntigen% sens% specPPVNPVAntibodies assessed during Rx and at follow-up?Evidence of seropositivity/increase in titres
      Daines [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ]
      2610.0–12.01st lifetimeELISA<100: −ve

      >100: +ve
      ExoA

      AP

      Ela

      3/3



      ExoA

      AP

      Ela

      3/3
      AT CHRONIC INFECTION: (6 mo)Baseline and follow up

      (annual samples over 3 yr period)
      Yes at 6 mo

      No at 12 mo
      39.1

      24.9

      23.7

      10.7
      77.6

      74.8

      84.1

      95.6
      47.8

      34.1

      44.0

      56.2
      70.7

      65.4

      67.7

      67.0
      AT CHRONIC INFECTION (12 mo)
      35.6

      23

      19.2

      8.4
      77.6

      74.8

      84.1

      95.6
      56.4

      42.6

      49.5

      61.1
      59.7

      54.4

      56.1

      56.2
      Kappler [
      • Kappler M.
      • Nagel F.
      • Felicke M.
      • Heilig G.
      • Grimmelt A.-C.
      • Pawlita I.
      • et al.
      Predictive values of antibodies against Pseudomonas aeruginosa in patients with cystic fibrosis one year after early eradication treatment.
      ]
      580.4–25.9One yr post first detection of PaELISA-ve <1:500

      +ve >1:500
      AP

      Ela

      ExoA

      cumulative
      43

      52

      48

      71
      94

      90

      97

      84
      82

      79

      91

      75
      71

      74

      74

      82
      Antibodies one yr post Pa detectionPa antibodies predict success of early eradication Rx at 1 yr
      Dōgru [
      • Dōgru D.
      • Pekcan S.
      • Yalçin E.
      • Özçelik
      • Kiper N.
      • Gurcan N.
      • et al.
      The role of serum Pseudomonas aeruginosa antibodies in the diagnosis and follow-up of cystic fibrosis.
      ]
      908–26.34 grps: negative Pa; intermittent; chronic; mucoidELISANRAP

      Ela

      ExoA
      84

      80

      80
      40

      44.4

      43.3
      100

      100

      100
      77.8

      80.0

      78.4
      1st visit and 2 yr follow upNo at 2 year follow up
      Anstead [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ]
      3031.0–12.01st lifetimeELISAfor E 15 ELISA:

      <1:500: −ve

      >1:500: +ve
      AP3

      Ela

      ExoA

      MC antigens
      Antigens produced by the Medical College of Wisconsin (MCW): ExoS + PopB, Cell lysate PAo1 and ExoA.


      PopB +ExoS

      PAO1
      Pooled across 4 time-points4 time-points: wks 0,22,46 and 70Seropositivity to AP and ExoA sig assoc. with increased risk of recurrent Pa during 60-week post eradication follow up

      (p = 0–003, p = 0.001)
      45

      52

      67

      69

      48

      60
      81

      78

      58

      65

      83

      72
      38

      38

      29

      33

      42

      35
      85

      86

      87

      89

      86

      88
      Cruz [
      • Cruz A.C.
      • Neves B.C.
      • Higa L.Y.S.
      • Folescu T.
      • Marques A.
      • Milagres L.G.
      Type III apparatus of Pseudomonas aeruginosa as a tool to diagnose pulmonary infection in cystic fibrosis patients.
      ]
      272.5–16.8N = 11 initial Pa −ve

      (N = 16 chronic Pa)
      Western Blot
      Antigen commercialized by Statens Serum Institute (Copenhagen, Denmark).
      ExoS ExoT PopB

      PopD
      NRNRNRNRAssessed at 4 time-pointsYes

      At mean 21 mo
      Douglas [
      • Douglas T.A.
      • Brennan S.
      • Berry L.
      • Winfield Wainwright C.E.
      • Grimwood K.
      • et al.
      Value of serology in predicting P.aeruginosa infection in young children with CF.
      ]
      1310.1–7.1Established persistent PaELISA<3.1 AU: −ve

      >3.1 AU: +ve



      1:256
      1:256 dilution of serum.
      St-Ag:1–17





      ExoA in house
      91
      Patients undergoing BAL.
      53
      Patients of the Australasian BAL Trial.


      82
      Patients undergoing BAL.
      93
      Patients of the Australasian BAL Trial.
      64
      Patients undergoing BAL.
      82
      Patients of the Australasian BAL Trial.


      57
      Patients undergoing BAL.
      52
      Patients of the Australasian BAL Trial.
      14
      Patients undergoing BAL.
      26
      Patients of the Australasian BAL Trial.


      11
      Patients undergoing BAL.
      19
      Patients of the Australasian BAL Trial.
      99
      Patients undergoing BAL.
      94
      Patients of the Australasian BAL Trial.


      98
      Patients undergoing BAL.
      98
      Patients of the Australasian BAL Trial.
      Baseline and exacerbationsNo
      Pressler [
      • Pressler T.
      • Karpati F.
      • Granström M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ]
      7910.3–73.23 grps: Free of Pa,

      Intermittent,

      and chronic Pa
      CIE



      ELISA
      >2 precipitins: +ve



      >110 EU < 4 yrs.

      >160 EU for >4 yrs.

