Fungal infections in patients with cystic fibrosis 1 ProFessional article Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia Korespondenca/ Correspondence: rok tomazin, e: rok.tomazin@mf.uni- lj.si Ključne besede: cistična fibroza; glivne okužbe; aspergillus; candida; scedosporium Key words: cystic fibrosis; fungal infections; aspergillus; candida; scedosporium Citirajte kot/Cite as: Zdrav Vestn. 2017; 86(1–2):42–52 received: 18. 5. 2016 accepted: 7. 12. 2016 Zdrav Vestn | januar – februar 2017 | letnik 86Professional article Microbiology and immunology Fungal infections in patients with cystic fibrosis Glivne okužbe pri bolnikih s cistično fibrozo rok tomazin, tadeja Matos Abstract In the last two decades prevalence of fungal infections is increasing for various reasons. One of them is the advance of medical science and the associated longer life expectancy in some patient groups. This includes cystic fibrosis patients who encounter fungal diseases already in their childhood. Fun- gal pathogens isolated in high frequencies from the respiratory tract include Aspergillus fumigatus, Candida albicans and Scedosporium apiospermum. In the case of cystic fibrosis, these organisms normaly colonise the respiratory and intestinal mucosae and can cause hipersensitivity reactions and invasive diseases. The fungus-patient interactions are complex and depend on several different factors which determine what course will the colonisation/infection take. Izvleček V zadnjem času se pojavnost glivnih okužb povečuje iz različnih razlogov. Eden od njih je napredek v medicinski znanosti in z njim povezana daljša pričakovana življenjska doba določenih skupin bol- nikov. Sem spadajo tudi bolniki s cistično fibrozo, ki se s problematiko glivnih patogenov srečujejo že v otroštvu. Največjo vlogo imajo glive Aspergillus fumigatus, Candida albicans in Scedosporium apiospermum. Pri cistični fibrozi so ti organizmi kolonizatorji dihal in prebavil, povzročitelji pre- občutljivostnih reakcij in v redkih primerih povzročitelji invazivnih okužb. Do kakšnega poteka bo prišlo, je odvisno od značilnosti posameznega bolnika, glive in okolja ter izredno kompleksnih inte- rakcij med njimi. Cystic fibrosis and fungi Cystic fibrosis (CF) is one of the most common autosomal recessive genetic diseases. One in every twenty-seven people carries a recessive allele. The dis- ease occurs as the result of mutations in the CFTR gene (Cystic Fibrosis Trans- membrane Conductance Regulator), which affects the irregular synthesis of the protein involved in the transport of chloride ions across the membrane of the epithelial cells. Consequently, many organ systems are affected, especially the respiratory tract  (1). Disturbances in the transport of ions on the respira- tory epithelium and the resulting change in viscosity of the secretions hinder the functioning of the mucociliary escala- tor which creates favourable conditions for the development of various infec- tions. They are most frequently caused by Staphylococcus aureus, Haemophi- lus influenzae, Burkholderia cepacia and Pseudomonas aeruginosa. The latter is considered one of the main causes for the progressive reduction in pulmonary functions in many CF patients  (1,2). In 2 Zdrav Vestn | januar – februar 2017 | letnik 86 Microbiology and iMMunology recent times, more interest has been paid to infections caused by opportunistic pathogenic fungi. Fungi are ubiquitous eukaryotic or- ganisms, which may colonise the respir- atory and digestive tracts and causehy- persensitivity reactions and infections in CF patients (3). The most common types of fungi isolated in CF patients include Aspergillus fumigatus, Candida albicans and Scedosporium apiospermum  (3). Initially, disorders or weakening of the immune system are not typical for CF patients, therefore the localisation of fungi is limited to the respiratory epithe- lium. Irregular mucus secretion from the lower respiratory tract causes long-term exposure to fungal antigens, which leads to the development of hypersensitivity reactions. Consequently, chronic coloni- sation and hypersensitive reactions are the main types of relationship between fungi and a CF patient. In immunosup- presed state, which normally occurs af- ter lung transplantation, the relationship between opportunistic pathogens and the patient can become more severe. In such cases, though very rare in CF pa- tients, fungi can invade the lung paren- chyma and cause an invasive pulmonary mycosis, which, in the absence of an im- mune response, can progress into multi- organ disseminated infection associated with high mortality. Clinical mycology in cystic fibrosis Every day, CF patients are exposed to an environment inhabited by fungi, including moulds and yeasts. A wide variety of