FUNGAL COLONIZATION AND INFECTION IN CYSTIC FIBROSIS – PREVALENCE, CONSEQUENCES AND INTERVENTION

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From DEPARTMENT OF CLINICAL SCIENCE, INTERVENTION AND TECHNOLOGY, DIVISION OF

PEDIATRICS

Karolinska Institutet, Stockholm, Sweden

FUNGAL COLONIZATION AND INFECTION IN CYSTIC FIBROSIS – PREVALENCE, CONSEQUENCES AND INTERVENTION

Mahasin Al Shakirchi

Stockholm 2022

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All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet.

Printed by Universitetsservice US-AB, 2022

Cover illustration: Reprinted by permission from Springer Nature:

Mycopathologia, Emerging Fungal Threats in Cystic Fibrosis by C. Schwarz et al. COPYRIGHT (2021).

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Fungal colonization and infection in Cystic Fibrosis - prevalence, consequences and intervention

THESIS FOR DOCTORAL DEGREE (Ph.D.)

By

Mahasin Al Shakirchi

The thesis will be defended in public at Karolinska University Hospital Huddinge on October the 21^st, 2022

Principal Supervisor:

PhD Isabelle de Monestrol Karolinska Institutet

Department of Clinical Science,

Intervention and Technology- CLINTEC Division of Pediatrics

Co-supervisors:

Professor Lena Hjelte Karolinska Institutet

Department of Clinical Science,

Intervention and Technology- CLINTEC Division of Pediatrics

Professor Peter Bergman Karolinska Institutet

Department of Laboratory Medicine Division of Clinical Immunology

Associate Professor Lena Klingspor Karolinska Institutet

Department of Laboratory Medicine Division of Clinical Microbiology

Opponent:

Professor Vanda Friman

Sahlgrenska Academy, University of Gothenburg, Institute of Biomedicin, Department of Infectious Diseases

Examination Board:

Associate Professor Marita Gilljam

Sahlgrenska Academy, University of Gothenburg, Institute of Medicine

Department of Respiratory Medicine

Senior consultant Ingvild Nordöy, MD, PhD Oslo University Hospital, Rikshospitalet Department of Rheumatology,

Dermatology and Infectious diseases Section for Clinical Immunology and Infectious Diseases

Professor Magnus Svartengren Uppsala University

Department of Medical Sciences, Occupational and Environmental Medicine

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To ammo Jafer and bibi Zakia

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POPULAR SCIENCE SUMMARY OF THESIS

Introduction

Cystic fibrosis (CF) is one of the most common life-shortening inherited diseases in people of European descent. The disease affects several organ systems, with most severely affected being the lungs. The mucus in the CF lungs is thick and sticky which prevent clearing of inhaled microorganisms. These microorganisms remain causing inflammation which results in lung damage and eventually respiratory failure. Bacteria are well studied in CF. Fungi on the other hand have received little attention despite the fact that they are frequently isolated from the CF airways. The role of fungi in the development of lung damage in CF is still unclear. The purpose of this thesis was to increase our knowledge on fungi in CF by investigating how common it is (prevalence), consequences and what happens when a new effective CF treatment is introduced.

Study I

This was a 16-year study (2000–2015) including 133 CF patients who attended solely the Stockholm CF center. During the study period 42 different fungal species were identified.

The fungal prevalence increased over time. Interestingly, individuals who had any of the most common fungi C. albicans, C. dubliniensis or A. fumigatus for three consecutive years exhibited lower lung function. The decline in lung function was most pronounced in individuals who had C. dubliniensis.

Study II

In this study, we focused on the most common mold in CF; A. fumigatus using the same data set as in study I. The annual expected decline in lung function was steeper after the first finding of A. fumigatus. In addition, CF patients who were colonized (growth in sputum culture) with A. fumigatus had poorer lung function compared to non-colonized.

Furthermore, eradication of A. fumigatus was associated with better lung function.

Surprisingly, co-colonization with P. aeruginosa (a common bacterium in CF airways) presented as a protective factor against the potential negative effect of A. fumigatus on lung function.

Study III

This was a Scandinavian study in which 548 CF patients from 4 CF centers participated.

Patients who were chronically colonized with A. fumigatus (recurrent findings of A.

fumigatus in sputum cultures) were older and had poorer lung function. Inhalation antibiotics was a strong risk factor for chronic colonization with A. fumigatus. For each month of use of

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inhalation antibiotics, the risk of becoming chronically colonized with A. fumigatus increased by 11%.

Study IV

CFTR modulators are a group of relatively new treatments that correct the basic defect in CF at a cellular level. The treatments are mutation specific. Lumacaftor/ivacaftor or so-called Orkambi® is suitable for patients who have two copies of the most common CF-causing mutation F508del. In total 135 CF patients attending Stockholm, Lund and Gothenburg CF centers were included and followed up one year after the onset of the treatment. Lung function, nutritional status and sweat chloride levels improved one year after treatment with lumacaftor/ivacaftor. Furthermore, the need for treatments with antibiotics and antifungal agents decreased. Regarding the bacteriological findings, a reduction of growth of some less common bacteria such as Streptococcus pneumoniae and Stenotrophomonas maltophilia was observed. On the other hand, the growth of some fungi such as C. albicans, Scedosporium apiospermum and Penicillium species increased. The most common and key bacteria (P.

aeruginosa and S. aureus) and mold (A. fumigatus) did not change after the start of lumacaftor/ivacaftor.

In summary

These four studies have increased our knowledge on fungi in the CF airways. Fungi became more common which emphasizes the importance to clarify this emerging topic. The first two studies showed a negative association between repeated findings of the most common fungi (C. albicans, C. dubliniensis and A. fumigatus) over time and impaired lung function.

However, these studies cannot determine whether the fungi themselves were the cause of impaired lung function or whether the presence of these fungi was a consequence of poorer lung function. In addition, we have demonstrated that eradication of A. fumigatus has a beneficial effect on lung function. We have also reported that inhaled antibiotics was a strong risk factor for chronic colonization with A. fumigatus. Despite the improvement of a number of clinical parameters and the reduction of the use of antibacterial and antifungal treatment with lumacaftor/ivacaftor, no significant changes in the presence of the most relevant key microorganisms in CF were noted one year after initiation of the treatment.

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POPULÄRVETENSKAPLIG SAMMANFATTNING

Introduktion

Cystisk fibros (CF) är en av de vanligaste ärftliga sjukdomar med förkortad livslängd hos individer med europeiskt ursprung. Sjukdomen drabbar flera organ med de svåraste symptomen från lungorna. Slemmet i CF-lungorna är tjockt och segt, vilket förhindrar rensningen av inhalerade mikroorganismer. Dessa mikroorganismer stannar kvar i CF lungan och orsakar inflammation vilket leder till lungskador som så småningom resulterar i andningssvikt. Bakterier är väl studerade vid CF. Svampar däremot har fått lite uppmärksamhet trots att de ofta förekommer i CF-luftvägar. Vad svamp har för roll i utvecklingen av lungskador vid CF är fortfarande oklart.