      1.501 EU = value of 1
      St-Ag:1–17



      ExoA





      St-Ag: 1–17
      96



      93





      97
      89



      89





      83
      87



      86





      80
      97



      95





      98
      Baseline and 1 yearYes

      Seropositivity demonstrated risk factor for developing chronic Pa (p < 0.05)
      Ratjen [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]
      3751.0–52.03 grps: free of Pa, intermittent, and chronic PaELISA1000

      285

      300
      ExoA

      AP

      Ela

      3 combined
      72.0

      85.4

      76.2

      92.7
      97.5

      97.5

      97.5

      93.3
      95.9

      96.6

      96.2

      92.1
      80.8

      88.8

      83.2

      93.8
      Grp 1: baseline and after intervention therapy over last 8 yrs.

      Grp 2: patients who failed to clear Pa
      Decrease in titres against AP and ExoA in patients clearing Pa infection, increasing in patients failing to eradicate Pa
      Kappler [
      • Kappler M.
      • Kraxner A.
      • Reinhardt D.
      • Ganster B.
      • Griese M.
      • Lang T.
      Diagnostic and prognostic value of serum antibodies against Pseudomonas aeruginosa in cystic fibrosis.
      ]
      1830.4–413 grps: free of Pa, intermittent and chronic PaELISA<1:500: −ve

      >1:500: +ve
      3 combined86.195.697.180.23 monthly samples over 2 yrsSeropositivity strongly linked to Pa infection
      Proesmans [
      • Proesmans M.
      • Balinska-Miskiewicz Dupont L.
      • Bossuyt X.
      • Verhagen J.
      • Høiby N.
      • De Boeck K.
      Evaluating the Leeds criteria for Pseudomonas aeruginosa infection in a cystic fibrosis Centre.
      ]
      1621.3–52.84 grps: never, free of Pa, intermittent, chronicELISA17 AU for chronicitySt-Ag: 1–1788.096.092.193.91 serum sample outside resp. exacerbationSeropositivity sig high in chronic Pa
      Tramper-Stranders [
      • Tramper-Stranders G.A.
      • van der Ent C.K.
      • Slieker M.G.
      • Trehaggen-Lagro S.W.J.
      • Teding van Berkhout F.
      • Kimpen J.L.L.
      • et al.
      Diagnostic value of serological tests against Pseudomonas aeruginosa in a large cystic fibrosis population.
      ]
      2200.0–65.03 grps: not colonised, intermittent, and chronic PaELISA1:500 borderline +ve

      1:1250 + ve
      ExoA

      AP

      Ela
      79

      76

      87
      8944–83

      (range)
      85–97

      (range)
      At end of study periodYes

      Seropositivity demonstrated risk factor for developing chronic Pa
      Burns [
      • Burns J.L.
      • Gibson R.L.
      • McNamara S.
      • Yim D.
      • Emerson J.
      • Rosenfeld M.
      • et al.
      Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis.
      ]
      422.5–15.5‘initial’ isolation within study periodELISA



      Immunoblot
      NR



      1 band
      ExoA



      Whole-cell proteins
      78



      90
      NR



      NR
      NR



      NR
      NR



      NR
      Baseline and 3-monthly until 3rd bronchoscopyCombined culture and serology more effective in determining infection than culture alone
      Ramsey [
      • Ramsey B.W.
      • Wentz K.R.
      • Smith A.L.
      • et al.
      Predictive value of oropharyngeal cultures for identifying lower airway bacteria in cystic fibrosis patients.
      ]
      430.4–25.02 grps: expectorators; non-expectoratorsELISA>1:800ExoA75A

      46B
      80A

      93B
      90A

      83B
      57A

      70B
      During 2 yr study periodExoA good predictor of Pa colonisation, not sig when included with OP culture in regression model
      Pedersen [
      • Pedersen S.S.
      • Espersen F.
      • Høiby N.
      Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay.
      ]
      243 CF

      164 controls
      0.9–38

      0–64
      Chronic Pa + ve monthly sputum for 6 consecutive moELISA

      CIE
      ≥1.65 EU: +ve

      ≥2 precipitins: +ve
      St-Ag:1–17

      St-Ag:1–17
      90

      100
      100

      98
      NR

      NR
      NR

      NR
      During study periodSig increase in precipitating antibodies with duration of infection
      Döring [
      • Döring G.
      • Høiby N.
      Longitudinal study of immune response to Pseudomonas aeruginosa antigens in cystic fibrosis.
      ]
      108.0–29.0Chronic Pa + ve monthly sputum for 6 consecutive moCIE



      RIA
      ≥2 precipitins: +ve



      1:10

      1:10
      St-Ag:1–17



      AP

      Ela
      100



      100

      90
      NRNRNRMultiple samples over 10 yr periodFollowing antibody response: AP response 15 mo

      Ela response 11 mo

      St-Ag response 6 mo

      an increase in titres and precipitins was observed in all patients
      Høiby [
      • Høiby N.
      Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey.
      ]
      133>4 yrs.