fungi can be isolated from respiratory samples but the two most documented and studied agents are A. fumigatus and C. albicans. Other com- mon and interesting isolates include S. apiospermum, Exophiala dermatitidis and Rasamsonia argillacea, whose role in CF pathobiology has yet to be defined. Fungi of the genus Candida Species of the genus Candida are as- comycetous yeasts, which can normally be found in the oral and intestinal mu- cosa, moist areas of the skin, the upper respiratory tract and genital mucosa. They are present in people with a normal immune response, as well as in patients with immunodeficiencies. Table 1 sum- marises interactions between candida and a CF patient. Factors associated with the isolation of yeasts or candida include pancreatic insufficiency, osteopenia, diabetes mel- litus and the concurrent colonisation with the bacteria P. aeruginosa (4,5). The high proportion of CF patients colo- nised with candida (49.4 %) is mainly due to the progression of the underlying disease (6). The most frequently isolated species is C. albicans, followed by Can- dida glabrata, Candida parapsilosis, Can- dida krusei and Candida tropicalis. They are often recovered from the sputum, but the clinical significance of these isolates is questionable because of the natural occurrence of yeasts in the oral cavity and pharynx; there is no proof support- ing the efficiency of antifungal treatment in these cases (7,8). In certain situations, the commensal relationship in CF patients turns into parasitism. The most commonly en- countered are superficial infections that affect the oral and/or genital mucosa. There have been documented cases of oropharyngeal candidosis, the develop- ment of which is enhanced by the im- paired functioning of the salivary glands, CF-related diabetes, steroid treatment and broad-spectrum antibiotic treat- ment (6). Since there may also be other causes for a similar symptomatology in Fungal infections in patients with cystic fibrosis 3 ProFessional article CF patients (e.g. vitamin B complex de- ficiency), microbiological confirmation is always advised (6). Female CF patients often suffer from fungal vulvovaginitis; its occurrence is probably underesti- mated as very little attention is paid to this type of fungal infections in CF (9). Most superficial candidoses are success- fully treated with fluconazole, while se- rious cases and chronic recurrences of the infection are treated with itracona- zole (10,11). In addition to the aforementioned su- perficial infections, Candida spp. can also cause candidaemia and invasive can- didosis, which can develop into septic shock if not treated properly. The origin of the infection is usually endogenous – candida are part of the normal gastro- intestinal microbiota. The origin of the infection can also be the various vascu- lar catheters on which yeasts form bio- films. Lung transplant patients who re- ceive immunosuppressives after surgery are at particularly high risk for invasive candidosis  (5,6). The haematogenous dissemination of candida mainly affects the kidneys, spleen, skin, eyes, heart and meninges (12). Candidaemia that devel- ops into sepsis is associated with a high mortality rate ranging between 30 and 60 %  (13). In order to monitor the pro- gression of candidaemia and determine the damage to the organs, a fundoscopy and a transoesophageal echocardiog- raphy are advised for neutropenic pa- tients (14). The first choice for treatment are antifungals from the group of echi- nocandins, usually caspofungin  (10,11). The choice of antifungal medication de- pends on the type of pathogen and the site of infection, among other factors. Fungi of the genus Aspergillus Species of the genus Aspergillus are ubiquitous filamentous ascomycete fun- gi. They are found in the air, water, dust and decomposing plants. Respiratory in- fection or colonisation occurs through the inhalation of aerosolised conidia. The latter are small enough (2–5 µm) to enter the lower respiratory tract, but in a healthy person they are expelled regularly by the clearance and defensive functions of the respiratory epithelium. In CF patients these two functions are compromised, therefore various types of Table 1: summary of the roles of different species of the genus Candida in cF patients. Infection, colonisation with Candida spp. Colonisation Surface infection Systemic infections Risk factors in CF diabetes, osteopenia, colonisation with Pseudomonas aeruginosa diabetes, treatment with corticosteroids, treatment with broad-spectrum antibiotics immunosuppression after transplant surgery Clinical significance short-term: / long-term: progression of the underlying disease candidosis of the oral cavity and pharynx, fungal vulvovaginitis candidaemia, disseminated