Author: lowes1

If you or someone you know suffers from Severe Asthma, then it will be worth your time to learn about the world of Asthma and Fungi. While this paper is pretty technical and lengthy by design, it will provide you with a glimpse of how fungi and asthma are linked.

The World Asthma Foundation (WAF) salutes the Department of Paediatric Respiratory Medicine, Royal Brompton Hospital Harefield NHS Foundation and Paediatric Respiratory Medicine, National Heart and Lung Institute, Imperial College, Sydney Street for their contribution to medicine and those that suffer from Asthma. The WAF will cover this topic in more depth in future post.

Fungi have many potential roles in paediatric asthma, predominantly by being a source of allergens (severe asthma with fungal sensitization, SAFS), and also directly damaging the epithelial barrier and underlying tissue by releasing proteolytic enzymes (fungal bronchitis). The umbrella term ‘fungal asthma‘ is proposed for these manifestations. Allergic bronchopulmonary aspergillosis (ABPA) is not a feature of childhood asthma, for unclear reasons. Diagnostic criteria for SAFS are based on sensitivity to fungal allergen(s) demonstrated either by skin prick test or
specific IgE. In children, there are no exclusion criteria on total IgE levels or IgG precipitins because of the rarity of ABPA. Diagnostic criteria for fungal bronchitis are much less well established. Data in adults and children suggest SAFS is associated with worse asthma control and greater susceptibility to asthma attacks than non-sensitized patients. The data on whether antifungal therapy is beneficial are conflicting. The pathophysiology of SAFS is unclear, but the epithelial alarmin interleukin-33 is implicated. However, whether individual fungi have different pathobiologies is unclear. There are many unanswered questions needing further research, including how fungi interact with other allergens, bacteria, and viruses, and what optimal therapyshould be, including whether anti-neutrophilic strategies, such as macrolides, should be used.

Considerable further research is needed to unravel the complex roles of different fungi in severe asthma.

1. Introduction
   The important role of fungi in worsening asthma has long been appreciated. In 2006, the term
   ‘severe asthma with fungal sensitization (SAFS)’ was first proposed in a review article that rightly
   acknowledged the historical evidence implicating fungi in the pathophysiology of asthma going
   back to the seventeenth century [1]. The role of fungi in asthma remains controversial to the present
   day, and these issues are reviewed below. What is certainly beyond dispute is that (a) although
   most acute attacks of asthma are precipitated by a viral infection, a sudden heavy aeroallergen load,
   such as grass pollen (“thunderstorm asthma”) [2] or soya bean (ships unloading in the docks of
   Barcelona) [3], can precipitate severe attacks, which might be eosinophilic rather than neutrophilic
   [4]; and, (b) fungal allergens can also cause acute attacks of asthma [5–7].
   The term SAFS focuses on allergic sensitization, but this is quite restrictive, because allergy is
   not the only mechanism whereby fungi can modulate asthma. Additional to allergic sensitization,
   which does not necessarily require airway fungal infection, is the release of tissue damaging
   proteases and other enzymes, which might disrupt the airway epithelial barrier and cause mucosal
   damage and airway remodeling [8]. For this to happen, a chronic fungal bronchitis needs to be
   J. Fungi 2019, 6, 55 2 of 17
   established. Sensitization and tissue damage both may co-exist. Here, I propose that the more
   general term ‘fungal asthma’ is used to encompass allergic sensitization (SAFS), fungal bronchitis,
   and combined sensitization/bronchitis (Figure 1). In adults, this would also include ABPA, not
   discussed here because of the rarity of this condition in children with asthma. All three entities may
   potentially benefit from anti-fungals, but fungal bronchitis without sensitization should not require
   the intensification of anti-Type 2 inflammatory medications. Of course, the pro-inflammatory
   effects of tissue damaging enzymes may merit treatment (as, for example, the anti-inflammatory
   strategies that may be used in cystic fibrosis (CF) [9,10] to counter the effects of infection driven,
   neutrophilic tissue destruction. The picture might also be dynamic; increasing inhaled steroids may
   cause topical immunosuppression (discussed in more detail below) and, thus, predispose to fungal
   bronchitis as a secondary phenomenon.
   Figure 1. Schematic of fungal involvement in asthma in children, in whom the diagnosis of allergic
   bronchopulmonary aspergillosis (ABPA) is rarely made.