      Range NR
      Patients colonised with Pa

      Chronic Pa: n = 38
      CIE≥2 precipitins: +veSt-Ag:1–1793NRNRNRAssessed at monthly clinic visitsNumber of precipitins increased sig in 1st year of colonisation (p < 0.01); high number of precipitins assoc. with poorer VC (p < 0.002)
      AP: alkaline protease; AU: area under the ROC curve; CIE: Crossed immune electrophoresis; ELA: elastase, ELISA: Enzyme-linked immunosorbent assay; EU: ELISA unit; Exo/ExoA/S/T: exotoxin A/S/T; grp/s: group/s; IgG: immunoglobulin G, MAg: multiple antigenic blend; MC: monoclonal antibody; mo: month/s; n: number; NPV: negative predictive value; NR: not reported; OP: oropharyngeal; p: p-value; pa: Pseudomonas aeruginosa; PopB: PopB protein; PopD; PopD protein; PPV: positive predictive value; RIA: radioimmunoassay; ROC: receiver operator characteristic curves; sig: significant; St-Ag: standard antigen; yr/s: year/s; VC: vital capacity; −ve: negative; +ve: positive; A: expectorators; B: non-expectorators.
      a Patients undergoing BAL.
      b Patients of the Australasian BAL Trial.
      c Antigens produced by the Medical College of Wisconsin (MCW): ExoS + PopB, Cell lysate PAo1 and ExoA.
      d Antigen commercialized by Statens Serum Institute (Copenhagen, Denmark).
      e 1:256 dilution of serum.

      3.3.2 Predictive validity

      3.3.2.1 Diagnosis of early infection

      A substantial overlap has been described between anti-Pa antibody titres in those patients who did subsequently acquire Pa and in those patients who did not [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ]. An immune response to Pa can occur earlier than culture positivity [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ,
      • Hayes D.
      • Farrell P.M.
      • Li Z.
      • West S.E.
      Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.
      ,
      • Tramper-Stranders G.A.
      • van der Ent C.K.
      • Slieker M.G.
      • Trehaggen-Lagro S.W.J.
      • Teding van Berkhout F.
      • Kimpen J.L.L.
      • et al.
      Diagnostic value of serological tests against Pseudomonas aeruginosa in a large cystic fibrosis population.
      ]. These data highlight the need for anti-Pa antibodies as a complementary test to cultures in the early stages of infection. Moreover, elevated levels of anti Pa antibodies (ExoA and AP) prior to AET may be associated with higher risk of Pa re-isolation during follow-up [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ] (Table 5).
      Table 5Anti-Pa antibodies at initial isolation of Pa infection.
      First author yearPatients (n)Age range (yrs)Pa time samplingMethodTitre cutoffAntigen% sens% specPPVNPVAntibodies assessed during Rx and at follow-up?Evidence of seropositivity/increase in titres
      Daines [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ]
      2610.0–12.01st lifetimeELISA<100: −ve

      >100: +ve
      ExoA

      AP

      Ela

      3/3
      54.4

      35.3

      32.4

      14.7
      75.9

      75.0

      84.2

      95.6
      32.7

      23.3

      30.6

      41.7
      88.6

      84.3

      85.3

      83.9
      Baseline and follow up

      (annual samples over 3 yr period)
      Yes at 6 mo

      No at 12 mo
      Anstead [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ]
      3031.0–12.01st lifetimeELISA<1:500: −ve

      >1:500: +ve


      AP3

      Ela

      ExoA

      MC antigens*

      PopB +ExoS

      PAO1
      Pooled across 4 time-points4 time-points: wks 0,22,46 and 70Seropositivity to AP and ExoA sig assoc. with increased risk of recurrent Pa during 60-week post eradication follow up (p = 0–003, p = 0.001)
      45

      52

      67

      69

      48

      60
      81

      78

      58

      65

      83

      72
      38

      38

      29

      33

      42

      35
      85

      86

      87

      89

      86

      88
      Ratjen [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ]
      3751.0–52.03 grps: free of Pa, intermittent and chronic PaELISA1000

      285

      300
      ExoA

      AP

      Ela

      3 combined
      72.0

      85.4

      76.2

      92.7
      97.5

      97.5

      97.5

      93.3
      95.9

      96.6

      96.2

      92.1
      80.8

      88.8

      83.2

      93.8
      Grp 1: baseline and after intervention therapy over last 8 yrs.

      Grp 2: patients who failed to clear Pa
      Decrease in titres against AP and ExoA in patients clearing Pa infection, increasing in patients failing to eradicate Pa
      Kappler [
      • Kappler M.
      • Kraxner A.
      • Reinhardt D.
      • Ganster B.
      • Griese M.
      • Lang T.
      Diagnostic and prognostic value of serum antibodies against Pseudomonas aeruginosa in cystic fibrosis.
      ]
      1830.4–413 grps: free of Pa, intermittent and chronic PaELISA<1:500: −ve

      >1:500: +ve
      3 combined86.195.697.180.23 monthly samples over 2 yrsSeropositivity strongly linked to Pa colonisation
      Burns [
      • Burns J.L.
      • Gibson R.L.
      • McNamara S.
      • Yim D.
      • Emerson J.
      • Rosenfeld M.
      • et al.
      Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis.
      ]
      422.5–15.5‘initial’ isolation within study periodELISA