candidosis, invasive candidosis Medication of choice / Fluconazole, itraconazole (10,11) echinocandins, fluconazole, amphotericin b or based on the type of fungus and the results of susceptibility testing (10,11) 4 Zdrav Vestn | januar – februar 2017 | letnik 86 Microbiology and iMMunology fungi, most commonly A.fumigatus, can be isolated from the respiratory tract of a large number of patients (5). Judging by the cultivation results, the prevalence of A. fumigatus is between 6 and 57 % (15). The isolation of this fungus from respir- atory samples can indicate colonisation, allergic reactions or an infection (13). Ta- ble 2 summarises interactions between aspergilli and a CF patient . The inhalation of aspergillus conidia leads to different clinical manifestations, which are difficult to distinguish from each other because they overlap to some extent (15,16): a. intermediate colonisation, when the patient is only temporarily colonised, b. allergic sensitisation, when a patient becomes sensitive to aspergillus anti- gens, c. chronic colonisation, when a patient is colonised over a long period of time with one or more strains, d. allergic bronchopulmonary aspergil- losis (ABPA), which represents the intermediate state between colonisa- tion and infection, e. aspergilloma, f. invasive pulmonary aspergillosis, and g. aspergillus bronchitis. The leading factors related to the iso- lation of moulds in CF patients are: the age above 18 years, diminished pulmo- nary function and inhalation treatment with antibiotics. Long-term exposure to mould aller- gens, which is the result of inadequate clearance of the respiratory epithelium, leads to the occurrence of various hy- persensitivity reactions. These are most frequently caused by moulds Alternaria alternata, Cladosporium herbarum, Pen- icillium spp. and A. fumigatus, the latter playing the most important role in CF patients. Allergens are either components of the cell wall or secondary metabolites that are secreted by the fungus into its immediate surroundings  (17). Classi- fied as A. fumigatus allergens are vari- ous polysaccharides, elastases, catalase, superoxide dismutase, glycopeptides and nucleo- and proteolytic enzymes. Hypersensitivity reactions are mostly chronic in nature and are not related to a patient’s deteriorated immunity; they can lead to the aggravation of the under- lying disease in CF patients (17,18). With the onset of immunosuppression, some of the aforementioned allergens take on the role of virulence factors, which en- able the development of an invasive in- fection. Ranked first among the diseases caused by A. fumigatus is allergic bron- chopulmonary aspergillosis (ABPA), which affects between 7 and 9 % of CF patients (19). It mostly affects adults (15). In ABPA the conidia typically germinate and the mycelium colonises the bron- chial tree without invading the lung pa- renchyma. An inflammatory response to aspergillus antigens develops, regulated by CD4 Th2 cells, which secrete various inflammatory cytokines, especially IL-4 and IL-10 (17,20). This is followed by the invasion of vasoactive substances and the emergence of specific antibodies, es- pecially of the IgE class. Such an inflam- matory reaction is clinically manifested as a productive cough, the emergence of pulmonary infiltrates and central bron- chiectases, and typically also peripheral and pulmonary eosinophilia and an in- crease in the concentration of IgE in the serum  (17,20). Possible complications include pulmonary fibrosis and chronic cavitary aspergillosis, which can develop into aspergilloma (21). ABPA diagnosis is based mainly on the clinical features and immunological findings . ABPA mostly Fungal infections in patients with cystic fibrosis 5 ProFessional article affects patients with bronchial asthma and CF patients; in the latter a correct di- agnosis is more difficult to make because the symptoms of the underlying disease the manifestations of ABPA overlap. The minimum diagnostic criteria for ABPA in CF patients are (19): a. subacute or acute clinical aggravation of the disease in absence of any other possible cause; b. elevated total serum IgE levels of > 500 IU/mL, except in patients re- ceiving corticosteroid treatment who need to repeat testing after comple- tion of treatment. If the concentra- tion of IgE is between 200 and 500 IU/mL, tests are repeated after one to three months; c. immediate skin sensitivity to A. fu- migatus antigens if the patient is not receiving antihistamine treatment or in vitro presence of serum IgE specific for A. fumigatus; d. detection of precipitating antibodies or in vitro presence of serum IgG spe- cific for A. fumigatus, or e. presence of new or recent radiologi- cal irregularities in the thorax that persist despite antibiotic