Syftet med denna avhandling var att fördjupa vår kunskap om svamp vid CF genom att undersöka förekomsten av svampar, konsekvenser och vad som händer när en ny effektiv CF-behandling introduceras

Studie I

Detta var en 16-årig studie (2000–2015) med 133 CF-patienter tillhörande Stockholm CF- center. Under studieperioden identifierades 42 olika svamparter. Förekomsten av svamp ökade med tiden. Individer som hade haft någon av de vanligaste svamparterna C. albicans, C. dubliniensis eller A. fumigatus tre år i rad uppvisade en försämring av lungfunktionen som var mest uttalad hos individer som hade fynd av C. dubliniensis.

Studie II

I den här studien fokuserade vi på den vanligaste mögelsvampen vid CF; A. fumigatus. Vi använde oss av samma dataset som i studie I. Den årliga förväntade minskningen i lungfunktionen vid CF blev brantare efter det första fyndet av A. fumigatus. Vidare hade CF- patienter som var koloniserade (växt i sputumodling) med A. fumigatus sämre lungfunktion jämfört med icke-koloniserade. Dessutom hade patienter som lyckats bli av med A. fumigatus bättre lungfunktion. Till vår förvåning visade samtidig kolonisering med P. aeruginosa (en vanlig bakterie i CF-luftvägarna) en skyddande faktor mot den potentiella negativa effekten av A. fumigatus på lungfunktionen

Studie III

Detta var en skandinavisk studie där 548 patienter från 4 CF-centra deltog. Patienter som var kroniskt koloniserade med A. fumigatus (återkommande fynd av A. fumigatus i odling) var äldre och hade sämre lungfunktion. Inhalationsantibiotika var en stark riskfaktor för kronisk kolonisering med A. fumigatus. Varje månads användning av inhalationsantibiotika ökade risken att bli kroniskt koloniserad med A. fumigatus med 11 %.

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Studie IV

CFTR-modulatorer är en grupp relativt nya behandlingar som korrigerar den underliggande defekten vid CF på cellnivå. Behandlingarna är mutationsspecifika. Lumacaftor/ivacaftor, som heter Orkambi®, är lämplig för patienter som har två kopior av den vanligaste CF- mutationen F508del. Totalt 135 CF-patienter från Stockholm, Lund och Göteborg CF-center inkluderades och följdes upp ett år efter start av behandlingen. Lungfunktion, nutritionsstatus och svettkloridnivåer förbättrades efter ett års behandling med lumacaftor/ivacaftor. Vidare minskade behandlingar med antibiotika och svampmedel. Avseende de bakteriologiska fynden observerades en minskning av mindre vanliga bakterier som Streptococcus pneumoniae och Stenotrophomonas maltophilia. Däremot ökade fynden av vissa svampar såsom C. albicans, Scedosporium apiospermum och Penicillium species. Fynden av de vanligaste och viktigaste bakterierna (P. aeruginosa och S. aureus) eller svampen (A.

fumigatus) ändrades inte efter Orkambi®-behandlingen.

Sammanfattning

Dessa fyra studier har ökat vår kunskap om svamp i CF-luftvägarna. Förekomsten av svamp har ökat med åren vilket understryker vikten av att klargöra detta framväxande ämne. De första två studierna pekade på ett negativt samband mellan upprepade fynd av de vanligaste svamparna (C. albicans, C. dubliniensis och A. fumigatus) och lungfunktionen. Dessa studier kan dock inte fastställa om svamparna i sig var orsaken till försämringen eller om förekomsten av dessa svampar var en konsekvens av sämre lungfunktion. Dessutom har vi visat att undanröjande av A. fumigatus har en gynnsam effekt på lungfunktion. Vi har också rapporterat att inhalationsantibiotika var en stark riskfaktor för kronisk kolonisering med A.

fumigatus. Trots förbättring av en rad kliniska parametrar och minskning av användning av antibiotika och antisvampbehandling efter insättning av lumacaftor/ivacaftor, noterades ingen signifikant ändring i förekomsten av de mest relevanta mikroorganismerna vid CF ett år efter insatt behandling.

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ABSTRACT

Background

Cystic fibrosis (CF) is an inherited progressive disease affecting multiple organs. The main cause of death in CF is related to the respiratory complications. Inflammation and infection, triggered by colonizing microorganisms in CF airways, cause structural lung damage resulting in respiratory failure and death. Isolation of bacteria and fungi from the CF respiratory tract is common. While the role of bacteria has been studied extensively, fungi have received less attention and their impact on the CF progressive lung disease has not been established yet. In this thesis we aimed to thoroughly study the fungal epidemiology, consequences and parameters that may impact the fungal outcomes in CF.

Methods

Study I and II were 16-year longitudinal retrospective single center studies. Study I consisted of two parts. The descriptive part of the study encompassed data on the fungal prevalence, diversity and variation over time. In the second part, the impact of the most common fungi;

C. albicans, C. dubliniensis and A. fumigatus on lung function was assessed using linear mix model. In study II, we determined the impact of A. fumigatus colonization and eradication on lung function. The impact of the first acquisition of A. fumigatus on the annual predicted decline in lung function was analyzed by a using linear mixed model and the natural logarithm of the percentage of predicted forced expiratory volume in 1 second (ppFEV1).

The impact of colonization and eradication was analyzed by calculating the ratios of ppFEV1 for a specific condition divided with ppFEV1 of the compared condition. Study III was a prospective one-year multicenter study. A multivariate regression analysis was used to identify risk factors associated with chronic colonization with A. fumigatus. Study IV was also a prospective multicenter study aimed to determine the impact of lumacaftor/ivacaftor on the microbiological outcomes as well as on a number of clinical parameters. Linear models were used for continuous outcomes, logistic models for dichotomous variable and Poisson models for count variables.

Results

The three most common fungi were C. albicans (63%), A. fumigatus (22%) and C.

dubliniensis (11%). During the study period (16 years), the fungal prevalence and diversity increased. Interestingly, persistent colonization with C. albicans, A. fumigatus or C.

dubliniensis for three consecutive years was associated with a significant decline in lung function with 2.6%, 4.9% and 7.6% decrease in ppFEV1 respectively. Regarding A.

fumigatus, the most common mold, the annual predicted decline in lung function increased

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from –1.8% per year to –2.3% after the first acquisition. Furthermore, within the same individual lung function was 4.3% and 7.9% lower when A. fumigatus persisted for two and three consecutive years. Patients who eradicated A. fumigatus, with or without treatment, and remained free from A. fumigatus in the respiratory cultures for two consecutive years displayed 9.9% and 14.5% higher lung function compared with patients who continued to culture A. fumigatus two and three years in a row. Inhaled antibiotics was a risk factor for chronic colonization with A. fumigatus with 11% increasing risk for each month of use of inhaled antibiotics.