      Immunoblot
      >1:800



      1
      ExoA



      Whole-cell proteins
      30



      50
      NRNRNRBaseline and 3-monthly until 3rd bronchoscopyCombined culture and serology more effective in determining infection than culture alone
      AP: alkaline protease; AUROC: area under the ROC curve; CIE: Crossed immune electrophoresis; Ela: elastase, ELISA: Enzyme-linked immunosorbent assay; EU: ELISA unit; Exo/ExoA/S: exotoxin A/S; grp: group; IgG: immunoglobulin G, MAg: multiple antigenic blend; MC: monoclonal antibody; mo: month/s; n: number; NPV: negative predictive value; NR: not reported; Pa: Pseudomonas aeruginosa; PopB: PopB protein; PopD; PopD protein; PPV: positive predictive value; RIA: radioimmunoassay; ROC: receiver operator characteristic curves; sens: sensitivity; Spec: specificity; St-Ag: standard antigen; yrs.: years; −ve: negative; +ve: positive.

      3.3.2.2 Predictive value of anti-Pa titres following AET

      There is a slight discordance between culture results and serology in the post-treatment period. “Culture decline” (i.e. culture conversion from Pa-positive to Pa-negative) shows a different trend in comparison with “serology decline” (i.e. conversion from Pa-antibody positive to Pa-antibody negative) [
      • Tramper-Stranders G.A.
      • van der Ent C.K.
      • Slieker M.G.
      • Trehaggen-Lagro S.W.J.
      • Teding van Berkhout F.
      • Kimpen J.L.L.
      • et al.
      Diagnostic value of serological tests against Pseudomonas aeruginosa in a large cystic fibrosis population.
      ].
      Nevertheless, a decline in anti-Pa antibody titre over one year can predict success of AET [
      • Kappler M.
      • Nagel F.
      • Felicke M.
      • Heilig G.
      • Grimmelt A.-C.
      • Pawlita I.
      • et al.
      Predictive values of antibodies against Pseudomonas aeruginosa in patients with cystic fibrosis one year after early eradication treatment.
      ]. Similarly, an increase in titres over time is described in AET failure [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ,
      • Pressler T.
      • Karpati F.
      • Granström M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ,
      • Dolce D.
      • Ravenni N.
      • Mergni G.
      • Braggion C.
      • Campana S.
      • Taccetti G.
      Anti-P.aeruginosa antibodies and microbiological outcome in not chronically infected patients.
      ] or evolution towards chronic infection [
      • Pressler T.
      • Karpati F.
      • Granström M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ] (see Table 5).

      3.3.2.3 Cost

      The costs of commercial kits may vary from country to country as will laboratory overheads (e.g. staff costs, utilities, etc.). A standardised method of Pa antibody testing would be required. This would either be the same kit/method in several labs or one test in a single central laboratory. The latter would then require additional costs for transport of serum samples.

      4. Discussion

      Analysis of Pa microbiological status used in literature on patients with initial or chronic Pa infection, as well as eradication clinical trials, has revealed great variability of definitions.