   The justification for this sort of phenotyping is that it is clinically useful, because defining it
   leads to a change in management. Unfortunately, much of the paediatric guidance has had to be
   extrapolated from work in adults. The aim of this review is to assess the clinical utility of current
   concepts of fungal asthma (as defined above) in children, and suggest new approaches and where
   future work is needed. Although this review will focus as far as possible on children, it inevitably
   has to supplement this with adult experience and animal and cellular models where paediatric data
   are not available. Prior to writing this manuscript, a literature search was performed while using
   the search term limited to English Language papers,
   which was supplemented from the author’s personal archive of references.
2. Definition of SAFS and Fungal Bronchitis
   2.1. SAFS in Adults
   SAFS was first defined in adults [11], and it has been suggested that it is a more severe
   phenotype than seen in unsensitized patients. For the purposes of the definition of SAFS, severe
   asthma is defined as treatment with 500 mcg Fluticasone/day or equivalent, or continuous oral
   corticosteroids, or four prednisolone bursts in the previous 12 months or six in the previous two
   years (as with so many definitions of severity, the figures are fairly arbitrary). The immunological
   J. Fungi 2019, 6, 55 3 of 17
   criteria for SAFS in adults also include a total immunoglobulin (Ig)-E < 1000, and negative IgG
   precipitins to Aspergillus fumigatus (AF) because allergic bronchopulmonary aspergillosis (ABPA) is
   a diagnostic consideration in adults, and in order to differentiate between ABPA and SAFS.
   Additionally, there needs to be evidence of sensitization (skin prick test wheal (SPT) ? 3 mm,
   specific IgE (sIgE) ? 0.4) to at least one of seven fungi, namely AF, Cladosporium herbarum, Penicillium
   chrysogenum (notatum), Candida albicans, Trichophyton mentagrophytes, Alternaria alternate, and Botrytis
   cinerea. The question as to whether sensitization is best determined by sIgE or SPTs was addressed
   in 121 patients with severe asthma (British Thoracic Society/SIGN steps 4 and 5) who underwent
   both tests to all the above fungi, except Trichophyton mentagrophytes [12]. Fungal sensitivity was very
   common, but concordance between skin prick tests and sIgE tests was poor (77% overall, but only
   14–56% for individual fungi). Hence, both of the tests need to be undertaken to rigorously diagnose
   SAFS.
   2.2. SAFS in Children
   There is no consensus definition of SAFS in children. Empirically, we define SAFS as severe,
   therapy resistant asthma [13] (STRA, with any pattern of symptoms), and we have used the same
   sensitization criteria as in adults, although in fact in a clinical setting we usually can only test for
   AF, Cladosporium and Alternaria alternate. For reasons that are unclear, ABPA is rarely, if ever, seen
   in children with asthma, despite being relatively common in children with CF [14], and so we do
   not adopt the IgE and IgG precipitin criteria of the adult definition. It is likely, but unproven, that
   there will also be discordance between sIgE and SPT results in children also [15], so both tests are
   needed.
   Table 1 contrasts the diagnosis of SAFS in adults and children.
   Table 1. Diagnostic criteria for severe asthma with fungal sensitization (SAFS) in adults and
   children.
