MYCOLOGY – Aspergillus fumigatus

The gradual introduction of more aggressive antibiotic therapy during the Eighties and the resulting increase in survival have been associated with a marked increase in these publications on aspergillus, both on allergic bronchopulmonary aspergillosis (ABPA) and aspergillus colonisation and infection.  Although many of these publications are repetitive, it is clear that just as P. aeruginosa followed the reduction in S. aureus infections so Aspergillus infection and ABPA have followed the more aggressive  and successful treatment of P. aeruginosa. Also the use of immunosuppression following transplantations has given added importance to the presence of and potential problems from fungal infections and ABPA (Iversen M et al. Eur J Clin Microbiol Infect Dis 2007; 26:879-886.[PubMed]).

Although both corticosteroids and antifungal drugs have a role in the management of these problems, it is disappointing that after so many publications on the subject of Aspergillus and cystic fibrosis, a Cochrane review recently concluded “there are no randomised controlled trials to evaluate the use of antifungal therapies for the treatment of ABPA in people with cystic fibrosis. Trials with clear outcome measures are needed to properly evaluate this potentially useful treatment for cystic fibrosis” (Elphick HE, Southern KW Cochrane Database Syst Rev 2012 Jun 13;6:CD002204. [PubMed]).

Initially most attention was paid to ABPA but it is now realised that infection both local within the airways and invasive can be a serious problem. Some of the following studies mentioned relate to ABPA and others to the presence of chronic A. fumigatus infection in the airways.

1965 Mearns MB, Young W, Batten JC. Transient pulmonary infiltrations in cystic fibrosis due to allergic aspergillosis. Thorax 1965; 20:385-392.
This was the first description of allergic bronchopulmonary aspergillosis (ABPA) occurring in two children with cystic fibrosis. A subsequent study from this group showed precipitating antibody to Aspergillus to be present in 31% of 122 children with CF compared with in only 7% in 60 asthmatic children (Mearns M et al. Lancet 1967;i:538-539.below )(also see Brueton et al, 1980 below for typical chest X-ray).

1967 Mearns M, Longbottom J, Batten J. Precipitating antibodies to aspergillus fumigatus in cystic fibrosis. Lancet 1967;1(7489):538-539[PubMed]

1975 Warren CP, Tai E, Batten JC, Hutchcroft BJ, Pepys J. Cystic fibrosis – immunological reactions to A. fumigatus and common allergens.  Clin Allergy. 1975; 5:1-12. [PubMed]

1980 Brueton MJ, Omerod LP, Shah KJ, Anderson CM. Allergic bronchopulmonary aspergillosis complicating cystic fibrosis in childhood. Arch Dis Child 1980; 55:348-353. [PubMed] Full free article available.

Allergic bronchopulmonary aspergillosis

Martin Brueton

This report in the leading, widely read UK paediatric journal, by Martin Brueton (figure) when working in Prof. Charlotte Anderson’s unit in Birmingham, was important as it brought to the attention of general paediatricians (most of whom still cared for a few children with CF) the complication of allergic bronchopulmonary aspergillosis (ABPA) in children with CF. The problem had been described previously by Margaret Mearns in older patients with CF (Mearns et al, 1965 above).

This paper certainly made us more aware of ABPA – the condition for which steroids and a not antibiotics were required. The complication came to be suspected in the presence of increasing asthmatic symptoms accompanied by major chest X-ray changes ranging from total or lobar lung collapse to extensive but changing areas of consolidation (figure) associated with eosinophilia, positive skin tests and positive serum precipitins for Aspergillus; also there was not the expected clinical response to intravenous antibiotics. Sometimes the extent of the often major segmental x-ray changes was greater than and quite out of keeping with the outward clinical disturbance.

Large doses of oral steroids were recommended and are still used but few would now agree with the authors of the present article that there was no place for anti-fungal agents, for these now have an increasing place in treatment to reduce the load of Aspergillus antigen present. As more intravenous antibiotics were used during the Eighties the incidence of ABPA increased – this was the experience both in Copenhagen and Leeds.

1990 Simmonds EJ, Littlewood JM, Evans EG. Cystic fibrosis and allergic bronchopulmonary aspergillosis. Arch Dis Child 1990; 65:507-511.  [PubMed] Free PMC article
Over a three year period eight patients were identified, an incidence of 5.8%. Patients were clinically well at the time of diagnosis (Shwachman scores 70-90, Chrispin-Norman chest x ray scores 2-15) and they responded rapidly to treatment with oral prednisolone. There had been little deterioration in their respiratory function and nutrition over the study period.    We concluded that allergic bronchopulmonary aspergillosis is not uncommon in patients with cystic fibrosis. It is a potential cause of lung damage and prospective screening could lead to earlier detection and treatment.