      4.1 Respiratory sampling

      The methods used for the definition of Pa microbiological status of CF patients, may be subject to various technical issues. Diagnosis of infection is based on respiratory sample analysis, usually performed on expectorated or induced sputum or on OP swabs [
      • ECFS-CTNSOP 2.5/002
      ,
      • ECFS-CTNSOP 2.4/002
      ,
      • ECFS-CTNSOP 2.4/001
      ,
      • Mogayzel P.J.
      • Naureckas E.T.
      • Robinson K.A.
      • Brady C.
      • Guill M.
      • Lahiri T.
      • et al.
      Cystic Fibrosis Foundation pulmonary guideline. Pharmacologic approaches to prevention and eradication of initial Pseudomonas aeruginosa infection.
      ] Therefore, sampling methods strongly influence the sensitivity of infection diagnosis. Any definition of first lifetime/early/new as well as chronic Pa infection should clearly describe sampling frequency. Sample types, collection frequency and correct identification of the pathogen can be standardised by the application of ECFS-CTN SOPs which must be adopted by all CTN centres.
      A minimum standard of four respiratory samples per annum must be applied in order to produce a homogenous study population. This is of the utmost importance as chronic infection may be mistakenly labelled as ‘intermittent’ as currently defined by Leeds Criteria [
      • Lee T.W.R.
      • Brownlee K.G.
      • Conway S.P.
      • Denton M.
      • Littlewood J.M.
      Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients.
      ]. Indeed, it has been reported that over half of patients categorised as ‘intermittent’ using Leeds criteria may actually have underlying chronic Pa infection [
      • Lee T.W.R.
      • Brownlee K.G.
      • Conway S.P.
      • Denton M.
      • Littlewood J.M.
      Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients.
      ]. Moreover, mucoidy is an important characteristic to consider in the definition of chronic infection as Heltshe [
      • Heltshe S.L.
      • Khan U.
      • Beckett V.
      • Baines A.
      • Emerson J.
      • Sanders D.B.
      • et al.
      Longitudinal development of initial, chronic and mucoid Pseudomonas aeruginosa infection in young children with cystic fibrosis.
      ] demonstrated that the majority of children (87%) developed mucoid infection before meeting the ‘Leeds’ definition of chronic infection.
      The sensitivity and the positive predictive value of OP cultures in detecting Pa infection is low when compared to BAL [
      • De Boeck K.
      • Southern K.
      The early cystic fibrosis years.
      ,
      • Rosenfeld M.
      • Emerson J.
      • Accurso F.
      • Armstrong D.
      • Castile R.
      • Grimwood K.
      • et al.
      Diagnostic accuracy of oropharyngeal cultures in infants and young children with cystic fibrosis.
      ,
      • Ramsey B.W.
      • Wentz K.R.
      • Smith A.L.
      • et al.
      Predictive value of oropharyngeal cultures for identifying lower airway bacteria in cystic fibrosis patients.
      ]. This limited sensitivity might result in missed detection of initial Pa infection. To increase sensitivity of detection in non-expectorating paediatric patients with CF, induced sputum samples have recently been proved to provide additional microbiological information in comparison to OP swabs [
      • Ho S.A.
      • Ball R.
      • Morrison L.J.
      • Brownlee K.G.
      • Conway S.P.
      Clinical value of obtaining sputum and cough swab samples following inhaled hypertonic saline in children with cystic fibrosis.
      ] and very importantly BAL samples do not seem to be more sensitive than induced sputum in this population [
      • Ronchetti K.
      • Tame J.D.
      • Paisey C.
      • Thia L.P.
      • Doull I.
      • Howe R.
      • et al.
      The CF-sputum induction trial (CF-SpIT) to assess lower airway bacterial sampling in young children with cystic fibrosis: a prospective internally controlled interventional trial.
      ,
      • Blau H.
      • Linnane B.
      • Carzino R.
      • Tannenbaum E.L.
      • Skoric B.
      • Robinson P.J.
      • et al.
      Induced sputum compared to bronchoalveolar lavage in young, non-expectorating cystic fibrosis children.
      ]. There is therefore no clear evidence to support the routine use of BAL for the diagnosis and management of pulmonary infection in children with CF [
      • Mogayzel P.J.
      • Naureckas E.T.
      • Robinson K.A.
      • Brady C.
      • Guill M.
      • Lahiri T.
      • et al.
      Cystic Fibrosis Foundation pulmonary guideline. Pharmacologic approaches to prevention and eradication of initial Pseudomonas aeruginosa infection.
      ,
      • Jain K.
      • Wainwright C.
      • Smyth A.R.
      Bronchoscopy-guided antimicrobial therapy for cystic fibrosis.
      ]. Moreover, Pa qPCR might improve sensitivity of infection diagnosis on culture negative OP swabs [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ,
      • Le Gall F.
      • Le Berre R.
      • Rosec S.
      • Hardy J.
      • Gouriou S.
      • Boisramé-Gastrin S.
      • et al.
      Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.
      ,
      • Héry-Arnaud G.
      • Nowak E.
      • Caillon J.
      • David V.
      • Dirou A.
      • Revert K.
      • et al.
      Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.
      ]. However, this will require further comparative validation studies using BAL samples [
      • Waters V.
      • Grimwood K.
      Defining chronic Pseudomonas aeruginosa infection in cystic fibrosis.
      ].

