Tacjana Pressler *, Birgitte Frederiksen, Marianne Skov, Peter Garred, Christian Koch, Niels Høiby

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1 Journal of Cystic Fibrosis 5 (2006) Early rise of anti-pseudomonas antibodies and a mucoid phenotype of Pseudomonas aeruginosa are risk factors for development of chronic lung infection A case control study Tacjana Pressler *, Birgitte Frederiksen, Marianne Skov, Peter Garred, Christian Koch, Niels Høiby Rigshospitalet, The Copenhagen Cystic Fibrosis Centre, Denmark Received 28 July 2005; received in revised form 10 October 2005; accepted 11 November 2005 Available online 10 January 2006 Abstract P. aeruginosa is the most significant pathogen in CF lung disease. Chronic infection is preceded by a period of intermittent colonization. Early aggressive antimicrobial treatment at initial detection of P. aeruginosa in lower respiratory tract (LRT) secretions can prevent transition to chronic infection in approximately 80% of the patients, while the rest progress to chronic infection in spite of treatment. To analyze risk factors for development of chronic infection, a cohort of 89 CF patients free of chronic infection at the study period start was followed for 10 years. 28 of the patients (study group) developed chronic infection in spite of early treatment and 28 age-matched patients who did not (controls) were included in the analysis. During the 3 years period prior to onset of chronic infection, P. aeruginosa-positive cultures were more frequent in the study group than in the controls (2.2 vs. 0.5 per year, p <0.0001). Growth of mucoid strains of P. aeruginosa was more frequent in study group than in controls (11.5% vs. 0%, p < ). Most important, specific anti-pseudomonal IgG serum antibodies were significantly higher in the study group than in controls (0.98 Elisa Units vs. 0.53, p =0.04) already 3 years prior to onset of chronic infection and increased 0.44 EU pr year in the study group but remained at the initial level in the control group ( p < 0.005). Occurrence of Aspergillus-positive cultures were significantly more frequent in the study group than in controls ( p =0.01). The strongest risk factor for development of chronic P. aeruginosa infection was increasing levels of specific anti-pseudomonal antibodies, specifically of IgG1 and IgG4 subclass and total anti-pseudomonas IgG, 3 years prior to onset of chronic infection, with odds ratio (OR) 8.9, 7.7 and 7.4, respectively ( p <0.005), and growth of mucoid P. aeruginosa strains with OR of 7.4, p =0.006). Occurrence of Aspergillus was also a risk factor for developing chronic P. aeruginosa infection with the OR of 4.7 ( p =0.008). D 2005 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Specific anti-pseudomonal antibodies; Risk factors; Mucoid P. aeruginosa; Chronic P. aeruginosa infection; Cystic fibrosis 1. Introduction * Corresponding author. Tel.: ; fax: address: tania@rh.dk (T. Pressler). The bronchopulmonary disease in cystic fibrosis (CF) evolves predominantly as a lower respiratory infection leading to progressive obstructive lung disease [1]. The lungs of CF infant are nearly normal at birth but subsequently there is rapid onset of infection and inflammation. Inflammation begins at an early age and progresses throughout life, ultimately leading to lung tissue destruction [1,2 6]. The initial pathogens are often Staphylococcus aureus and Haemophilus influenzae but bronchoscopic studies have confirmed that P. aeruginosa infection may also be an early event [7]. Chronic pulmonary infection with P. aeruginosa develops in most patients with CF; by adulthood 80% of patients are infected in most CF centers and chronic P. aeruginosa infection is the primary cause of increased morbidity and mortality in CF [8 11] /$ - see front matter D 2005 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi: /j.jcf

2 10 T. Pressler et al. / Journal of Cystic Fibrosis 5 (2006) 9 15 Chronic infection is, in most patients, preceded by a stage of intermittent colonization for an average duration of 12 months [12]. P. aeruginosa strains found early in CF patients resemble those in the environment being relatively sensitive to antibiotics, having smooth lipopolysaccharide (LPS) containing O-chains and being of non-mucoid phenotype. The bacterial population adaptates to the environments within the respiratory and conductive zones of the lungs of CF patients [13]. The P. aeruginosa phenotype becomes LPSrough and highly mucoid due to production of alginate and in consequence the production of biofilm which protects the microcolonies from action of antibiotics and host immune response [13,14 16]. Attempts have been made to prevent or postpone chronic bronchopulmonary P. aeruginosa infection in CF [17 22] and the results are very encouraging. In a Danish study, after an observation period of 44 months only 16% of the patients became chronically infected with P. aeruginosa compared with 72% of historical controls [18]. Since 1989 early intervention at the time of first acquisition of P. aeruginosa and again each time P. aeruginosa was isolated from the airway secretions has been a standard treatment in Copenhagen CF centre. The purpose of the present case control study is to define the possible risk factors for development of chronic P. aeruginosa infection despite vigorous antimicrobial treatment [18]. 