   Fungal Sensitization
   (Positive Skin Prick Test
   and/or Specific IgE to One or
   More Fungus)
   Other Adult Criteria
   Other Paediatric
   Criteria
   Aspergillus fumigatus
   Cladosporium herbarum
   Penicillium chrysogenum
   (notatum)
   Candida albicans
   Trichophyton mentagrophytes
   Alternaria alternate
   Botrytis cinerea
   Treatment with 500 mcg Fluticasone
   Propionate/day, or Continuous oral
   corticosteroids, or 4 prednisolone bursts in
   12 months or 6 bursts in 24 months
   Severe, therapy
   resistant asthma
   (ERS/ATS Task Force
   criteria)
   IgE < 1000 IgE can be any level
   Negative IgG precipitins to Aspergillus
   fumigatus
   IgG precipitins to
   Aspergillus fumigatus
   can be positive or
   negative
   J. Fungi 2019, 6, 55 4 of 17
   2.3. Beyond SAFS: Fungal Detection in the Airway, Fungal Bronchitis and Asthma
   There is no requirement to detect fungi within the airway in order to diagnose SAFS, although
   fungal infection might be part of the syndrome. However low-grade fungal infection might drive
   asthma without inducing sensitization, for example, by the release of tissue damaging enzymes
   disrupting epithelial barrier function (below). In CF, AF bronchitis is associated with worse
   outcomes [16–18], giving biological plausibility to this mechanism in asthma. The isolation of
   fungus from airways of SAFS patients is unsurprisingly very common. AF sputum positivity by
   PCR was 70% in SAFS patients not taking anti-fungals [19], but the frequency was reduced in those
   prescribed these medications, and in a small subgroup in whom serial samples were obtained,
   itraconazole therapy resulted in sputum reverting from a positive to negative PCR. The
   sensitization to multiple molds is also common in asthma. In one study, 60% of patients were polysensitized,
   most frequently to Aspergillus fumigatus (32%) and A. Alternata (28%), Penicillium
   chrysogenum, Penicillium brevicompactum, Cladosporium cladosporioides, and Cladosporium
   sphaerospermum [20,21]
   There is also the issue of how intensively the presence of fungi should be sought. CF
   definitions are largely based on positive cultures, although whether repeated cultures or a single
   culture is needed for diagnosis is controversial. Much of the focus has been on AF, not least because
   it grows at 37 degrees (body temperature) and the spores are aerodynamically well suited to
   lodging in the lower respiratory tract, but as already stated, many other fungi may be important. In
   a study in which 69 adults underwent FOB and BAL, no fewer than 86% had fungi detectable by
   PCR on BAL, 46% of which were AF. Although a positive BAL was associated with increased BAL
   and plasma cytokines, there was no relation to asthma severity [22]. Molecular techniques may be
   even more sensitive. This study suggests that, the harder fungi are sought, the more they will be
   found. This group reported no increased asthma severity in SAFS adults; and importantly,
   potentially broadened the spectrum of fungi to which the patient may be sensitized.
   2.4. Fungal Asthma or Fungal Asthmas?
   It should be noted that the danger of umbrella definitions is that it could be taken to assume
   that all fungi have equal effects. The magnitude of the effects might be different, and will likely also
   be dependent on levels of exposure, but, more importantly, the pathophysiological pathways may
   be different. Clearly, if the approach is treatment with anti-fungals, this is irrelevant, but any
   molecular therapies may need to be fungus-specific (below).
3. Paediatric and Adult Severe Asthma and the Atopies: Important Differences Relevant to
   Fungal Asthma
   The vast majority of children with severe asthma are markedly atopic [23], with multiple
   sensitizations to aeroallergens, such as house dust mite, grass and tree pollens, cockroach, and furry
   pets. By contrast, much severe adult asthma is neutrophilic, often in the obese and with other comorbidities,
   and with a female preponderance [24,25]. It is also increasingly being realized that
   atopy is not ‘all-or-none‘ and can be quantified [26]. Different atopies have differing significances
   [27–29]. Furthermore, complex interactions between allergens may be more important than
   individual results [30]. Sensitization to fungi is one part of the atopies; the question is, whether
   there is a discrete entity of SAFS in children, or whether fungal sensitization is one facet of asthma
   with polysensitzation to aeroallergens; to some extent, this remains unresolved. It might also be
   that the significance of fungal sensitization will be different in adults, and more likely to be a
   discrete entitity rather than mark of multiple sensitization, and this needs further exploration.
   However, the issue of anti-fungal treatment for paediatric SAFS is more one of ‘does it work?‘
   rather than ‘should it work?‘
   J. Fungi 2019, 6, 55 5 of 17
4. Epidemiological Data: Associations between Fungi and Asthma Severity
   4.1. Cross-Sectional Studies
   Most of the big studies are in adults. The European Community Respiratory Health Survey
   [31] studied 1132 adults aged 20–44 years with current asthma. The frequency of mold sensitization
   (Alternaria alternata or Cladosporium herbarum, or both) increased significantly with increasing
   asthma severity across Europe, but there was no association between asthma severity and
   sensitization to pollens or cats. However, Dermatophagoides pteronyssinus sensitization was also
   positively associated with asthma severity. Thus, mold sensitization was highly associated with
   severe asthma in adults, but not uniquely so. In a systematic review and meta-analysis of 20 studies
   from 13 African countries [32] the mean asthma prevalence was 6%. The prevalence of fungal
   sensitization, mostly on skin prick testing, ranged from 3% to 52%, mean 28% with a pooled
   estimate of 23.3%. Aspergillus species were commonest. The prevalence of ABPA was estimated at
   1.6–21.2%. A similar study related fungal allergy to asthma severity, and there were no paediatric
   data.