      4.2 Anti-Pa antibodies

      Anti-Pa antibodies have demonstrated success as a useful adjunct for assessment of early eradication therapy [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ] and to distinguish intermittent from chronic Pa [
      • Kappler M.
      • Kraxner A.
      • Reinhardt D.
      • Ganster B.
      • Griese M.
      • Lang T.
      Diagnostic and prognostic value of serum antibodies against Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Brett M.M.
      • Ghoneim A.T.
      • Littlewood J.M.
      Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis.
      ]. Daines [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ] was the first to report the utility of Pa serology in predicting subsequent first isolation from cultures of Pa negative patients [
      • Daines C.
      • VanDeVanter D.
      • Khan U.
      • Emerson J.
      • Heltshe S.
      • McNamara S.
      • et al.
      Serology as a diagnostic tool for predicting initial Pseudomonas aeruginosa acquisition in children with cystic fibrosis.
      ], and Hayes [
      • Hayes D.
      • Farrell P.M.
      • Li Z.
      • West S.E.
      Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.
      ], Ratjen [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ] and Pressler [
      • Pressler T.
      • Karpati F.
      • Granström M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ] have all reported high diagnostic accuracy of serology [
      • Ratjen F.
      • Walter H.
      • Haug M.
      • Meisner C.
      • Grasemann
      • Döring G.
      Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis.
      ,
      • Hayes D.
      • Farrell P.M.
      • Li Z.
      • West S.E.
      Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.
      ,
      • Pressler T.
      • Karpati F.
      • Granström M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ]. The presence of raised titres of anti-Pa antibodies (anti-alkaline protease and anti-Exotoxin A) before AET is associated with an increased risk of Pa recurrence [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ]. Therefore, studies excluding participants with raised anti-Pa antibodies might be expected to achieve higher eradication rates and furthermore prevent enrolment of ineligible patients [
      • Langton Hewer S.C.
      • Smyth A.R.
      Antibiotic strategies for eradicating Pseudomonas aeruginosa in people with cystic fibrosis.
      ].
      The use of serology raises many problems regarding the interpretation and the validity of anti-Pa antibodies, both in the early stages of infection and in monitoring AET. There is a need for a standardised method of Pa antibody testing. This would either require the same kit/method to be used in several labs or testing performed in a central laboratory with the same kit/method. The latter requires transport of blood samples, potentially over long distances.
      The two kits mostly used in Europe for the detection of anti-Pa antibodies are the Pseudomonas -CF-IgG ELISA kit by Statens Serum Institut, Denmark, and the Mediagnost anti-Pseudomonas aeruginosa IgG Enzyme Immuno Assay E15, Reutlingen, Germany. Since the antigens used (for example, ExoA, Ela, AP and St-Ag: 1–17) in different anti-Pa antibody detection kits are not consistent, their use may give variable results. In the absence of Pa infection, the optimum time interval for repeated checking of Pa antibody dynamics is not known or standardised. There is also inconsistency in the levels of anti-Pa antibody response detected by different kits as patients move between the different phases of Pa infection e.g. from time of initial Pa infection to chronic infection (Table 5).
      A decrease in anti-Pa antibody titres after AET has been described. However, there may not be an immediate decline in antibody titres even after successful eradication because of the persistence of an antigen stimulus for a long period. This is linked to Pa antigens in obstructed airways despite negative Pa cultures. This makes anti-Pa antibody titres in the context of eradication definition and “next infection” or “intermittent infection” difficult to interpret. Host response to initial phases of Pa infection is variable and the chronology of the immune response to Pa antigenic determinants is not completely clear. The immune response to the virulence factors produced by Pa (Exotoxin A, Elastase and alkaline protease) seems to occur over a longer period in comparison with St-Ag, which may usually be expressed earlier [
      • Mauch R.M.
      • Levy C.E.
      Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis as a diagnostic tool: a systematic review.
      ,
      • Döring G.
      • Høiby N.
      Longitudinal study of immune response to Pseudomonas aeruginosa antigens in cystic fibrosis.
      ].
      Longitudinal immunological studies would therefore need to be undertaken to assess the utility of anti-Pa antibodies in monitoring response to repeated eradication treatment.
      Cut-off values in the initial phases of Pa infection need to be determined by receiver operating characteristic (ROC) curve analyses [
      • Tramper-Stranders G.A.
      • van der Ent C.K.
      • Slieker M.G.
      • Trehaggen-Lagro S.W.J.
      • Teding van Berkhout F.
      • Kimpen J.L.L.
      • et al.
      Diagnostic value of serological tests against Pseudomonas aeruginosa in a large cystic fibrosis population.
      ]. These have only been agreed with certainty for chronic Pa infection. All these considerations highlight that unless sampling and laboratory issues are properly addressed it will be difficult to apply definitions in a robust fashion. This points to the need to establish a central laboratory capable of providing standardised anti-Pa antibody testing for all trial centres or the provision of the same standardised method for testing across a network of laboratories. No specific training requirements are needed if a central reference laboratory is used for Pa antibody testing.

      4.3 Considerations for proposing a definition regarding initial (first lifetime/early/new) Pa infection to be used in clinical trials

      Traditional methods of Pa identification reliant upon microbiology present inherent difficulties. Decision regarding the use of serology in defining Pa microbiological status introduces a greater level of complexity in terms of patient enrolment and laboratory testing. Therefore, in the initial phase of Pa infection (Table 2), serology should be considered an adjunct to, rather than a replacement for cultures.
      Thus, the proposed definitions to describe initial (first lifetime/early/new) Pa infection in CF patients for application in clinical trials are:
      “First lifetime documented Pa in a respiratory culture with anti-Pa antibody titres below the cut-off for chronic infection” (level of evidence 1+, grade of recommendation A) OR, if the patient has previously grown Pa “New Pa detection in a respiratory culture after at least four negative cultures regularly (quarterly) performed over one year, with anti-Pa antibody titres below the cut-off for chronic infection” (level of evidence 2+, grade of recommendation C).