1994 Simmonds EJ, Littlewood JM, Hopwood V, Evans EG. Aspergillus fumigatus colonisation and population density of place of residence. Arch Dis Child 1994; 70:139-140. [PubMed]
The relation between antibody titres of aspergillus in patients with cystic fibrosis and the population density of their place of residence was investigated by Dr Eddie Simmonds – then our CF Research Fellow. Patients with high titres of antibodies to Aspergillus fumigatus were significantly more likely to live in an area of low population density.So living in a rural environment may predispose to A. fumigatus colonisation. “Mucking out” stables seems to markedly increase exposure to airborne fungal spores.

1996 Egan JJ, Yonan N, Carroll KB, Deiraniya AK, Webb AK, Woodcock AA.  Allergic bronchopulmonary aspergillosis in lung allograft recipients. Eur Respir J 1996; 9:169-171.[PubMed]
Following lung transplantation for end-stage cystic fibrosis, two male patients presented with shortness of breath, peripheral blood eosinophilia and segmental lung collapse. At bronchoscopy, each had bronchial mucous plugging containing Aspergillus fumigatus. This finding was associated with a systemic eosinophilia and skin test positivity to Aspergillus. Augmented steroid therapy resulted in the successful resolution of the symptoms.

The authors consider these to be the first reported cases of allergic bronchopulmonary aspergillosis in lung allograft recipients.

2001 Clifton IJ, Whitaker P, Metcalfe R, Phillip M, Shaw N, Conway SP, Peckham DG. Pharmacokinetics of oral voriconazole in patients with cystic fibrosis. J Antimicrob Chemother 2011; 66:2438-40[PubMed]

2001 Mastella G, Rainisio M, Harms HK, Hodson ME, Koch C, Navarro J, Strandvik B, McKenzie SG. Allergic bronchopulmonary aspergillosis in cystic fibrosis. Eur Respir J 2001 May;17(5):1052-3.[PubMed] Free full text available
Because of the difficulties of recognizing allergic bronchopulmonary aspergillosis (ABPA) in the context of cystic fibrosis (because of overlapping clinical, radiographic, microbiologic, and immunologic features), advances in our understanding of the pathogenesis of allergic aspergillosis, new possibilities in therapy, and the need for agreed-upon definitions, an international consensus conference was convened. Areas addressed included fungal biology, immunopathogenesis, insights from animal models, diagnostic criteria, epidemiology, the use of new immunologic and genetic techniques in diagnosis, imaging modalities, pharmacology, and treatment approaches. Evidence from the existing literature was graded, and the consensus views were synthesized into this document and recirculated for affirmation. Virulence factors in Aspergillus that could aggravate these diseases, and particularly immunogenetic factors that could predispose persons to ABPA, were identified. New information has come from transgenic animals and recombinant fungal and host molecules. Diagnostic criteria that could provide a framework for monitoring were adopted, and helpful imaging features were identified. New possibilities in therapy produced plans for managing diverse clinical presentations.

2002  Skov M, Hoiby N, Koch C. Itraconazole treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. Allergy 2002; 57:723-728.[PubMed]
In a retrospective follow-up of 21 CF patients from a total of 250 treated once or twice within a five-year study period (1994-98), 9 patients were treated with systemic glucocorticosteroids in combination with itraconazole and 12 patients were treated with itraconazole (200-600 mg/day) as monotherapy. During treatment the percentage of Aspergillus fumigatus (AF)-positive sputum cultures significantly reduced (P < 0.05); precipitating antibodies to AF decreased significantly in all patients (P < 0.05); forced expiratory volume (FEV1) increased to pre-exacerbation level; total IgE levels decreased in 42% of patients on monotherapy and in 56% on combination therapy. Specific IgE (radioallergosorbant; RAST) level decreased in 6 of 21 patients. Eleven patients had transient increased levels of alanine transaminase (ALAT). One patient had isolated increase in alkaline phosphatase and another in aspartate transaminase (ASAT).

The authors concluded that high dose itraconazole as monotherapy or in combination with systemic glucocorticosteroids seems effective in CF patients with ABPA. No hepatotoxicity was observed during long-term therapy.

2006 Thomson JM, Wesley A, Byrnes CA, Nixon GM. Pulse intravenous methylprednisolone for resistant allergic bronchopulmonary aspergillosis in cystic fibrosis. Pediatr Pulmonol 2006; 41:164-170.[PubMed]
This is the first reported use of pulse intravenous methylprednisolone in the treatment of ABPA in CF. The authors present the clinical course of four children with CF and severe ABPA, in whom pulse methylprednisolone was used to manage the disease because of relapses or marked side effects on high-dose oral corticosteroids. Methylprednisolone pulses achieved disease control in 3 of the 4 children. However, troublesome side effects were experienced, in some cases necessitating discontinuation of therapy.