   Another such study in severe asthma (GINA step 4 or 5 treatment) [33] enrolled 124 patients. A
   variety of markers were collected, including spirometry, exhaled nitric oxide, serum cytokines, and
   IgE. Fungal sensitization was assessed from IgE specific to fungal allergens (AF, Alternaria, Candida,
   Cladosporium, Penicillium, and Trichophyton species and the Schizophyllum commune). Thirty-six of
   124 patients (29%) were sensitized to at least one fungal allergen, most commonly Candida (16%),
   AF (11%), and Trichophyton (11%). Early-onset asthma (<16 years of age) was more common in
   patients with fungal sensitization (45% vs 25%; p = 0.02, see below). Interleukin-33 levels were also
   higher in patients with fungal sensitization, as discussed in more detail in the sections on
   pathophysiology. Asthma Control Test scores were worse in patients with multiple when compared
   with single fungal sensitizations and non-sensitized controls.
   4.2. SAFS and Control of Asthma
   Adult SAFS patients are more likely to have uncontrolled symptoms [34–39]. In a retrospective
   review of urban adult asthma patients, total serum IgE was highest in the 53 patients (17.3%) with
   fungal sensitization (median, 825 IU/mL vs. 42 non-atopic (n=137, 44%) vs. 203 other allergen
   sensitized (n=117, 38.1%), p < 0.001). The fungal sensitized patients were more likely to have been
   admitted to the intensive care unit (ICU) admission and been ventilated (13.2% vs. 3.7% non-atopic
   vs. 3.4% other sensitization p = 0.02; and 11.3%, 1.5%, and 0.9%, respectively, for ventilation, p <
   0.001). There are two possible interpretations of these data; firstly, polysensitized atopic asthmatics
   do worse, or that fungal sensitization is a discrete entity and an independent risk factor for bad
   outcomes.
   A study [40], which evaluated 206 adults with severe asthma (GINA step 4 or 5 treatment,
   mean age 45 ± 17 years, 99 [48%] male), of whom 78% had a positive SPT to one or more allergens.
   The most common allergen reported was house dust mites (Blomia tropicalis, Dermatophagoides
   pteronyssinus and Dermatophagoides farinii), but 11.7% were sensitized to Aspergillus species, and this
   was associated with uncontrolled asthma. In particular, Aspergillus sensitization was independently
   associated with the need for ?2 steroid bursts in the past year (odds ratio 3.05, 95% confidence
   interval 1.04–8.95). There was no association between asthma control and corticosteroid bursts with
   sensitization to any other allergen. Importantly, this study suggests that all fungi do not necessarily
   have equivalent effects.
   4.3. SAFS and Asthma Attacks: Children
   Paediatric data are much scantier, but the conclusions are very similar. A German group
   reviewed 207 children with a diagnosis of asthma of varying severity (25% had mild, 31%
   moderate, and 44% severe; 26% had a previous history of hospitalization for an asthma attack [35]).
   Alternaria was the leading mold causing sensitization, but this did not correlate with hospitalization
   J. Fungi 2019, 6, 55 6 of 17
   due to asthma attacks or other parameters of asthma severity. The prevalence of Alternaria
   sensitization increased with age and there was a significant association with the sensitization to
   other molds and aeroallergens, grass pollen, and cat epithelia. Alternaria sensitization in this study
   was thus not a risk factor for severe asthma and hospitalization. However, it should be noted that
   the risk might be a composite, both of sensitization, but also level of exposure; to take an absurd
   example, a sensitized patient who never subsequently encountered the allergen could not have an
   asthma attack triggered by that allergen.