      4.4 Considerations for proposing a definition of Pa eradication to be used in clinical trials

      ‘Successful’ eradication may be difficult to define as it requires ‘negative’ cultures over a certain period of time, but its appropriate length is unknown. The Artimino Consensus Definition (11), “repeated Pa negative respiratory samples (at least three) in the six months after completion of the eradication regimen, in conjunction with negative serum anti-Pa antibody titres”, had a clear time frame and was not solely reliant on cultures [
      • Döring G.
      • Høiby N.
      Consensus report: early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ]. It also clarified the frequency of sampling required. However, it included the requirement for standardised antibody testing and the need to collect blood samples as previously highlighted.
      Other purely culture-based definitions, such as the UK CF Trust Definition (2004) “at least three consecutive negative respiratory cultures spread over a six-month period”, can be simple, have clear time frames, defined frequency of sampling and the absence of problems associated with antibody testing [
      • The UK Cystic Fibrosis Trust Infection Control Group
      Pseudomonas aeruginosa infection in people with cystic fibrosis.
      ]. However, these are solely dependent on culture results and therefore may miss some therapeutic failures, particularly in non-expectorating patients.
      Despite both definitions of eradication mentioned above being based on an observation period of six months, the concept of “sustained eradication” has been introduced to indicate that particular category of patients who maintained Pa negative cultures for 12 months after AET [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ]. Those “sustained eradicators” proved to have a reduced risk (74%) of developing chronic infection with less anti Pa antibiotic usage and a reduced risk (57%) of mucoid Pa phenotype during a 5-year observation period, compared with non-sustained eradicators. During the same period of time, no statistically significant differences were observed with respect to other clinical parameters (lung function decline and pulmonary exacerbation) [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ].
      For use in clinical trials, the definition of Pa eradication, must therefore include both culture and PA serology.
      “Pa negative cultures for 12 months after treatment (having performed quarterly cultures) in conjunction with a titre of anti-Pa antibody below the cut-off for chronic infection one year after treatment.” [
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ] (level of evidence 2+, grade of recommendation C).
      Serological studies performed one year after treatment are able to predict AET success or the evolution towards chronicity (Table 4).
      Future studies should evaluate anti-Pa antibody dynamics over time as well as the molecular characteristics of bacterial isolates in order to distinguish new infections from re-colonisation with strains, which have been suppressed but not eradicated. Molecular genotyping, such as that described in the study by Kalferstova et al. [
      • Kalferstova L.
      • Villimovska Dedeckova K.
      • Antuskova M.
      • Melter O.
      • Drevinek P.
      How and why to monitor Pseudomonas aeruginosa infections in the long term at a cystic fibrosis Centre.
      ], should be conducted to examine all Pa isolates obtained after early eradication treatment, to evaluate the recurrence of the same strain and the possible evolution towards chronic infection [
      • Kalferstova L.
      • Villimovska Dedeckova K.
      • Antuskova M.
      • Melter O.
      • Drevinek P.
      How and why to monitor Pseudomonas aeruginosa infections in the long term at a cystic fibrosis Centre.
      ]. Studies on phenotypic and genotypic characteristics of bacterial strains responsible for initial infection and associated with treatment failure should be conducted. The molecular mechanisms of bacterial adaptation to the CF airways need more in-depth studies and the role of the paranasal sinuses in the development of chronic Pa infection should also be clarified. Paranasal sinuses are considered a reservoir for lung infection, therefore longitudinal studies aimed at clarifying the clinical relevance of paranasal sinuses' Pa microbiological status in determining chronic lung infection are necessary. Clinical trials to assess the efficacy of paranasal sinuses' medical/surgical treatments on the evolution towards chronicity also need to be performed [
      • Aanaes K.
      • Eickhardt S.
      • Johansen H.K.
      • von Buchwald C.
      • Skov M.
      • Høiby N.
      • et al.
      Sinus biofilms in patients with cystic fibrosis: is adjusted eradication therapy needed?.
      ,
      • Rudkjøbing V.B.
      • Aanaes K.
      • Wolff T.Y.
      • von Buchwald C.
      • Johansen H.K.
      • Thomsen T.R.
      An exploratory study of microbial diversity in sinus infections of cystic fibrosis patients by molecular methods.
      ,
      • Aanaes K.
      • Johansen H.K.
      • Skov M.
      • Buchvald F.F.
      • Hjuler T.
      • Pressler T.
      • et al.
      Clinical effects of sinus surgery and adjuvant therapy in cystic fibrosis patients-can chronic lung infections be postponed?.
      ].
      Moreover, the role of lung damage in predisposing the patients to bacterial infection and the effect of modulators in reducing airways' receptivity to Pa infection require better assessment.
      Future studies should also focus on diagnostic accuracy of induced sputum in comparison with other sampling methods in non-expectorating patients. Application of non-routinely used molecular techniques, such as qPCR, must also be incentivized, as this might help to discriminate between intermittent and chronic Pa infections [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ,
      • Le Gall F.
      • Le Berre R.
      • Rosec S.
      • Hardy J.
      • Gouriou S.
      • Boisramé-Gastrin S.
      • et al.
      Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.
      ,
      • Héry-Arnaud G.
      • Nowak E.
      • Caillon J.
      • David V.
      • Dirou A.
      • Revert K.
      • et al.
      Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.
      ].