treatment or physiotherapy. The fungus A. fumigatus can be iso- lated from 60 % of CF patients, but the cultivation has no diagnostic signifi- cance. ABPA is traditionally treated with corticosteroids, which are used in acute aggravation and as maintenance therapy to prevent progressive advancement of the damage to the lung tissue  (19,22). Currently, apart from corticosteroid therapy, recombinant monoclonal anti- bodies (e.g. omalizumab) are being in- troduced to prevent IgE from binding to effector immune cells  (22-24). Because this treatment option for ABPA has only been used for a few years, all the pros and cons of this treatment remain un- known. Apart from the aforementioned groups of medication used to treat ABPA, various antifungal drugs are also used, lessening the load of aspergilli and thus reducing episodes of exacerbation and improving the pulmonary func- tion (22,25). CF patients also exhibit the so-called allergic sensitisation (AS), which is still poorly defined. This term is used to de- scribe hypersensitity reactions which do not meet all of the criteria necessary for making a diagnosis of ABPA (17). AS supposedly affects between 20 and 65 % of CF patients (16). The problems of es- tablishing the prevalence of AS lie in its overlapping clinical features with ABPA and the use of different diagnostic mi- crobiological methods. Another problem is poor comparability of commercially available tests and in-house tests  (16). Recently, differences have been identi- fied that might enable a relatively simple differentiation between ABPA and AS based on the determination of CDC203c basophils concentration, galactomannan antigen in lower respiratory tract sam- ples and pecific IgG in serum. In con- trast with ABPA patients, patients with AS have a lower plasma concentration of CDC203c basophils, a negative ga- lactomannan test (< 0.5) and a positive A.fumigatus-specific serum IgG (< 75µg/ mL) (13,16,27). In addition to colonisation and hy- persensitivity reactions, fungi also cause infections that can affect several organ systems. They are very aggressive and associated with high mortality, but are fortunately relatively rare. Unlike hyper- sensitivity reactions, invasive mycoses normally affect immunocompromised patients. The most significant risk factor for the development of invasive mycoses 6 Zdrav Vestn | januar – februar 2017 | letnik 86 Microbiology and iMMunology in CF patients is lung transplantation and the accompanying immunosuppres- sive treatment. The most common infection is inva- sive pulmonary aspergillosis (IPA) and only rarely disseminated invasive asper- gillosis (8). The most significant risk fac- tors for the development of IPA in CF pa- tients are colonisation of the respiratory tract with A. fumigatus before transplant surgery and a positive intraoperative cul- ture (28,29). Post-transplant CF patients are exposed to an increased risk for IPA as a large proportion of patients are col- onised with A. fumigatus (up to 57 %) even before transplantation  (15,29). The occurrence of the infection is enabled by different virulence factors, especially hydrolytic enzymes (elastases, proteases, phospholipases, catalase) and gliotoxin, which further weaken the functioning of the mucociliary escalator, damage the respiratory epithelium and participate in the formation of biofilms (30,31). IPA usually occurs approximately a month and a half after lung transplan- tation and affects about one fifth of the patients (29). The infection starts in the lower respiratory tract with invasion of hyphae into the lung parenchyma. Typi- cal of the fungus A. fumigatus is angioin- vasion, resulting in thrombosis, infarc- tion and necrosis of lung tissue (12). CF patients also exhibit a non-angioinvasive type of IPA, which can occur in the ab- sence of neutropenia (6). IPA is associ- ated with a relatively high mortality (13), which in the case of CF normally does not exceed 16 % (29). Diagnosing IPA is difficult; it demands the consideration of clinical features, and the use of diagnos- tic imaging – especially CT scans of the thorax – and microbiological methods, such as the cultivation of bronchoscopic samples, detection of galactomannan and β-D-glucan antigenaemia and de- tection of aspergillus DNA in the af- fected tissues, bronchoalveolar lavage and blood. The gold standard for diag- nosis remains the detection of aspergilli in the primarily sterile clinical sample with cultivation and histopathological evidence of the invasion in the affected tissue (32). Voriconazole is the drug of choice in CF patients with IPA. When voricona- Table 2: summary of the roles of different species of the Aspergillus genus in cF patients Infection, colonisation with Aspergillus spp. Colonisation Hypersensitivity