   The Melbourne Air Pollen Children and Adolescent study [41] recruited 644 children and
   adolescents (aged 2–17 years) that were hospitalized for asthma and showed that exposure to
   Alternaria, less well known taxa, including Leptosphaeria, Coprinus, and Drechslera, and total spore
   counts were significantly associated with admissions for asthma independent of rhinovirus
   infection. Surges of spores of Alternaria, Leptosphaeria, Cladosporium, Sporormiella, Coprinus, and
   Drechslera were associated with significant effects delayed for up to three days, and Cladosporium
   sensitization was associated with significantly greater effects than the other fungi. Importantly, this
   study broadens the range of fungi that might need to be considered as part of fungal asthma,
   although the alternative explanations are that the effects were not mediated by allergic
   sensitization, or less likely, that that these spores were merely possibly markers of some
   unidentified root cause.
   4.4. SAFS and Lung Tissue Destruction
   In a cross-sectional study [42], 329 (76.3%) of adult asthmatics were sensitized to at least one
   fungus and this was related to the development of lung destruction, as assessed by postbronchodilator
   spirometry and computed tomographic (CT) scans. The sensitization to AF and/or
   Penicillium chrysogenum was associated with a lower first second forced expired volume (FEV1)
   when compared with those not sensitized, independent of atopic status, and an increased frequency
   of CT abnormalities, bronchiectasis, tree-in-bud, and collapse/consolidation. Cluster analysis
   identified three clusters: (i) hypereosinophilic hypothetically, true SAFS; (ii) high immunological
   biomarker load and high frequency of radiological abnormalities (hypothetically, fungal bronchitis
   dominant; and, (iii) low levels of fungal biomarkers (fungi not relevant). The authors concluded
   that AF sIgE was a risk factor for lung damage irrespective of ABPA.
   4.5. Fungi and Risk Assessment
   GINA and other guidelines have rightly stressed the importance of risk assessment as well as
   asthma control. There is no question that fungal sensitization is a marker of future risk of poor
   control and asthma attacks. Whether this is true for fungal bronchitis, as it is in CF, has yet to be
   explored.
5. Clinical Features of SAFS in Children
   We have reported the largest, most detailed series of children with SAFS [43]. We studied 82
   children (median 11.7 years) with severe, therapy resistant asthma (STRA), who had undergone a
   protocolised series of investigations [44–46], including fibreoptic bronchoscopy (FOB),
   bronchoalveolar lavage (BAL), and endobronchial biopsy (EBx). Thirty eight were defined as SAFS,
   with a specific IgE or SPT to AF, Alternaria alternate or Cladosporium (in practice, we do not have
   access to testing for other fungi). We also found that children with SAFS had an earlier onset of
   symptoms (0.5 as compared with 1.5 years), a higher IgE (637 vs. 177) and were sensitized on
   testing sIgE to more non-fungal inhalant allergens, when compared with non-SAFS STRA. They
   were more likely to be prescribed maintenance oral corticosteroids (42% vs. 14%, p = 0.02).
   However, on BAL and EBx, the severity of airway inflammation and remodelling (absolute
   thickness of reticular basement membrane thickness and airway smooth muscle mass) did not
   differ between SAFS and control STRA, despite the greater use of anti-inflammatory medications.
   J. Fungi 2019, 6, 55 7 of 17
   Eight of 10 (80%) SAFS children responded to omalizumab, similar to STRA controls (11/18, 84%, p
   = NS). Mepolizumab was not licensed in children at the time of this study.
   Another paediatric study enrolled 64 children, of whom 25 (39%) had evidence of sensitization
   to at least one fungus [47]. Nineteen of 25 (76%) sensitized children had severe persistent asthma
   when compared to 13 of 39 (33%) non-sensitized (p = 0.0014). Nineteen of 32 (59%) severe persistent
   asthmatics had fungal sensitization, and these also had higher serum IgE and worse spirometry.
   Bronchial biopsy of sensitized children revealed that these children exhibited basement membrane
   thickening and eosinophil infiltration on bronchial biopsy.
   In a USA study of 126 children, Alternaria skin test reactivity was associated with severe,
   persistent asthma. Importantly, this was an independent risk factor to that of the total positive skin
   tests, suggesting there is an independent effect of this fungus unrelated to degree of atopy [48].
6. Treatment of SAFS and Fungal Asthma
   The possible aspects of treatment are: (a) the reduction of allergic inflammation; (b) reduction
   of fungal burden; (c) reduction of tissue damage; and, (d) modulating the pro-inflammatory effects
   of tissue destruction. Most focus has been on the reduction of fungal burden, but without
   necessarily ensuring that there is a fungal infection.