2. Materials and methods 2.1. Study design In January 1990, 225 CF patients were followed at Copenhagen CF centre. 89 patients were without any chronic lower respiratory tract infection. CF was diagnosed with standard criteria [23]. All patients were controlled on a regular monthly basis in the out-patient clinic. At each visit the clinical condition, pulmonary function and sputum bacteriology were examined and have thus been recorded prospectively since Serum samples were collected at intervals of 3 4 months and evaluated for anti-p. aeruginosa antibody levels [24,25]. All patients have since 1989 been treated according to the protocol of early eradication of initial colonization with P. aeruginosa. The strategy employed is based upon regular monthly control, which includes sampling of sputum or nasolaryngeal secretions for microscopy and culture and with treatment whenever P. aeruginosa is identified using a combination of inhaled colistin and oral ciprofloxacin [18]. In the 10 years period, 28 of 89 patients included in this study developed a chronic infection with P. aeruginosa. Chronic P. aeruginosa infection was defined as persistent presence of P. aeruginosa in sputum obtained by expectoration or nasolaryngeal suction for 6 consecutive months or less when levels of precipitating anti-pseudomonas antibodies were 2 precipitins [24]. An age-matched control group of 28 CF patients who, at the end of the study period, were still free of chronic infection were randomly selected from a study population. The patients with the age closest to the age of index patients were selected. Study period for each index patient and the matched control patient was a period of three years prior to development of chronic P. aeruginosa infection for the index patient. CF genotype, MBL genotype, sex, forced expiratory volume in one second (FEV1) [26], weight and height expressed as BMI z-score [27], occurrence of S. aureus, Aspergillus fumigatus and P. aeruginosa in lower respiratory tract in whole study period were recorded from the medical records Bacteriology Sputum or lower respiratory tract secretion obtained by endo-laryngeal suction was processed for Gram-staining and microscopy and culture as detailed previously [28]. P. aeruginosa and other microorganisms were identified as previously described [28] Specific anti-pseudomonas antibodies Specific IgG anti-pseudomonas antibodies were measured at least once a year by crossed immunoelectrophoresis [29]. Furthermore, the Pseudomonas specific IgG and IgG subclass 1 4 were assayed by ELISA [25]. Water soluble antigens were produced by sonication from each of 17 serotypes of the international antigen typing scheme of P. aeruginosa, equal volumes of each were mixed and designated standard antigen (St-Ag), protein concentration 9 g/l. The assay was calibrated by using a standard of pooled serum from 10 chronic infected patients with CF. Results were expressed as ELISA units (EU) and calculated in relation to the standard, which was given an arbitrary value of 100 EU [25]. In order to establish normal values for the ELISA assays sera from 143 healthy subjects (mean age 10.1 years; range years) and 64 CF patients (mean age 9.4 years; range years) without history of P. aeruginosa positive LRT secretions were investigated Mannose-binding lectin (MBL2) genotypes MBL2 genotypes were investigated by a PCR sequence specific priming method [30]. Structural variant alleles in codons 52 (D), 54 (B) and 57 (C), respectively, were pooled into an O allele, because of the similar reducing effect on the mannose-binding lectin serum concentration. The normal allele is named A Statistics Comparison between case and controls were made using Wilcoxon matched pairs signed rank sum test for

3 T. Pressler et al. / Journal of Cystic Fibrosis 5 (2006) continuous data and Fisher exact test for categorical data. A stepwise logistic regression model was used to determine risk factor for acquisition of chronic P. aeruginosa infection. A p value 0.05 was considered statistically significant. For significant variables on stepwise logistic regression, the odds ratio (OR) with 95% confidence intervals were calculated. 3. Results 3.1. Patients The gender, CFTR mutations and MBL2 genotypes are shown in Table 1b. No significant differences were found, however, 12 (43%) of 28 index patients were carriers of MBL variant alleles compared to 6 (21%) of 28 controls (n.s). The nutritional status of both groups was not different from a healthy population [27] (Table 1a). The lung function (average of all measurements in a one year) was significantly lower in the study group 3 years prior to onset of chronic infection, median FEV1 was 75% of predicted in study group versus 87% in controls ( p =0.04). Lung function improved in the study group and at onset of chronic infection FEV1 was 82% in the study group versus 87% in the controls (n.s) Bacteriology The median age at first isolate of P. aeruginosa was 5.1 and 5.9 years, respectively, in study group and controls (n.s.). The number of P. aeruginosa positive culture was summed up for all study patients, in the three years period prior to the start of chronic infection for the case group. The P. aeruginosa-positive cultures were more frequent in the study group, median 2.2 (22% of all Table 2 Levels of specific anti-pseudomonas antibodies Mean Mean+1SD Median 90th percentile a) CF patients without history of P. aeruginosa positive culture IgG IgG IgG IgG IgG b) Healthy controls IgG IgG IgG IgG IgG sputum samples) per year versus 0.5 (5% of all samples) per year in controls ( p < ). Growth of mucoid strains of P. aeruginosa was more frequent in the study group than in controls (11.5% of isolates versus 0%, p <0.0001) (Table 1a). Logistic regression analysis showed that the occurrence of mucoid phenotype of bacteria was a risk factor for developing chronic P. aeruginosa infection with the OR of 7.4 (95% CI , p = 0.006). Positive cultures with S.aureus were equally distributed in both investigated groups (Table 1). On the other hand occurrence of Aspergillus-positive cultures were significantly more frequent in the study group than in controls ( p = 0.01) (Table 1a). Logistic regression analysis showed that the occurrence of of Aspergillus is a risk factor for developing chronic P. aeruginosa infection with the OR of 4.7 (95% CI , p = 0.008). Allergic bronchopulmonary aspergillosis (ABPA) was diagnosed in two cases in the study group and in one control patient. Table 1 Baseline demographics of the CF study group and CF control group a) Study group (N =28) Control group (N =28) p value Median Range Median Range Age (years) at start of chronic infection for the study group Ns BMI z-score 2 years before Ns BMI z-score 1 year before Ns BMI z-score at infection start Ns Frequency of S. aureus positive culture per patient in 3 years study period Ns Frequency of Aspergillus positive culture pr patient in 3 years study period Percent mucoid strains of P. aeruginosa < Age at first isolate of P. aeruginosa (years) Ns Frequency of P. aeruginosa positive culture in 3 years study period < b) Study group Control group p value Gender M/F total 11/17 15/13 Ns MBL variant alleles 12 6 Ns CFTR mutation mild/severe 0/28 3/25 Ns

4 12 T. Pressler et al. / Journal of Cystic Fibrosis 5 (2006) 9 15 Table 3 Development of IgG and IgG subclass anti-pseudomonas specific antibodies in the CF study group and the CF control group Study group Control group p value Median Range Median Range IgG 3 years before years before year before At infection start <0.001 IgG1 3 years before years before < year before <0.001 At infection start <0.001 IgG2 3 years before years before year before At infection start IgG levels two years before onset of chronic infection with OR 7.4 (95% CI , p =0.004). 4. Discussion P. aeruginosa infection in patients with CF is associated with deteriorating lung function and increased morbidity [1,31 34]. The attempt has been made to define the risk factors for initial acquisition of P. aeruginosa in children with CF [35 38] and the risk factors turned out to include S. aureus infection [38] and chronic anti-staphylococcal antibiotic treatment [36,37], female gender, delta F508 homozygous genotype [38] and attendance at cystic fibrosis clinic [35,36], and viral infections [12]. In our population the sex of the patients has not shown to be a risk factor for acquisition of P. aeruginosa infection. The occurrence of severe CFTR mutation was not recog- IgG3 3 years before years before < year before At infection start <0.001 IgG4 3 years before years before < year before <0.001 at infection start <0.001 a) ELISA Units Specific anti-pseudomonas antibodies -1 There were no statistical significant differences between levels of specific anti-pseudomonas antibodies in healthy controls and CF patients without history of P. aeruginosa positive cultures (Table 2a and b). On the other hand the specific anti-pseudomonal serum antibodies of total IgG and each IgG subclasses were significantly higher in the study group than in controls already 3 years prior to onset of chronic infection and continued to increase through whole study period (Table 3). The anti-pseudomonas IgG and IgG1 increased with median 0.44 EU per year (range 0.4 to 2.0) and 0.3 EU per year ( 1.9 to 3.2), respectively, in the study group compared to 0.0 EU per year ( 0.4 to 1.4, and 0.2 to 0.3) for both IgG and IgG1 ( p <0.005) in control group (Fig. 1). Changes in IgG2, IgG3 and IgG4 levels were not significantly different between case and control groups. Logistic regression analysis showed that the strongest risk factor for developing chronic P. aeruginosa infection was elevated levels of specific antipseudomonal antibodies of IgG1and IgG4 subclasses already 3 years prior to onset of chronic infection, with OR 8.9 (95% CI , p =0.0035) and 7.7 (95% CI , p = 0.005), respectively, and elevated total anti-pseudomonas b) ELISA Units year - 3 year - 2 year - 1 year - 3 year - 2 year - 1 Fig. 1. Changes (EU per year) in specific anti-pseudomonal IgG levels in 3 years study period in a) the CF study group who developed chronic P. aeruginosa compared to the b) the CF control group (median and 95th percentile).