Treatment with lumacaftor/ivacaftor, improved lung function, nutrition status and inflammatory parameters. Furthermore, antibiotic and antifungal treatment decreased after initiation of the treatment with lumacaftor/ivacaftor. However, the key microorganisms such as P. aeruginosa, S. aureus and A. fumigatus did not change with lumacaftor/ ivacaftor treatment one year after the onset of the treatment. Other less common bacteria such as Streptococcus pneumoniae and Stenotrophomonas maltophilia became less common while

C. albicans, Penicillium species and Scedosporium apiospermum were more prevalent.

Conclusion

The results from our studies highlighted the potential impact of fungi in the course of CF lung disease. Unexpectedly, a negative association between Candida species, which have been considered as harmless colonizer, and lung function was observed. Because of the study design, causality cannot be determined. Despite the improvement in cardinal clinical outcomes after introducing lumacaftor/ivacaftor, it did not impact the prevalence of A.

fumigatus or other key pathogens in CF in short-term emphasizing the importance to continue monitoring patients treated with CFTR modulators with regards to microbiological outcomes.

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LIST OF SCIENTIFIC PAPERS

I. Al Shakirchi, M.; Klingspor, L.; Bergman, P.; Hjelte, L.; de Monestrol, I.

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. International Journal of Infectious Diseases 2020, Article. DOI:

10.1016/j.ijid.2020.05.063 From EBSCOhost ScienceDirect.

II. Al Shakirchi, M.; Sorjonen, K.; Klingspor, L.; Bergman, P.; Hjelte, L.; de Monestrol, I.

The Effects of Aspergillus fumigatus Colonization on Lung Function in Patients with Cystic Fibrosis. Journal of Fungi 2021, 7 (11). DOI:

10.3390/jof7110944.

III. Mahasin Al Shakirchi, Anders Lindblad, Lisa I. Påhlman, Gudmundur Vignir Sigurdsson, Karsten Kötz, Petrea Ericsson, Ferenc Karpati, Hanne V. Olesen, Tanja Pressler, Marianne Skov, Isabelle de Monestrol. 

Risk factor for chronic colonization with Aspergillus fumigatus in patients with Cystic fibrosis.

Manuscript

IV. Mahasin Al Shakirchi, Kimmo Sorjonen, Lena Hjelte, Lena Klingspor, Peter Bergman, Petrea Ericson, Marcus Svedberg, Ulrika Lindberg, Christine Hansen, Isabelle de Monestrol

Impact of lumacaftor/ivacaftor on the bacterial and fungal respiratory pathogens in Cystic Fibrosis: a prospective multicenter cohort study in Sweden.

Manuscript

Scientific paper not included in the thesis:

Blomquist, A.; Inghammar, M.; Al Shakirchi, M.; Ericson, P.; Krantz, C.; Svedberg, M.;

Lindblad, A.; Pahlman, L. I.

Persistent Aspergillus fumigatus infection in cystic fibrosis: impact on lung function and role of treatment of asymptomatic colonization - a registry-based case-control study. Bmc

Pulmonary Medicine 2022, 22 (1). DOI: 10.1186/s12890-022-02054-3.

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LIST OF ABBREVIATIONS

AR Autoregression

ABPA Allergic bronchopulmonary aspergillosis

BAL Bronchoalveolar lavage

BMI Body mass index

CBVAD Congenital bilateral absence of the vas deferens

CF Cystic fibrosis

CFF Cystic Fibrosis Foundation

CFRD Cystic fibrosis related diabetes CFRL Cystic fibrosis related liver disease

CFTR Cystic fibrosis transmembrane conductance regulator COPD Chronic obstructive lung disease

CT Computed tomography

DEXA Dual energy X-rays absorptiometry DIOS Distal intestinal obstruction syndrome ECFS European Cystic Fibrosis Society ENaC Epithelial sodium channel

ESS Endoscopic sinus surgery

FEV0.75 Forced expiratory volume in the first three-quarters of a second GLI Global Lung Function Initiative

GM Galactomannan

HIV Human immunodeficiency virus

HRCT High resolution chest tomography

Ig Immunoglobulin

IL Interleukin

IV Intravenous

IVF In vitro fertilization

LCI Lung clearance index

MALDI-TOF Matrix-assisted laser desorption/ionization time of flight mass spectrometry

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MESA Microepididymal sperm aspiration

MRI Magnetic resonance imaging

MRSA Methicillin-resistant Staphylococcus aureus

NaCl Natrium chloride

NLRP3 NOD-, LRR- and pyrin domain-containing protein 3

OP Oropharyngeal swab

OR Odds ratio

PCR Polymerase Chain Reaction

ppFEV1 The percentage of predicted forced expiratory volume in 1 second

ppFVC The percentage of predicted forced vital capacity

ROS Reactive oxygen species

SCFSC Scandinavian Cystic Fibrosis Study Consortium

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INTRODUCTION

Cystic Fibrosis (CF) is a genetic recessive disorder affecting vital organs and resulting in limited life expectancy. Morbidity and mortality in CF mainly derives from respiratory tract involvement (1). The mucus in the respiratory tract is sticky, thick and dehydrated resulting in impaired mucus clearance and insufficiency to clear inhaled microorganisms leading to their persistence, colonization and infection (2). The CF mucus is an excellent environment for inhaled organism to thrive. A vicious cycle of infection triggered by colonizing microorganisms results in an exaggerated and detrimental inflammatory response (2). This causes structural lung damage and bronchiectasis which leads to increased mucus production amplifying infection and inflammation (2). People with CF suffer from recurrent pulmonary infection which is a negative prognostic factor. The CF care is mainly based on preventing and treating pulmonary exacerbations to impede structural lung damage. Historically, the focus is on detecting bacteria from CF airways and treating them aggressively (3). A considerable part of the CF care consists of antibiotic treatments. Fungi, both yeast and mold, are frequently isolated from CF airways. The most common mold is Aspergillus fumigatus, and the most common yeast is Candida albicans (4, 5). Studies on the role of isolation of A.

fumigatus in CF respiratory tract showed conflicting results (6-9). Because of the lack of standardized detection methods and the absence of an agreement on isolation rate and the definition of colonization with A. fumigatus, it is difficult to compare studies. Yeasts are less studied and are traditionally considered as benign colonizers, however; few studies have challenged this notion (10, 11). Despite the high prevalence of A. fumigatus and other fungi in CF airways, there is no consensus on how to deal with these findings.

Given the uncertainties in the field, we aimed to study fungi in CF. To start with, we elucidated the fungal epidemiology in our center. Further, the impact of fungi on lung function, risk factors and parameters that may impact the fungal outcomes was assessed.

Hopefully, this thesis is a seed for future studies in the field in order to provide evidence based medical care for CF patients with fungal colonization.

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CYSTIC FIBROSIS

2.1 A HISTORICAL BACKGROUND

Although the identification of Cystic fibrosis (CF) as a disease was recognized as late as 1938, the salty sweat which is a hallmark of CF and its fetal event was mentioned at the end of the fifteen century, “Woe to that child which kissed on the forehead tastes salty, he is bewitched and soon must die” (12). Dorothy Andersen was the first to describe CF characteristics, named it cystic fibrosis in the pancreas, and ascertained that CF is a recessive genetic disorder more than 40 years before the identification of the gene (13, 14). Sweat test, the most reliable diagnostic method and up to present time the diagnostic golden standard in CF, was invented in 1953 and developed by Gibson and Cooke in 1959 (15, 16). Before 1983, the underlying defect that explains the CF manifestation in lungs, pancreas and sweat glands was unknown. The discovery of the defect in chloride and other ion channels in CF epithelial cells and sweat glands was crucial to the understanding of the pathophysiology in CF (17, 18). Ultimately, increasing knowledge about the basic defect led to the historical identification of the gene and its product, the CF transmembrane conductance regulator (CFTR) protein in 1989 (19, 20).