   6.1. Adult Data
   Most of the data on antifungal therapy are in adults, and the results are conflicting. The FAST
   study [11] enrolled 58 adults into a double blind, randomized controlled trial of oral itraconazole or
   placebo for 32 weeks, with a follow up period of 16 weeks. The primary end point was the Asthma
   Quality of Life Questionnaire (AQLQ), with secondary endpoints being rhinitis score, total IgE and
   respiratory function. The study was positive, with improvements in AQLQ and rhinitis scores, an
   improved morning peak flow (20.8 l/min.) with itraconazole, and total IgE dropped (-510 iU
   itraconazole when compared with +30 placebo). Seven patients in the itraconazole group, and two
   placebo patients discontinued treatment. Interestingly, 60% had big improvements in QoL with
   itraconazole. The benefits of itraconazole declined rapidly in the washout period. By contrast,
   EVITA3 was a randomized, double blind, placebo controlled trial of Voriconazole in SAFS [49]. Of
   note, Voriconazole does not increase steroid bioavailability, unlike itraconazole [50,51]. The study
   duration was three months with a nine-month follow period. Fifty-six adults with SAFS were
   recruited. The inclusion criteria were at least two severe asthma attacks (defined as the prescription
   of oral corticosteroids) in the previous year, and a positive specific IgE or skin prick test to AF. The
   voriconazole levels were measured to optimize therapy. The primary endpoints were quality of life
   and asthma attacks. The study was negative. Neither trial mandated a positive airway fungal
   culture. In another report, 41 patients were studied retrospectively [52]. In those who received
   treatment (n = 32), this was with any of terbinafine, fluconazole, itraconazole, voriconazole, or
   posaconazole combined with standard treatment, by comparison with nine patients who had
   standard asthma therapy only. Those that were treated with anti-fungals showed improvement in
   Asthma Control Test, peak flow rate, and IgE. The response was better with longer treatment
   periods, and it was well tolerated, but relapse was common after the discontinuation of treatment.
   It should be noted that all of these data largely predate the widespread introduction of biological
   and, therefore, should be interpreted with caution in light of new therapies [53].
   In another study [54], 110 STRA GINA stage 4 adult asthmatics were randomly assigned to 200
   mg itraconazole twice a day or 10 mg prednisolone once daily for four months. There was no
   requirement for the demonstration of fungal sensitization or any other manifestation of fungal
   asthma. The study was not blinded. 71% of the itraconazole group improved and there were very
   few side-effects, whereas there was minimal change with prednisolone.
   In terms of acute asthma, there is a single case report of an 83 years old woman [55], with a 33-
   year history of asthma prescribed inhaled and oral corticosteroids. She presented with an acute
   attack of wheeze that did not respond to oral corticosteroids and antibiotics. She was found to
   culture AF in her sputum, a positive AF sIgE and IgG precipitins, and a positive galactomannan.
   J. Fungi 2019, 6, 55 8 of 17
   Voriconazole was added with a good response. Perhaps the most likely explanation is that this was
   treating acute AF bronchitis in an immunosuppressed adult. There is no general role for antifungals
   in acute asthma.
   6.2. Paediatric Data
   There are no randomized controlled trials of treatment in children. On general principles, we
   try to minimize fungal exposure, especially advocating for rehousing if there is visible mold in the
   house; we would check any nebulizers which might be being used for fungal contamination; and
   we would advise against children going into stables and barns [56], where mold abounds.
   However, although the reducing the burden of fungal allergen exposure seems sensible, the
   relationship between mold exposure, mold sensitization, and asthma severity is complex.
   Approximately 90% of homes in one case control study were contaminated with mold [20]. The
   sensitization to AF, but not to other molds, was associated with asthma severity. Whether or not the
   child was sensitized, AF and Penicillium spp in dust was associated with severe asthma; the latter
   was associated with worse lung function. The lessons of this study are that environmental AF
   exposure should be minimized, and that not all molds have the same effects. However, although
   exposure to mold may limit airway infection, it should be borne in mind that allergen reduction
   strategies have sometimes had unexpected effects. In some cases, high level exposure might induce
   tolerance, and reduction in levels lead to increased sensitivity; and there is marked variation
   between allergens in the relationship between environmental concentrations and likelihood of
   sensitization or tolerance [57].