      4.5 Considerations for proposing a definition of chronic infection to be used in clinical trials

      Times to chronic Pa infection and to first appearance of mucoid Pa phenotype infection have been considered as clinically relevant indicators [
      • Mayer-Hamblett N.
      • Retsch-Bogart G.
      • Kloster M.
      • Accurso F.
      • Rosenfeld M.
      • Albers G.
      • et al.
      Azithromycin for early Pseudomonas infection in cystic fibrosis. The OPTIMIZE randomized trial.
      ,
      • Mayer-Hamblett N.
      • Kloster M.
      • Rosenfeld M.
      • Gibson R.L.
      • Retsch-Bogart G.Z.
      • Emerson J.
      • et al.
      Impact of sustained eradication of new Pseudomonas aeruginosa infection on long-term outcomes in cystic fibrosis.
      ].
      In this case, the follow-up period would be years rather than months. Historical data from Copenhagen suggests that AET can delay progression to chronic infection by at least 3.5 years [
      • Frederiksen B.
      • Koch C.
      • Høiby N.
      Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis.
      ,
      • Frederiksen B.
      • Koch C.
      • Høiby N.
      Changing epidemiology of Pseudomonas aeruginosa infection in Danish cystic fibrosis patients.
      ].
      There are a number of published definitions of chronic Pa infection. For example, the Copenhagen definition requires the isolation of Pa from sputum or respiratory samples on two or more occasions extending over six months or a shorter period if accompanied by a substantial rise in anti-Pa antibodies [
      • Aanaes K.
      • Johansen H.K.
      • Skov M.
      • Buchvald F.F.
      • Hjuler T.
      • Pressler T.
      • et al.
      Clinical effects of sinus surgery and adjuvant therapy in cystic fibrosis patients-can chronic lung infections be postponed?.
      ]. However, it requires a frequency of respiratory sampling (e.g. monthly) which many CTN centres do not currently perform. The requirement for antibody testing has previously been discussed. It would also require a clear definition of what is meant by a ‘substantial rise’ [].
      As another example, the Leeds Criteria definition does not require systematic sampling as practiced in Copenhagen or access to blood samples and antibody testing. However, the frequency of sampling required is not made clear and it may miss some chronically-infected patients, particularly if the majority of samples collected are OP swabs. Therefore, some chronically Pa infected patients are erroneously categorised by Leeds Criteria as ‘Intermittent’. The Leeds Criteria also make no allowance for raised anti-Pa antibodies or the appearance of the mucoid phenotype of Pa, both of which may indicate the transition from early colonisation to chronic infection. At present the UK and the Cystic Fibrosis Foundation Registries accept the Leeds Criteria definition [
      • Lee T.W.R.
      • Brownlee K.G.
      • Conway S.P.
      • Denton M.
      • Littlewood J.M.
      Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients.
      ,
      • The UK Cystic Fibrosis Trust Infection Control Group
      Pseudomonas aeruginosa infection in people with cystic fibrosis.
      ]. As there is a good correlation between chronic Pa infection and anti Pa-antibodies, the ECFS Registry accepts both the Leeds Criteria definition as well as definitions based on cultures associated with significantly raised anti-Pa antibodies according to local laboratory results [].
      Regarding chronic infection, the accepted definition appears to be the Leeds definition [
      • Lee T.W.R.
      • Brownlee K.G.
      • Conway S.P.
      • Denton M.
      • Littlewood J.M.
      Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients.
      ] (level of evidence 2++, grade of recommendation B). When the Leeds criteria for chronic infection are ambiguous, for instance, due to an insufficient number of samples to assess the immediate Pa status or when a patient has a questionable long-term history of intermittent Pa status, only patients with repeated culture positivity for Pa and a titre of anti-Pa antibodies above the cut-off for chronic infection should be included in the clinical trials.
      It is often difficult to distinguish the intermittent from the chronic phase. Therefore, as stated above, future studies should aim to evaluate phenotypic and genotypic characteristics of bacterial strains during their transition to chronic status [
      • Mayer-Hamblett N.
      • Ramsey B.W.
      • Kulasekara H.D.
      • Wolter D.J.
      • et al.
      Pseudomonas aeruginosa phenotypes associated with eradication failure in children with cystic fibrosis.
      ]. New methods (qPCR) could be useful in discriminating between intermittent and chronic Pa infection [
      • Boutin S.
      • Weitnauer M.
      • Hassel S.
      • Graeber S.Y.
      • Stahl M.
      • Dittrich A.S.
      • et al.
      One-time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.
      ,
      • Le Gall F.
      • Le Berre R.
      • Rosec S.
      • Hardy J.
      • Gouriou S.
      • Boisramé-Gastrin S.
      • et al.
      Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.
      ,
      • Héry-Arnaud G.
      • Nowak E.
      • Caillon J.
      • David V.
      • Dirou A.
      • Revert K.
      • et al.
      Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.
      ]. Recently, Jonckheere et al. [
      • Jonckheere L.
      • Schelstraete P.
      • Van Simaey L.
      • Van Braeckel E.
      • Willikens J.
      • Van Daele S.
      • et al.
      Establishing the diagnosis of chronic colonisation with Pseudomonas aeruginosa of cystic fibrosis patients: comparison of the European consensus criteria with genotyping of P.aeruginosa isolates.
      ] reported that genotyping was more sensitive than European Consensus Criteria in establishing the diagnosis of chronic infection [
      • Jonckheere L.
      • Schelstraete P.
      • Van Simaey L.
      • Van Braeckel E.
      • Willikens J.
      • Van Daele S.
      • et al.
      Establishing the diagnosis of chronic colonisation with Pseudomonas aeruginosa of cystic fibrosis patients: comparison of the European consensus criteria with genotyping of P.aeruginosa isolates.
      ].

      4.6 Advocacy for Pa serology

      Anti-Pa serology relating to first lifetime/early/new Pa infection and successful Pa eradication introduces a greater level of complexity in terms of patient enrolment, laboratory testing and outcome measure. Currently, the optimum method of antibody testing, and the interpretation of the antibody titres in the initial phase of infection are not clear. Specific research into these topics should be conducted.
      The follow-up of patients who had undergone AET may require repeated blood sampling to observe antibodies' dynamics and to ensure their correct interpretation. Therefore, an agreed approach regarding frequency of blood sampling and interpretation of results has to be established. On balance, th