2.2 EPIDEMIOLOGY AND DEMOGRAPHICS

CF is one of the most common congenital disease in people of European heritage. Worldwide, more than 80 000 people suffer from CF (21). The incidence ranges between 1:1353 newborns in Ireland to 1:6000 newborns in Portugal (22). In Sweden, the incidence is 1: 5600 newborns (23). In general, the majority of CF patients are diagnosed before the age of two (12). Due to the risk of missed diagnosis and delayed treatment, most countries with a high prevalence for CF have implemented newborn screening (24). It is evident that newborn screening prevents delayed and missed diagnosis and reduces disease severity (3, 25).

Unfortunately, in Sweden there is no newborn screening for CF.

The progression in CF care has improved the life expectancy in CF patients and has changed the demographics of the disease. In early 1940s, after Andersen´s identification of the disease, the median life expectancy was a few months. As a result of the constantly evolving therapeutic approach, the expected median age of survival has increased steadily to 50 years (26-28). Consequently, the demographic of CF has changed from being a pediatric disease to predominantly an adult disease at least in developed countries. Furthermore, the historically significant invention of CFTR modulator therapy targeting the underlying defect in CF probably will likely have a considerable impact on the CF care (29). Hopefully introducing

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next generation CF patients a greater opportunity to have a normal life expectancy, decrease the treatment burden and most importantly improve their quality of live.

2.3 THE CFTR GENE

Since the discovery of the gene, more than 2000 CFTR mutations have been identified, 360 of them are acknowledged as disease causing mutations (30-32). F508del is the most common CF-causing mutation accounting for almost 85 % of the CF population (30, 31, 33). Based on the CFTR mutations, the defect in the CFTR protein, which is a product of the CFTR gene, could occur at several levels: production, transportation, function, and stability. Therefore, the CFTR mutations are divided into seven classes based on their effect on the CFTR protein (34, 35), the seventh class have been recently suggested by De Boech and Amaral (21).

Class I A and B: premature RNA transient leading to a total absence of CFTR protein (stop mutation, e.g. G542X).

Class II: total misfolded CFTR protein and prematurely destroyed leading to total absence of CFTR protein at the apical cell membrane (e.g. F508del).

Class III: impaired gating of CFTR channel leading to less open activity (e.g. G551D).

Class IV: reduced conduction leading to decreased ion flow.

Class V: reduced synthesis of CFTR protein.

Class VI: reduced membrane stability.

Class VII or IA: total absence of mRNA transcription.

These gene classifications reflect the clinical phenotypes to a large extent. Class I, II, VII (or IA) and III have no residual function of the CFTR protein resulting in severe disease. On the other hand, class IV, V and VI have some residual function leading to pancreatic sufficiency and milder disease (36, 37). However, it has been noticed that within the same class there is a wide range of disease severity suggesting heterogeneity of the CFTR protein function. CF clinical outcome is influenced by prognostic factor such as environmental factors, modifier genes and pathogens colonized in the CF airways. (38).

2.4 DIAGNOSTIC CRITERIA IN CF

The diagnostic criteria are based on the presence of one or more clinical features compatible with CF, or a history of CF in a sibling, or a positive newborn screening alongside with evidence of CFTR dysfunction either by a positive sweat test, or two CF-causing mutations or a positive nasal potential difference (PD) (39). A level of sweat chloride concentration ≥ 60 mmol/L is consistent with CF. However, the test should be confirmed with a repeat sweat test. False positive sweat chloride tests have been noticed in critically ill and severely malnourished patients. An intermediate level of sweat chloride between 30-59 mmol/L may

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be associated with an atypical CF (40). Very rare CF-causing mutations are associated with a normal sweat test < 30 mmol/L (41-43). Conventionally, the gene test analyzes the 50 most common CFTR mutations covering 99% of the CFTR mutations in Sweden. In the case of a borderline sweat test and clinical manifestations compatible with CF, further CFTR gene analyses such as sequencing may be required (39).

2.5 PATHOPHYSIOLOGY, CLINICAL FEATURES AND TREATMENT

2.5.1 The pathophysiology of the CFTR channel

The main function of the CFTR protein is to regulate transportation of chloride and bicarbonate in the epithelial cells surface. Furthermore, the CFTR protein is a strong epithelial sodium channel (ENaC) inhibitor. A dysfunctional CFTR protein causes decreased chloride transportation to the cell surface and stimulates reabsorption of sodium from the epithelial cell surface which generates increased chloride ion transportation toward the interstitium by non-CFTR channel resulting in formation of natrium chloride (NaCl) and increased water absorption by increased gradient (44). This phenomenon results in dehydrated surface liquid and consequently a dysfunctional, viscous and sticky mucus resulting in mucus impactions and impaired mucociliary clearance (2). In addition, the dysfunctional CFTR protein inhibits transportation of bicarbonate to the epithelial cell surface which reduce the pH level. Given that the high pH level in the airway surface liquid is essential for the activation of antibacterial peptides, increased acidification of surface liquid will reduce it (2).

The consequences and clinical manifestations of a dysfunctional CFTR channel vary based on the affected organ. In the lung, the impaired mucociliary clearance and ineffective antibacterial peptides result in inability to clear inhaled pathogens leading to colonization, infection and inflammation (2). The mucus impaction in the pancreas duct prevents pancreatic enzymes from passing to the gut resulting in autodigestion and destruction of exocrine glands and eventually pancreas insufficiency (45). Contrary to the epithelial cells, the role of CFTR protein on ENaC in the sweat glands is to stimulate reabsorption of sodium from the lumen and its dysfunction depresses the ENaC activity leading to increased sodium and chloride in sweat which provide the rationale for sweat test as a diagnostic method (46).

In male CF patients, early in utero the mucus impaction clots the vas deferens preventing its formation and leading to congenital bilateral absence of the vas deferens (CBVAD) and infertility (47).

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2.5.2 Clinical features and management

CF is a multiorgan disease. The CFTR protein is expressed in epithelial cells in the airways, pancreas, intestine, liver and vas deferens as well as in sweat glands (37). However, the lungs and the pancreas are the most affected organs. In fact, CF’s respiratory and gastrointestinal manifestations dominantly impact patients’ quality of life and life expectancy (1).

Advancement in the understanding of the fundamental scientific basis of CF was crucial to understand the disease pathophysiology and consequently underpinned the treatment and management of the clinical features in CF. Centralized and multidisciplinary cystic fibrosis care is recommended and absolutely necessary to meet the disease complexity (37).