   Additionally, we would also optimize standard asthma management and treatment [6–8],
   including treatment with omalizumab and mepolizumab if indicated, before going on to ‘beyond
   guidelines’ therapy with antifungals. Anecdotally, a child with refractory asthma, persistently
   abnormal spirometry, total E >20,000 IU/mL, and severe airway eosinophilia was sensitized to
   multiple fungi and responded dramatically to itraconazole [58]. Additionally, anecdotally,
   omalizumab might effectively treat the occasional case of SAFS [59], alone or combined with
   itraconazole which may be used to reduce IgE levels into the omalizumab range [60]. Also
   anecdotally, we have seen the occasional SAFS child who appeared to improve with antifungals.
   6.3. Conclusions: What is the Role of Antifungals in SAFS?
   It is suggested that the individual facets, or treatable traits of fungal asthma, are determined on
   an individual basis and a bespoke treatment plan developed. Clearly, the evidence for the use of
   antifungals is conflicting and of low quality. Part of the reason might be that SAFS as
   conventionally declined does not require the presence of fungal bronchitis. It is difficult to see how
   anti-fungal therapy would benefit SAFS if there were no fungal infection, and symptoms were
   solely due to sensitization to fungal spores. Logically, future trials of anti-fungal therapy in
   SAFS/fungal asthma should mandate the presence of fungal bronchitis.
   Our current approach is to address environmental exposures and optimize standard therapy in
   children with SAFS. If asthma control is optimal and there are no other markers of ongoing risk,
   such as a persistently raised exhaled nitric oxide or a past history of really severe attacks, with no
   present side-effects, then we would not use antifungals. However, if asthma control remains
   suboptimal, or significant risks persist, then we would consider adding an antifungal, such as
   itraconazole. It is important to note that there is a potential interaction between corticosteroids and
   azoles at the cytochrome p450 level [61], such that the combination of itraconazole and inhaled
   budesonide has led to iatrogenic Cushing Syndrome [50,51].
7. Risk Factors for SAFS: Genetic Studies
   Although there is expanding literature on the genetic associations of ABPA, SAFS has been
   little investigated. There might indeed be genetic factors that are associated with SAFS, but, to my
   knowledge, there has been no large scale Genome Wide Association Study (GWAS) to confirm or
   J. Fungi 2019, 6, 55 9 of 17
   otherwise this suggestion. In a small, preliminary study [62], 325 haplotype-tagging single
   nucleotide polymorphisms (SNPs) in 22 previously suggested candidate genes were studied in
   SAFS (n = 47), atopic asthmatics (n = 152), and healthy control patients (n = 279). There were
   significant associations of Toll-like receptors (TLR) 3 and 9 (TLR3), C-type lectin domain family
   seven member A (dectin-1), IL-10, mannose-binding lectin (MBL2), CC-chemokine ligand 2 (CCL2)
   and CCL17, plasminogen, and the adenosine A2a receptor, different from those reported in asthma
   complicated by ABPA. Some of these hits are supported by cell and animal data (below). The main
   weakness of this study was the absence of a second validation cohort, without which the findings
   are, at best, preliminary. In an initial small study comprising 76 adults with chronic cavitatory
   pulmonary aspergillosis (n = 40), ABPA (n = 22), and SAFS (n = 14), no genetic associations of SAFS
   could be determined, unsurprisingly with such a small number of patients [63]. However, in one
   intriguing study, six SAFS children were heterozygous for a 24-base pair duplication in the CHIT1
   gene [64]. This duplication associates with an increased susceptibility to fungal infection and
   decreased circulating chitotriosidase levels [65]. Clearly there is a need for more work in this area.
8. Pathophysiology of SAFS and Fungal Asthma
   8.1. Introduction
   As discussed, there are two pathological mechanisms, whereby fungi, especially AF, can cause
   disease in children with asthma [8]. These are as a source of allergen(s) to which the child is
   sensitized, leading to wheeze on exposure, and driving a Type 2 inflammatory response; and, the
   release of tissue damaging enzymes by fungi that have infected the airway (not dissimilar to, for
   example, house dust mite, which is allergenic and tissue damaging), and that might also generate
   an allergic response. It should be noted that other proteins could generate an allergic response
   without requiring airway infection, for example, in sensitization to furry pets.