5 T. Pressler et al. / Journal of Cystic Fibrosis 5 (2006) nized as a risk factor, but the prevalence of mild CFTR mutations in our patient population is very low [39]. MBL2 variant alleles have previously shown to have an impact on the disease course of Danish CF patients, but not on the acquisition of P. aeruginosa [40]. In the present matched case control study we did not observe an increased frequency of index cases carrying MBL2 variant alleles compared with controls, though the power of the current study was limited by its small sample size. To evaluate this further, larger matched case control studies are needed before a conclusion can be drawn whether mannose-binding lectin plays a role in susceptibility for P. aeruginosa in CF patients. Maselli et al. [38] found that colonization with S. aureus were associated with an earlier acquisition of P. aeruginosa. Distinction between chronic infection and intermittent colonization was not made. In our study the study population harbored S. aureus as often as controls, in an average 11 times in the three-year study period (Table 1). All S. aureus isolated were treated with relevant antibiotics according to the current resistance pattern. The use of antistaphylococcal antibiotics was not different in both groups and neither isolation nor treatment of S. aureus came out as a risk factor. Attendance to the CF clinic not practicing cohort segregation and exposure to P. aeruginosa-positive patients have been reported as risk factors for early P. aeruginosa acquisition [35]. Evidence for transmission of P. aeruginosa between CF patients is well established [41 44]. Cohort isolation of patients with CF according to their colonization status has been the policy in our CF Center since 1981 [45]. It can explain why contact to the CF clinic is not anymore a risk factor in our CF population. In our study occurrence of Aspergillus-positive cultures were significantly more frequent in the study group than in controls. Logistic regression analysis showed that the occurrence of Aspergillus is a risk factor for developing chronic P. aeruginosa infection. A similar observation was made by Graff and Burns [46] when they studied factors affecting the incidence of Stenotrophomonas maltophilia in CF. The other factor they found having an impact on incidence of S. maltophilia was the use of antibiotics, particularly oral fluroquinolones. The occurrence of Aspergillus as a risk factor for P. aeruginosa chronic infection in our study may reflect more frequent use of antibiotics in the study group compared to the controls. As only 2 of the CF patients of the study group and one of the control group developed ABPA, steroid treatment of this disease cannot be responsible risk factor. P. aeruginosa were more frequently found in the study group than in the control group and because of our policy to treat P. aeruginosa every time this microorganism appears in respiratory tract secretions the study population had a significant higher antibiotic requirements. The observation of reduced lung function in a group of patients who subsequently developed P. aeruginosa infection have been made before [47,48], and confirmed in the present study. Poorer lung function in the study group indicates pre-existing lung damage when compared with patients who remained free of P. aeruginosa. It has been known for many years that chronic P. aeruginosa infection preceded by intermittent colonization [12,49] and the most characteristic feature of chronic P. aeruginosa infection is the production of alginate (responsible for the mucoid phenotype) and simultanously biofilm formation. The transition to the mucoid variant correlates with development of a pronounced antibody response and a poor prognosis [8,48,50,51]. Growth of mucoid strains of P. aeruginosa was more frequent in the study group than in controls and occurrence of mucoid phenotype of bacteria was a risk factor for developing chronic P. aeruginosa infection with the OR of 7.4. It has been shown in vitro, that P. aeruginosa growing in alginate biofilms is highly resistant to antibiotics and protected against phagocytosis [52 55]. The most important results of this study are the observation on differences between specific anti-pseudomonas antibody levels in case and control groups. Specific antipseudomonal IgG serum antibodies were significantly higher in the study group than in controls already 3 years prior to onset of chronic infection and increased significantly through the whole study period in the study group but remained at the initial level in the control group. The strongest risk factor for developing of chronic P. aeruginosa infection was increasing levels of specific anti-pseudomonal antibodies already 3 years prior to onset of chronic infection. The results show that antibody measurement have potential diagnostic value and can be used as a prognostic tool for identification of the group of CF patients at risk of establishing chronic P. aeruginosa infection. Specially increasing levels of specific anti-pseudomonas IgG antibodies seem to be a useful parameter to select the patients being at risk to develop a chronic infection. We recommend that specific anti-p. aeruginosa antibodies should be measured regularly and at least once a year in all CF patients and more frequently in individual patients when P. aeruginosa has been intermittently isolated. 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