2.5.2.1 In the lung

The lung manifestations are the main cause of morbidity and mortality in the CF population (1). Infection and inflammation are the hallmarks of the pathophysiology in CF airways. The dysfunctional CFTR channel in airways’ epithelial cells results in mucus impaction, impaired mucociliary clearance and reduced antibacterial activity leading to an inability to clear microorganisms (2). Furthermore, it has become more evident that CFTR is expressed in both innate and adaptive immune cells resulting in a deficient immune response in terms of reduced killing ability as well as generating an inadequate and excessive inflammatory process leading to bronchiectasis and extracellular matrix injury (45, 48). Consequently, microorganisms such as bacteria and fungi are trapped and colonize the CF airways triggering a cascade of inflammatory responses that causes structural lung damage and eventually respiratory failure (2). At an early age, the most common colonizing bacteria are Staphylococcus aureus and Haemophilus influenzae (37). Henceforward, the impaired epithelial surface become susceptible to gram-negative bacteria such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter xylosoxidans leading to a vicious circle of infection and inflammation resulting into further lung injury (2). The role of bacteria and how they should be managed have been studied extensively. Nevertheless, even though fungi such as Aspergillus fumigatus and Candida albicans are commonly isolated from the CF airways, the role of these organisms in the CF lung progression is not fully understood (49, 50).

The respiratory clinical manifestations comprise predominantly of cough, increased mucus production and episodes of pulmonary exacerbations. Lung function tests reveals progressive obstructive lung disease, and the radiologic imaging shows airway wall thickening and bronchiectasis. Treatments of lung manifestations are based on three cornerstones: Airway clearance (chest physiotherapy as well as mucus dissolving agents such as nebulized

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hypertonic saline, Dornas alfa and Mannitol), infection control with antibiotic treatment (maintenance, alternate and acute during lung exacerbations) and physical activity. Improved nutrition, including supplementation of fat-soluble vitamins and essential fatty acids, and managing complications such as CF related diabetes (CFRD) have a great impact on CF lung progression (37). At the end stage of life, CF patients end up in terminal respiratory failure requiring oxygen supplementation and, in some cases, non-invasive ventilatory support. In these cases, lung transplantation is the only rescue (51).

2.5.2.2 In the gastrointestinal tract

The CF manifestations in the pancreas involve deficiencies in both exocrine and endocrine function. Approximately 85% of CF patients are pancreatic exocrine insufficient. Unlike the lung, the pancreas is affected at birth (52). As described earlier, the mucus impaction in the pancreas duct prevents pancreatic enzymes from passing to the gut which leads to autodigestion and destruction of the exocrine pancreatic glands resulting in fat malabsorption (52). Notably, the radiological image confirms a total fatty replacement of the pancreas (14, 52). Treatment of exocrine pancreatic insufficiency is pancreatic enzyme replacement.

However, pancreatic enzymes correct the fat malabsorption only partially because of impaired bicarbonate secretion which is essential for enzymes activation (52). Furthermore, a high energy and high fat diet is recommended in CF patients due to fat malabsorption and a high metabolic rate as a consequence of increased respiratory effort and chronic inflammation. Because fat-soluble vitamins levels are low in CF secondary to the fat malabsorption, supplementation is necessary (53). Eventually, the destruction reaches beta- cells resulting in CFRD. About 30 % of adult CF patients develop CFRD which has crucial implications in both burden of treatment and CF lung progression (54). Insulin therapy replacement is the treatment of choice (55).

CF has a significant impact on the gut and liver. In the gut, the dehydrated viscous secretions and dysmotility shown in CF lead to several clinical manifestations such as meconium ileus, distal intestinal obstruction syndrome (DIOS) and obstipation (53). Whereas liver manifestations account for only 2-4 % of the CF mortality, about 10 % of CF patients have biliary cirrhosis (52, 56). However, it is common to have transient abnormal liver function tests (52). Several studies have shown that the likelihood of developing advanced CF liver disease (CFLD) decreases dramatically in adulthood (52). Yet, the available treatments for CFLD are very limited (52). Lever transplantation is an established treatment in CF patients with cirrhosis and decompensated portal hypertension (57).

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2.5.2.3 Other CF manifestations

Sino nasal polyposis, chronic sinusitis and anosmia are very common in CF (58). Although almost all CF patients have chronic rhinosinusitis and abnormalities in radiological imaging, only 10% of CF patients report sin nasal symptoms (58). The most dominant symptoms are headache, nasal obstruction and anosmia. Medical treatments include isotonic saline irrigations and nasal corticosteroids (59). Intranasal topical antibiotics, most studied tobramycin, is also an option (60). Symptomatic CF patients not responding to maximal medical treatment can be offered surgery with endoscopic sinus surgery (ESS) (58).

Decreased bone density is commonly observed in CF patients (61). Therefore, dual energy X-rays absorptiometry (DEXA) is recommended from the age of 8 and should be repeatedly performed (62). Vitamin D and K deficiency and low calcium intake as well as persistent respiratory inflammation are some of the contributing factors (61). Vitamin D, K and calcium supplementation is needed to treat and prevent low bone density. Furthermore, physical activity is recommended. Bisphosphonates should be considered in CF patients with osteoporosis or low trauma fracture (62).

Delayed puberty, male infertility and reduced female fertility are some of CF manifestations in the reproductive organs. More than 95% of men with CF are infertile due to CBVAD (63).

However, the majority have viable sperms and pregnancy is possible through using in vitro fertilization (IVF) with micro epididymal sperm aspiration (MESA). One third of CF women have difficulty conceiving and subfertility is more prevalent in patients with pancreas insufficiency. Thick cervical secretions, malnutrition and altered sex hormones are suggested as explanations to female subfertility (63). A recent study highlighted the possible direct role of CFTR on the reproductive organs as increased number of spontaneous pregnancies have been reported after introducing highly effective CFTR modulator therapy (64).

Subacute and chronic electrolyte disorders has been demonstrated in patients with CF (65).

Fluid volume depletion is the main cause of electrolytes abnormalities. Hyponatremia, hypochloremia, hypokalemia and metabolic acidosis can occur in both subacute and chronic cases (65). Parenterally supplementation is the first choice of treatment for subacute cases while oral salt supplementation is recommended in chronic cases.

Increased risk for gut malignancy has been reported (66). An observational 20 years follow up register study showed that the overall cancer risk in CF patients was not higher than non- CF patients. However, the risk of cancers arising from the digestive tract, testicles and lymphoid leukemia was higher (67). In addition, lung transplant recipients with CF had a higher overall risk to develop cancer than non CF recipient mainly from the digestive tract (68). Therefore, screening for colorectal cancer with colonoscopy from the age of 40 years

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for CF patients and from the age of 30 years in organ transplant CF patients is recommended (69).