reactions Infections Risk factors in case of CF age above 18, inhalation treatment with antibiotics, FeV1 reduction long-term exposure to fungal allergens lung transplant immunosuppression, uncontrolled/untreated abPa, colonisation of respiratory organs with aspergilli before a lung transplant Clinical significance long-term colonisation as a risk for the development of hypersensitivity reactions and infections abPa, as invasive (pulmonary) aspergillosis, chronic cavitary aspergillosis, aspergilloma, aspergillus bronchitis Medication of choice / corticosteroids, recombinant monoclonal antibodies, triazoles (voriconazole or itraconazole) (10,11,22) Voriconazole, amphotericin b (10,11) Key: ABPA – allergic bronchopulmonary aspergillosis, AS – allergic sensitisation, FEV1 – forced expiratory volume in one second Fungal infections in patients with cystic fibrosis 7 ProFessional article zole is not the optimal choice, ampho- tericin B is recommended (10,11). In CF patients, unlike in any other patient group, the fungus A. fumigatus creates unique clinical features, referred to as aspergillus bronchitis. It was first described in 2006 and defined six years later (33,34). Aspergillus bronchitis is de- scribed as chronic inflammation of the lower respiratory tract (bronchitis) with A. fumigatus confirmed in the culture of bronchoscopic samples or detected with molecular methods, and the pres- ence of increased specific IgG in the se- rum (31,33,34). The most significant fac- tor that helps to differentiate between colonisation and aspergillus bronchitis is clinical improvement in a patient receiv- ing antifungal treatment; the medication proves ineffective in A.fumigatus coloni- sation. The value of antifungal treatment in these cases is not yet well defined, but antifungals from the group of triazoles, especially itraconazole and voriconazole, should be considered (33,34). Exophiala dermatitidis Among the fungi often associated with the colonisation of the respiratory tract in CF patientswe also find the fun- gus Exophiala dermatitidis, which the general public usually refers to as »black yeast«. It is a fungus with a melaninated cell wall, which appears in yeast form as a young culture and becomes increas- ingly filamentous as it matures. In parts of the world with a moderate climate it is rarely isolated from nature; it is mostly associated with the human environment, such as spas, saunas and dishwashers. In short, it can be found in a warm and moist environment  (35,36). We assume that the original natural habitat of E. der- matitidis is the tropical rainforest (35). In medicine, E. dermatitidis is known as the cause of infections of the skin and subcutaneous tissue, especially in tropi- cal climates. There are also rare docu- mented cases of infections affecting the central nervous system (37). The role of E. dermatitidis in CF is still unknown. Table 3 summarises interactions between E. dermatitidis and a CF patient. In Europe, E. dermatitidis is isolated from 5 to 19 % of respiratory samples; descriptions of supposed invasive infec- tions are rare (38-41). None of the docu- mented cases meet the criteria for a con- firmed invasive mycosis – the described diagnoses are usually based on the isola- tion of E. dermatitidis from the sputum. Improvement was noted with antifungal treatment. The patients can be treated exclusively with amphotericin B or with a combination of amphotericin B and flucytosine. Treatment with itraconazole has proved to be the most efficient (36- Table 3: overview of the emerging fungal pathogens and their clinical significance in cF. Exophiala dermatitidis Scedosporium apiospermum Rasamsonia argillacea Risk factors in case of CF age above 12, pancreatic insufficiency, colonisation with A. fumigatus unknown. unknown. Clinical significance colonisation, invasive infections during immunosuppression colonisation, invasive infections during immunosuppression colonisation, invasive infections during immunosuppression Treatment amphotericin b ± flucytosine, itraconazole (36-39) Voricozanole, posacozanole (50-52) echinocandins? (54,56) 8 Zdrav Vestn | januar – februar 2017 | letnik 86 Microbiology and iMMunology 39). There are, however, reports of a long- term colonisation of the respiratory tract where even several months of itracona- zole treatment did not eliminate from the sputum (42). Most research indicates that E. der- matitidis has the role of a coloniser in CF and that the isolation from the respira- tory tract normally does not indicate an infection. At the moment, the identified risk factors for the colonisation with E. dermatitidis are the age above 12, pancre- atic insufficiency and the colonisation with A. fumigatus  (40,43). It is interest- ing that the occurrence of