   Any account of the potential role of exogenous infection of any cause must consider the
   possibility that this is iatrogenic, secondary to the use of corticosteroids. It is known that systemic
   corticosteroids are immunosuppressive, and also that mucosal immunity is essential for normal
   host defence [65]. It is biologically plausible that topical steroids would be immunosuppressive, and
   indeed their use is associated with increased prevalence of tuberculosis, [66] atypical Mycobacterial
   infection [67], and, in patients with COPD, pneumonia [68]. It is virtually impossible to dissect out
   the contribution of inhaled corticosteroids (ICS) to SAFS, because, by definition, all SAFS patients
   will be prescribed ICS. In one study [69], the fungal microbiome (mycobiome) was determined on
   bronchoscopic samples. The investigators reported that the mycobiome was highly varied with the
   biggest load in severe asthmatics. Healthy controls had low fungal loads; the most common fungus
   detected was the poorly characterized Malasezziales. AF was most the common in fungus in
   asthmatics and accounted for the increased fungal burden. Corticosteroid treatment was
   significantly associated with an increased fungal load. These interesting data cannot unravel
   whether inhaled corticosteroids caused SAFS, or SAFS led to the prescription of more inhaled
   corticosteroids.
   8.2. Cell and Animal Studies
   A number of different pathways have been implicated in SAFS, including the pattern
   recognition receptors (PRRs) TLR3, TLR9, and Dectin-1 and IL-7, Il-10, IL-22, CCL2, and CCL17
   [70,71]. IL-33 has been implicated in both adult [36] and paediatric SAFS [5]. IL-33 is an epithelial
   alarmin, together with IL-25 and TSLP. It is a member of the eleven member IL-1 family of
   cytokines. Of these, seven are proinflammatory (IL-1?, IL-1?, IL-18, IL-33, IL-36?, IL-36?, and IL-
   36?) and four probably immunomodulatory (IL-1 receptor antagonist [IL-1RA], IL-36Ra, IL-37, and
   IL-38). A recent manuscript [72] demonstrated that IL-1? and IL-1? are elevated in the BAL and
   sputum from adult SAFS patients. The same group used a murine model utilizing the AF challenge
   to show that IL-1R1 signaling promotes increased airway hyper-responsiveness and neutrophilic
   inflammation associated with type 1 (IFN-?, CXCL9, CXCL10) and type 17 (IL-17A, IL-22)
   J. Fungi 2019, 6, 55 10 of 17
   responses, each exacerbated in IL-1RA?/? mice. The administration of human recombinant IL-1RA
   (Kineret/anakinra) abrogated these responses, all suggesting that IL-1R1 signaling via type 1 and
   type 17 responses is an important and potentially treatable pathway of SAFS.
   A murine model further explored the links between Alternaria and asthma [73]. Wild-type and
   mice lacking the IL-33 receptor (ST2?/?) underwent inhalational challenge with inhaled house dust
   mite, cat dander, or Alternaria. Mice that were sensitized with house dust mite were subsequently
   challenged with Alternaria (with or without serine protease activity having been knocked down),
   and inflammation, remodeling, and lung function assessed 24 h after the challenge. Only Alternaria
   possessed intrinsic serine protease activity that led to the release of IL-33 into the airways via a
   mechanism that is dependent on the activation of protease activated receptor-2 and adenosine
   triphosphate signaling. This led to more pulmonary inflammation relative to that produced by the
   house dust mite challenge. IL-1? and matrix metalloproteinase (MMP) 9 release were also features
   of Alternaria challenge. Furthermore, Alternaria triggered a rapid, augmented inflammatory
   response, mucus hypersecretion, and airway obstruction. The effects of Alternaria were critically
   dependent on ST2 signaling. Hence, Alternaria-specific serine protease activity resulted in rapid IL-
   33 release, leading to TH2 inflammation and exacerbation of allergic airway disease.
   Alternaria proteases may have an important role. One study [74] used cells from normals or
   patients with severe asthma. They used both 16HBE cells and fresh bronchial epithelial cells
   cultured to air-liquid interface (ALI), and challenged them apically with extracts of Alternaria in
   order to further explore the role of Alternaria proteases. Alternaria extract protease activity was

Keywords: atopy; aspergillus bronchitis; fungal sensitization; itraconazole; severe asthma;
voriconazole