2.5.3 CFTR modulators…correcting the basic defect

Until recently, the therapeutic approach in CF was merely based on treating the complications of the disease even though the gene was cloned 30 years ago. Over the last decade, after years of disappointment, the CF community have witnessed the most astonishing achievement in CF history, a therapy targeting the basic defect, namely the CFTR modulators. These drugs improve the CFTR protein function either by facilitating trafficking of the protein through the cell to the surface (correctors) or by increasing the gating of the CFTR channel (potentiators) (19). One potentiator (ivacaftor) and three correctors (lumacaftor, tezacaftor and elexacaftor) are licensed and approved for clinical use (29). Ivacaftor (Kalydeco®) is the first CF modulator, approved in 2011 and suitable for the most common gating mutation G551D and some other rarer gating mutations (70, 71). Studies showed a more than 10%

increase in the percentage of predicted forced expiratory volume in 1 second (ppFEV1%), decreased exacerbation rate, improved gastrointestinal symptoms, and decreased sweat chloride (70, 71). However, this drug is indicated in only a small group of the CF population.

F508del is the most common CF mutation and 85% of the CF population has at least one copy of F508del and 40% have two copies. The first compound therapy, lumacaftor and ivacaftor (Orkambi®), showed a significant though modest improvement of the CFTR function in CF patients with two copies of F508del (72). The next corrector, tezacaftor compound with ivacaftor (Symkevi®) showed a similar effect in CF patients with two copies of F508del and in patients with one copy of F508del and one “residual function” mutation, covering 50% of the CF population (73, 74). However, the impact of these two drugs on clinical outcomes are modest compared to ivacaftor with only 2.6 - 4% improvement in ppFEV1 (72-74). The pursuit of optimized therapy to restore the CFTR function reached a milestone with the newer corrector, elexacaftor. Elexacaftor in combination with tezacaftor and ivacaftor (Kaftrio®/Trikafta®) has shown an impressive improvement in lung function with 10-13.8 % increase in ppFEV1, 63% reduction of exacerbation rate and decrease in sweat chloride by 41-45 mmol/L (75, 76). Furthermore, elexacaftor/tezacaftor/ivacaftor is suitable for CF patients with at least one copy of F508del and a “minimal function” mutation covering almost 85% of the CF population (76). The remaining obstacle to overcome is inventing a targeting therapy for CF patients with rare mutations and CF patients with nonsense mutations causing no protein production (stop mutation). Most likely, CF patients

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FUNGI IN THE CF AIRWAYS

Fungi is commonly isolated in CF airways. However, the fungal prevalence and diversity vary between studies and CF sites (4, 77). The isolation rate, microbiological detection methods and environmental factors have been suggested to influence the fungal epidemiology (4, 78). For example, a prospective multicenter study demonstrated that introducing a uniform fungal culture protocol increased recovery of filamentous fungi (79).

For this reason, researchers in the field pointed out the urgency of standard fungal culture and detection protocol in CF (5, 80, 81). Moreover, the impact of geographical factors on fungal diversity have been demonstrated in an international study in CF patients which reported that Scedosporium species are more prevalent in Southern Europe and A. fumigatus in the north (81). In the CF respiratory tract, C. albicans is the most common isolated yeast and A. fumigatus is the most common isolated mold (4, 5). C. albicans and other yeast in CF did not receive so much attention as they are traditionally considered as a harmless colonizer.

A number of A. fumigatus related diseases have been described, the most known are allergic bronchopulmonary aspergillosis (ABPA) and A. fumigatus colonization. Overall, the role of fungi in respiratory progressive CF disease is still unknown (4).

3.1 FUNGAL DETECTION METHODS

In CF, bacterial culture is recommended at least four times a year, for fungal cultures there is no such recommendation yet (3). Sputum culture is the golden standard to detect fungi from the CF lower respiratory tract (50, 82). In non-expectorating CF individuals, oropharyngeal swabs (OP) which is routinely used to detect bacterial findings, is not recommended for fungal outcomes (83). Bronchoscopy with BAL is invasive and often requires anesthesia. Consequently, in this group of patient fungal prevalence probably is underestimated.

The conventional methods to detect fungi in CF is direct microscopy and fungal culture.

Direct microscopy provides a quick information about the fungal outcomes. Sample material is applied on an object slide and mixed with Blankophor or Calcofour white and examined under fluorescence microscopy (84). The morphological features are used as a guide to distinguish between yeast and some mold species. Yeast cells and pseudo hyphae are the hallmark for Candida and other yeast species, while thin septated hyphae are typical for Aspergillus, Fusarium and Scedosporium species (85).

Culture-based methods are the most common detection methods. Specifically, fungal culture has the advantages of allowing for sensitivity testing. A number of variables such as prior

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82, 86, 87). Nagano et al. demonstrated an increase in the fungal prevalence from 18% to 78% when mycological media was used instead of bacteriological media (88). Masoud- Landgraf et al. reported that homogenizing sputum resulted in significant increase in the prevalence and diversity of the fungal outcomes in CF (86).

Non-Culture-based Molecular diagnostic methods is less used in CF. Molecular methods such as polymerase chain reaction (PCR) and sequencing are often more sensitive than cultures and may provide data of genus, species and sometimes subspecies level. Reece et al.

presented higher identification rate of A. fumigatus in sputum with the PCR method in CF children compared to conventional culture method (89). However, this method cannot distinguish between live or dead species.

In our mycological laboratory at Karolinska University Hospital, sputum and BAL fluid were treated with sputolysin (1:1) before further handling. Yeast culture is performed on Sabouraud's agar with antibiotics (chloramphenicol and/or gentamicin), and on chromogenic medium (CHROMagar®, CHROMagar Microbiology, Paris, France), and incubated at 35°C for 48 h followed by incubation at 30°C for 72 h. For the identification of Candida species, the assimilation pattern was determined by the API 32C® (bioMériux, Lyon, France). From 2005, green yeast colonies on CHROMagar® were also identified to species level by BICHRO-DUBLI FUMOUZE® (Fumouze Diagnostics, Levallois-Perret, France) latex agglutination tests. Latex agglutination tests were also used to detect C. albicans, C. glabrata and C. krusei. From 2015 onwards, the identification of yeast was done by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) MS (Biotyper database v3.1; Bruker Daltoniks, Germany). Aspergillus and other molds species were identified by macro and microscopy according to the laboratory standard protocol.

Specimens were cultured on Sabouraud-dextrose agar with antibiotics (gentamicin and/or chloramphenicol) at 30°C for 7 days and potato- dextrose agar at 42°C for 7 days before moved to 30°C for an additional 3 days.

3.2 ASPERGILLUS FUMIGATUS

Aspergillus fumigatus is a ubiquitous saprophytic fungus commonly found in soil, plants, water and organic material (90). Due to its high temperature tolerance and conidia`s small size facilitating alveolar deposition, A. fumigatus is the most common filamentous fungi in humans (91). Additionally, A. fumigatus is the most isolated mold in CF airways. The role of A. fumigatus in the CF lung progression is not fully understood and CF clinicians and researchers have differing opinions in this regard.

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3.2.1 The Pathophysiology of A. fumigatus in CF airways

Hundreds of A. fumigatus conidia are inhaled regularly each day and in healthy individuals cleared successfully (92). However, patients with CF are unable to eliminate A. fumigatus conidia because of the impaired fungal defense on several levels. Defective mucociliary clearance in CF impedes the mechanical removal of conidia which is the first line of defense (93). Moreover, the killing ability by CFTR defected epithelial cells and alveolar macrophage is diminished as a result of reduced adhesion and phagocytosis of airborne conidia (93).