E. dermatitidis drops after the age of 35 (41). The coloni- sation is supposedly also dependent on the patient’s genotype: it was most com- mon in patients with a mutation of the CFTR gene at position 508 on chromo- some 7 (genotype ∆F508/∆F508) (40). It is mostly a chronic colonisation, which can be established only after an extend- ed incubation period on classic and/or specific mycological media with the ad- dition of erythritol and chlorampheni- col (40). For the isolation of E. dermatiti- dis, clinical samples have to be incubated for 5 to 7 days on average (43). Fungi from the genus Scedosporium Apart from A. fumigatus, Scedospori- um apiospermum is another important filamentous fungus that causes invasive infections in CF patients; it causes ex- tremely rapid, aggressive infections quite resistant to antifungal treatment. Table 3 summarises interactions between S. api- ospermum and a CF patient. Scedosporium spp. are filamentous ascomycetes found mainly in water and soil all around the world. Their chang- ing taxonomy makes classification dif- ficult: today, the species once belonging to genus Scedosporium are reclassified into the genera Parascedosporium, Lo- mentospora, Petriella, Petriellopsis, Pseu- dallescheria and, of course, Scedospori- um  (44). Consequently, the term Pseudallescheria boydii/Scedosporium apiospermum complex is usedto refer to most of the frequently isolated, medically relevant species. For easier understand- ing, we will discuss S. apiospermum. Globally speaking, S. apiospermum is the second most frequent mould isolated from respiratory samples in CF patients and is associated with chronic colonisa- tion  (45,46). The prevalence is between 5.7 and 10 % (47). Invasive scedosporiosis is very rare in CF patients and there have been only eight documented cases since 1996; unfortunately, all of them ended in death  (47,48). Most of the patients had a previously detected colonisation with S. apiospermum. Therefore the isolation of this mould from the respiratory tract is a contraindication for lung transplan- tation  (47). Apart from invasive infec- tions, there have also been documented cases of allergic bronchopulmonary sce- dosporiosis (49). Scedosporiosis is difficult to treat be- cause the fungus is resistant to several antifungal drugs. S. apiospermum fungi are intrinsically resistant to ampho- tericin B and flucytosine, and usually re- act only to triazole treatment; voricona- zole and posaconazole may be used (50). In vitro activity against S. apiospermum is also exhibited by the more recent isa- vuconazole  (50). For now, voriconazole remains the drug of choice (52). Rasamsonia argillacea Rasamsonia argillacea is a filamen- tous ascomycete fungus. It belongs to the group of the so-called emerging patho- gens as its significance in medicine has been researched only in the past few years. R. argillacea is a thermotolerant mould, which morphologically resem- Fungal infections in patients with cystic fibrosis 9 ProFessional article bles the genera of Penicillium and Pae- cilomyces  (53,54). It is, therefore, often misidentified and its prevalence is thus probably underestimated. 2.6 % of CF patients are supposedly colonised  (55). Table 3 summarises interactions between R. argillacea and a CF patient. R. argillacea is most frequently iso- lated from respiratory samples in CF patients and in patients with chronic granulomatous disease (54). Similarly to E. dermatitidis, R. argillacea is isolated using dithiothreitol liquefaction of vis- cous clinical samples from the lower res- piratory tract (43). R. argillacea forms a complex of related, morphologically in- separable species, among which the most common are R. argillacea sensu stricto, R. piperina and R. aegroticola  (55). The clinical significance of the isolation of species from the R. argillacea complex in CF patients is still unknown, but it prob- ably just indicates colonisation of the respiratory epithelium (54,56). The correct identification of moulds is not only important to obtain epide- miologica information, but is also neces- sary to choose the correct antifungals in the event of an infection. It is typical for the R. argillacea fungus to exhibit high minimal inhibitory concentrations for amphotericin B and antifungals from the azole group, especially voriconazole, while the minimal inhibitory concen- trations for the echinocandin group are low (54,56). Conclusion People are constantly in contact with fungi as these are normally present in our environment. In CF patients the in- haled fungi are not expelled from the lower respiratory tract because of the impaired clearance function, which ena- bles their growth and leads to different clinical conditions. 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