The fungal prevalence, diversity and variation over time as well distribution between age groups were determined by using descriptive data analysis. We conducted linear mixed models with autoregressive (AR1) covariance matrix and subjects as random effect to assess the impact of colonization with C. albicans, C dubliniensis and A. fumigatus on lung function on short and long term. Adjustment was performed for age, CFTR genotype, chronic and intermittent colonization with P. aeruginosa and number of intravenous antibiotic courses per year. Finally, we performed a multivariate analysis to determine potential predictors for colonization with C. dubliniensis for three consecutive years.
Study II
The impact of the first colonization with A. fumigatus on the annual predicted lung function decline was assessed conducting linear mixed models using the natural logarithm of ppFEV1.
Further analysis on the impact of colonization and eradication on lung function was measured by calculating the ratios of ppFEV1 for a specific condition divided by ppFEV1 in the compared condition. A ratio <1 means that ppFFV1 was worse when the patient was colonized, and vice versa. Parameters that may influence the impact of colonization on lung function were assessed using odds ratio (OR). OR >1 was associated with a protection against the possible negative effect of A. fumigatus on ppFEV1. Analyses were conducted with R 4.0.2 statistical software (165) employing the lmerTest package (166) .
Study III
Statistical analyses were performed with the SPSS ver. 26 software (IBM Corp., Armonk, NY). We performed extended Fisher’s exact test to analyze differences in the categorical variables between the three different groups (non-colonized, intermittently colonized with A.
fumigatus and chronically colonized with A. fumigatus) and if the test was significant, Fisher’s exact test was performed. For continuous variables, Kruskal-Wallis one way ANOVA was performed and Mann-Whitney U-test if the former test was significant. A
multivariate logistic regression analysis was used to determine risk factor for chronic colonization with A. fumigatus. Six covariates were chosen: age, ppFEV1, nebulized hypertonic saline, inhaled corticosteroids, inhaled and intravenous antibiotics.
Study IV
Data was analyzed with multilevel piecewise linear spline regression, with a single knot placed at the start of the lumacaftor/ivacaftor treatment by employing R 4.1.0 statistical software (167) and the lmerTest (166) and lspline (168) packages. The slope (change of the outcome variables over time) before the onset of the treatment with lumacaftor/ivacaftor was compared with the slope before. The slope before indicated the natural course of the outcome variables without treatment and serve as control. Continuous outcome variables were analyzed with linear models, dichotomous outcome with logistic models and count variables with Poisson models.
RESULTS
6.1 STUDY I
6.1.1 The fungal prevalence and diversity
In total 133 patients were included, totally1499 patient-years. The mean follow-up period was 10.2 years. The fungal diversity is presented in Table 1 and the fungal prevalence in Figure 1. The alteration of the annual prevalence of the four most common fungi over time indicated a stability in the prevalence of Exophiala dermatitis while A. fumigatus and C.
dubliniensis became more frequent, Figure 2.
Table 2: The fungal diversity in the study cohort.
Candida species Yeast excluded Candida species
Aspergillus species Mold excluded Aspergillus species C. albicans Cryptococcus curvatus A. candidus Acremonium species C. dubliniensis Exophiala dermatitidis A. fumigatus Alternaria species
C. famata Geotrichum klebahnii A. flavus Cladosporium species
C. glabrata Geotrichum silvicola A. glaucus Doratomyces species C. guilliermondii Kloeckera apiculata A. niger Fusarium dimerum C. intermedia Rhodotorula mucilaginosa A. nidulans Rasamsonia argillacea
C. krusei Trichosporon asahii A. ochraceus Rhizopus microsporus
C. lambica A. terreus Paecilomyces variotii
C. lusitaniae A. versicolor Penicillium species
C. lypolytica Scedosporium
apiospermum
C. norvegensis Scytalidium hyalinum
C. parapsilosis Syncephalastrum
racemosum
C. sphaerica
C. tropicalis
Reproduced from: A 16-year retrospective study on fungal prevalence and diversity in patients with cystic fibrosis: Candida dubliniensis was associated with a decline in lung function, by Al Shakirchi et al., 2020.
6.1.2 The impact of C. albicans, C. dubliniensis and A. fumigatus on lung function
One and two years after colonization with C. dubliniensis a significant decline in lung function was detected, -3.8% (p=0.002) and –4.1% (p=0.017), respectively. Furthermore, the persistence of any of C. albicans, C. dubliniensis or A. fumigates for three years in a row was associated with a significant decline in lung function most pronounced for C. dubliniensis.
The decline in lung function was 7.6% for C. dubliniensis (p=0.001), 4.9 % for A. fumigatus (p=0.007) and 2.6% for C albicans (p=0.014).
Figure 1: The fungal prevalence for the whole study period 2000-2015. Reproduced from: A 16-year retrospective study on fungal prevalence and diversity in patients with cystic fibrosis: Candida dubliniensis was associated with a decline in lung function, by Al Shakirchi et al., 2020.
Figure 2: The fungal prevalence of the four most common fungi as well as P. aeruginosa and S. aureus during the study period 2000-2015. Reproduced from: A 16-year retrospective study on fungal prevalence and diversity in patients with cystic fibrosis: Candida dubliniensis was associated with a decline in lung function,
6.1.3 The impact of co-colonization on lung function
Patients who cultured positive for P. aeruginosa, A fumigatus, C. albicans or C. dubliniensis exhibited no alteration in lung function the year of detection, however, colonization with these four pathogens at the same time was associated with 5.1 % decrease in lung function (p=0.019). In addition, patients who were cultured positive for C. albicans and C.
dubliniensis three years in a row and at the same time were colonized with A. fumigatus and P. aeruginosa displayed 9.7 % reduction in ppFEV1 (p=0.001).
6.1.4 Parameters associated with colonization with C. dubliniensis three years in a row
As the reduction in lung function was most pronounced in patients who were colonized with C. dubliniensis three years in a row, we aimed to assess parameters that may predict this condition. We found that only high age (p=0.001) and ESR (p<0.001) were significantly higher in CF patients colonized with C. dubliniensis three years in a row.