Further, the secretion of Pentraxin 3, a soluble pattern recognition essential for A. fumigatus clearance is reduced in CF (94).

A number of studies in vitro and in CFTR deficient animal models showed that the host antifungal inflammatory response induced by A. fumigatus per se is detrimental (95-97). For instant, exposing CFTR defected epithelial bronchial cells to A. fumigatus conidia was associated with increased secretion of proinflammatory cytokines, interleukin (IL) 6 and IL8 (97). Further, neutrophil recruitment and interleukin-1β secretion was increased in CF defected murine models and human CF cells as response to A. fumigatus challenge and the dysregulation of NLRP3 ( NOD, LRR-and pyrin domain-containing protein 3) with anakinra inhibited this pathogenic inflammatory process (95). Further, treatment with Kynurenines in CF defected murine inhibited the aberrant activation of Th17 and the inflammatory response (96). Studies conducted in humans demonstrated a higher level of reactive oxygen species (ROS) produced by neutrophils as a response to A. fumigatus challenges in CF patients, compared to healthy individuals as well as pre-treatment with CFTR modulators reduced its level (98, 99). Finally, germinated A. fumigatus released proteases and gliotoxins causing further lung damage (93).

3.2.2 Epidemiology of A. fumigatus in CF

In general, the prevalence of A. fumigatus isolates in CF airways is relatively high. Studies demonstrated a prevalence ranging between 29 to 55.9 % (100, 101). On the other hand, at an early age the prevalence of A. fumigatus is reported to be low (102, 103). However, several studies point out that the prevalence of A. fumigatus in CF children is probably underestimated (6, 8). In a longitudinal study in CF infants diagnosed by newborn screening (age 0-2 year), the prevalence of Aspergillus species in BAL was 12.5 % (8). Similarly, a recently published cross-sectional study reported a prevalence of 17.9 % of A. fumigatus in BAL in CF children before the age of 5 (6).

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3.2.3 A. fumigatus related diseases in CF

A. fumigatus can cause a variety of diseases in CF, the most recognized one is ABPA (104).

In literature, several conditions have been described, namely: A. fumigatus colonization, A.

fumigatus infection, A. fumigatus bronchitis and A. fumigatus sensitization (104-107).

However, there is no agreement on the definition of these conditions and even less on how to deal with them. In 2013, Baxter et al. proposed a new classification of A. fumigatus related disease in CF based on serological response, sputum galactomannan (GM) and real time PCR (108). They defined four classes:

Non-disease (class 1): patients with or without positive PCR, no immunological response and negative GM.

Serologic ABPA (class 2): positive PCR and GM, elevated total immunoglobulin (Ig) E and A. fumigatus specific IgE and IgG.

Aspergillus sensitized (class 3): elevated A. fumigatus specific IgE (not IgG), positive or negative PCR and negative GM.

Aspergillus bronchitis (class 4): elevated A. fumigatus specific IgG (not IgE), positive PCR and GM.

Armstead et al. propose two models to explain the above-mentioned classification (109). One model implies that A. fumigatus sensitization is a precursor to ABPA and A. fumigatus colonization to A. fumigatus bronchitis (109). The second model considers the four diseases to be independent of each other (109).

The limitation of the classification of Baxter et al. is that it does not take into consideration the clinical and radiological characteristics. Furthermore, GM in sputum is associated with high frequency of false positivity as it cross-reacts with beta-lactam antibiotics which are widely used in CF (110). On the other hand, instead of fungal cultures, which is the most common detection method, the authors used molecular methods (PCR) known to be a more sensitive detection method (89). Baxter et al. define A. fumigatus bronchitis based on microbiological and serological outcomes. While other authors define A. fumigatus bronchitis as lung exacerbation that does not respond to antibacterial treatment and recovery of A. fumigatus in sputum (49, 111). The inconsistency in the definition of A. fumigatus related diseases in CF impedes the possibility to make a comparison between studies in the field thereby limiting the possibility of achieving an agreed consensus guideline.

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3.3 ASPERGILLUS FUMIGATUS COLONIZATION

3.3.1 The clinical implication

Most Researchers and clinicians in the field regard isolation of A. fumigatus in a sputum fungal culture without immunological response as colonization. However, distinguishing between an innocent colonization and infection in the presence of A. fumigatus in CF airways is yet not possible. Further, there is no united definition of A. fumigatus colonization. Some studies distinguished between transient (one positive sputum culture in a given year) and persistent colonization (≥2 positive sputum culture in a given year) (7, 112, 113). Others adopted Leeds definition criteria used for P. aeruginosa colonization in CF (114) (9). While some studies adopted at least one positive sputum culture as definition for A. fumigatus colonization (106, 115, 116).

The impact of A. fumigatus colonization on CF lung progression and clinical outcomes has not been established yet. Studies in the field presented conflicting results (6-9). Three retrospective longitudinal studies using a similar definition of A. fumigatus colonization in a similar cohort presented different conclusions (7, 9, 113). The first one reported lower lung function in CF patients chronically colonized with A. fumigatus compared to non-colonized (7). Nonetheless, after adjustment for lung function at base line the difference become insignificant. The second one reported accelerated decline in lung function in patients chronically colonized with A. fumigatus even after adjustment for lung function at baseline (113). Interestingly, the authors suggested that CF patients with poor lung function were more susceptible for A. fumigatus persistence as patients chronically colonized with A. fumigatus had lower lung function at base line and four years before the enrolment (113). The third study also showed lower lung function in patients colonized with A. fumigatus compared to non-colonized (9). However, the difference was not significant after adjusting for confounders (BMI and age at baseline, number of hospitalizations and years of inhaled antibiotic use). The authors concluded that colonization with A. fumigatus was not associated with lung function decline (9). Similarly to the second study, the authors concluded that lower lung function in CF patient colonized with A. fumigatus was explained by lower lung function at base line (9).

Few studies investigated the role of A. fumigatus in the very young CF population (6, 8). An Australian longitudinal study showed a negative association between isolation of Aspergillus species in BAL before the age of 2 years and lung function measured as forced expiratory volume in the first three-quarters of a second (FEV 0.75) conducted later at school age (8).

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cross-sectional design and ppFEV1 as lung function measurement, compared to the longitudinal design and use of FEV 0.75 in the former study (6, 8).

Beyond lung function, the impact of A. fumigatus colonization on other outcomes such as quality of life, exacerbation rate, hospitalization and radiological changes has also been examined (7, 112, 117, 118). Worse quality of life in the respiratory domain has been reported in CF patients who were cultured positive for A. fumigatus in sputum compared to CF patient who did not (117). Furthermore, lung exacerbation and hospitalizations rate were more frequent in patients colonized with A. fumigatus (7, 118). Early changes in chest computed tomography (CT) prior to altered lung function have been shown in patients who were colonized with A. fumigatus (6, 112).

The above-mentioned studies used sputum culture as a fungal detection method. Other studies adopted different microbiological detective methods (107, 108). A French study reported a decline in lung function in CF patients who were positive for A. fumigatus specific IgG or cultured positive for A. fumigatus during the two years follow up period (107).

Another study using PCR as a detection method presented no impact on lung function for the same follow-up period (108).

Identifying patients at risk to be colonized with A. fumigatus in CF is of great importance. A number of parameters and clinical characteristics have been investigated in this topic. Older age, inhaled corticosteroid, macrolides use and antibiotic treatment (inhaled, intravenous (IV) or continuously) demonstrated as risk factors for A. fumigatus colonization in CF (119-121).

Lifestyle has also been examined as risk factor for colonization with A. fumigatus. CF patients living in urban areas have higher rate of A. fumigatus isolation (122). However, all these studies have a retrospective design.

3.3.2 Antifungal treatment of A. fumigatus colonization… should we or should we not

Yet there is no recommendation as to whether A. fumigatus colonization should be treated or not. However, in a survey study in the UK, 66% of adult physicians and pediatricians believed that A. fumigatus colonization is harmful, and they would treat it (123). Clinical trials addressing treatment benefits are very scarce. The only randomized study in CF patients chronically colonized with A. fumigatus was with itraconazole (124). No beneficial outcomes were reported regarding lung function, quality of life or the number of exacerbations that required IV antibiotics (124). The main limitation of this study was that 50% of the patients treated with itraconazole did not reach a therapeutic concentration level in serum (124). A prospective study on the effectiveness of posaconazole in A. fumigatus related disease in CF

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children including ABPA showed an improvement in lung function (125). Other studies in this field were case reports. Authors described beneficial clinical effects after receiving antifungal agents in CF patients presented with clinical deterioration which did not respond to antibacterial treatment (111, 126, 127). The existing evidence is insufficient to provide clinicians with tools to make an adequate treatment decision. There are neither serological, microbiological nor radiological markers to guide clinicians to determine who, when and how to treat. Treatment indications, choice of antifungal agents, adequate dosage (as CF patients have altered pharmacokinetics), therapeutical monitoring and treatment duration are some of the aspects that are needed to be clarified.

3.3.3 The pharmacokinetics of posaconazole (an antifungal agent)

During the last decade, posaconazole have been widely used in CF patients with A. fumigatus related disease in our center because of the difficulty with reaching therapeutic concentrations of itraconazole and the side effects of voriconazole. Posaconazole is a triazole antifungal agent approved initially as an oral formulation in 2005 and later as a delayed-release tablets formulation in 2014 (128, 129). In general, the drugs pharmacokinetics in CF patients is impaired due to the altered absorption ratios, a larger volume of distribution, and increased renal and non-renal clearance (130). For this reason, CF patients require higher dosages of antibiotics (131). Such recommendation is lacking regarding antifungal agents in CF. Studies assessing whether higher dosages of antifungal agents is also required in CF are scarce.

Clinical trials have reported low posaconazole concentration level in serum in lung transplanted CF patient (132, 133). A recent study concluded that higher dose posaconazole (400 mg twice a day followed by a dose of 400 mg once a day ) is required to achieve a therapeutic concentration level in CF teenagers (age of 12-17 years) (134).

To accomplish efficacy and reducing risk for adverse events and developing resistance, the drug concentration needs to reach a therapeutic level particularly in the targeted organ. A pharmacokinetic study performed on posaconazole in healthy individuals in plasma, epithelial cells liquid and alveolar cells obtained by bronchoscopy demonstrated high intrapulmonary posaconazole concentration (48-fold greater in alveolar cells than in serum) concluding god posaconazole penetration in the lungs (135). However, whether the intrapulmonary pharmacokinetics of posaconazole in CF airways is as high as in non-CF individuals and whether serum concentration reflects the concentration in the airways particularly in CF patients with advanced lung damage has not yet been addressed.

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3.4 ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS (ABPA)

ABPA is the most known A. fumigatus related disease in CF. The disease is a result of IgE mediated hypersensitivity to A. fumigatus peptides. The disease affects only patients with CF or asthma. There are several proposed criteria for ABPA, the most commonly used is the one proposed by the Cystic Fibrosis Foundation Consensus Conference published in 2003 (136).

The diagnosis criteria are presented in Table 1. The prevalence of ABPA in CF reported to be underestimated as symptoms and radiological features related to ABPA overlap with those seen in CF (109). Therefore, annual screening is recommended (136). Untreated ABPA result in progressive lung disease (137, 138). The main goal of treatment is to suppress the immunological response and reduce the fungal burden. According to the Cystic Fibrosis Foundation Consensus Conference recommendation, the first line of treatment is high dose systemic corticosteroids (0.5 mg- 2 mg/kg) for the first 1-2 weeks, then gradually reduced and phased out within 12 weeks. Unfortunately, there is a considerable risk to develop adverse events related to the high dose and prolonged treatment with corticosteroid. In patients who do not respond to systemic corticosteroids or experience relapse, adding an antifungal agent is recommended. Nonetheless, monotherapy with antifungal treatment have been reported to have a beneficial effect (125, 139-141). Lewington-Gower et al. proposed a new approach: using either medium dose corticosteroids or antifungal agents as monotherapy as first line treatment (142). However, systemic corticosteroids is still recommended in patients with mucoid impaction or those with a decline in lung function (143).

Table 1: Consensus Conference Proposed Diagnostic Criteria for ABPA in CF

Classic case Minimal diagnostic criteria

1) Acute or subacute clinical deterioration not attributable to another etiology.

2) Total serum IgE concentration of 1500 IU/mL (1200 ng/ mL).

3) Immediate cutaneous reactivity to Aspergillus or positive A. fumigatus specific IgE antibody.

4) Precipitating antibodies to A. fumigatus or elevated Aspergillus specific IgG antibody to A. fumigatus by an in vitro test.

5) New or recent abnormalities on chest imaging.

4) One of the following:

a. Precipitating antibodies to A. fumigatus or elevated Aspergillus specific IgG antibody to A. fumigatus by an in vitro test.

Or

b. New or recent abnormalities on chest imaging.

FUNGAL COLONIZATION AND INFECTION IN CYSTIC FIBROSIS – PREVALENCE, CONSEQUENCES AND INTERVENTION

From DEPARTMENT OF CLINICAL SCIENCE, INTERVENTION AND TECHNOLOGY, DIVISION OF

PEDIATRICS

Karolinska Institutet, Stockholm, Sweden

FUNGAL COLONIZATION AND INFECTION IN CYSTIC FIBROSIS – PREVALENCE, CONSEQUENCES AND INTERVENTION

Mahasin Al Shakirchi

Stockholm 2022

Fungal colonization and infection in Cystic Fibrosis - prevalence, consequences and intervention

THESIS FOR DOCTORAL DEGREE (Ph.D.)

Mahasin Al Shakirchi

POPULAR SCIENCE SUMMARY OF THESIS

POPULÄRVETENSKAPLIG SAMMANFATTNING

ABSTRACT

LIST OF SCIENTIFIC PAPERS

Scientific paper not included in the thesis:

CONTENTS

LIST OF ABBREVIATIONS

INTRODUCTION

CYSTIC FIBROSIS

FUNGI IN THE CF AIRWAYS