Pulse methylprednisolone may represent a treatment option for children with CF and ABPA, where ABPA fails to respond adequately to routine therapy.

2006 Shoseyov D, Brownlee KG, Conway SP, Kerem E. Aspergillus bronchitis in cystic fibrosis. Chest. 2006 ;130:222-226. [PubMed]
Aspergillus fumigatus, a widely distributed spore-bearing fungus, is commonly grown in sputum cultures of patients with cystic fibrosis (CF). A fumigatus may cause allergic bronchopulmonary aspergillosis (ABPA), a complex condition that leads to worsening of airway inflammation and progressive damage and is diagnosed by specific criteria.

In this report, the authors present six CF patients with respiratory deterioration that did not respond to appropriate antibiotic treatment. All had had A. fumigatus in sputum cultures but did not fulfill the criteria of ABPA. Treatment with antifungal agents was followed by improvement in clinical condition.

The authors suggest that in patients with CF, A. fumigatus should be considered as a pathogen that may directly cause respiratory exacerbations. Antifungal therapy should be considered when deteriorating respiratory function is not responding to antibacterial therapy and A. fumigatus is growing in sputum cultures. Thsi was an important new concept – of Aspergillus as a primary pathogen.

2008 Barton RC, Hobson RP, Denton M, Peckham D, Brownlee K, Conway S, Kerr MA. Serologic diagnosis of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis through the detection of immunoglobulin G to Aspergillus fumigatus. Diag Microbiol Infect Dis 2008; 62:287-291.[PubMed]
Allergic bronchopulmonary aspergillosis (ABPA) is seen in approximately 10% of patients with cystic fibrosis (CF) and can be difficult to diagnose. Consensus criteria require the presence of multiple elevated immunologic markers such as total immunoglobulin E (IgE), Aspergillus IgE and Aspergillus IgG, or precipitins for a robust diagnosis. There is some degree of standardization of total IgE and Aspergillus IgE levels, but there is no standardization in the measurement of IgG antibodies or precipitins to Aspergillus. The interpretation of results may, therefore, be confusing.

Eighty-seven patients with CF were categorized as having ABPA or as controls, using the consensus criteria and an in-house enzyme immunoassay to measure IgG levels to Aspergillus. All sera from patients were then analyzed by commercial fluorescent immunoassay (FEIA) for the quantitative detection of anti-Aspergillus IgG. FEIA results were analyzed against the consensus conference minimum diagnostic criteria to ascertain a cutoff point, which could predict a diagnosis of ABPA in CF. Eighty patients with CF and with no or incomplete evidence of ABPA had a mean FEIA score of 51.1 mg/L, whereas 7 CF patients with ABPA had a mean FEIA score of 132.5 mg/L. Using receiver operator characteristic curve analysis of the ImmunoCAP (Phadia) IgG score on ABPA versus all other patients gave an area under the curve of 0.933 (estimated SE, 0.027).

This analysis provisionally suggested that a score of 90 mg/L may be used as a cutoff point, which would give a sensitivity of 91% and specificity of 88.0% for the diagnosis of ABPA, though this requires further validation. This quantitative approach to Aspergillus IgG measurement in patients with CF along with the results of other tests will hopefully provide a more accurate approach to the diagnosis of ABPA.

2009 Cohen-Cymberknoh M, Blau H, Shoseyov D, Mei-Zahav M, Efrati O, Armoni S, Kerem E. Intravenous monthly pulse methylprednisolone treatment for ABPA in patients with cystic fibrosis. J Cyst Fibros 2009; 8:253-257. [PubMed]
Nine patients with CF and allergic bronchopulmonary aspergillosis (ABPA) (4 male, 5 female, ages 7-36 years) received high dose intravenous methyl prednisolone (HDIVPM) (10-15 mg/kg/d), for 3 days per month, and itraconazole, until clinical and laboratory resolution of their ABPA.
All patients showed clinical and laboratory improvement, (FEV(1) increase, serum IgE levels and total eosinophil counts decrease) and treatment was discontinued after 6-10 pulses. Adverse effects were minor and disappeared shortly after each IV pulse therapy. The authors suggest that high-dose IV-pulse methylprednisolone is an effective treatment for ABPA in CF with minor side effects.

This appears to be an effective way of avoiding unwelcome side effects of prolonged courses of oral corticosteroids (prednisolone) which are often severe during treatment of ABPA – particularly the altered facial features distress patients when repeat or prolonged courses are required.

2009 Roux AL, Catherinot E, Ripoll F, Soismier N, Macheras E, Ravilly S, Bellis G, Vibet MA, Le Roux E, Lemonnier L, Gutierrez C, Vincent V, Fauroux B, Rottman M, Guillemot D, Gaillard JL, Jean-Louis Herrmann for the OMA Group. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France. J Clin Microbiol 2009; 47:4124-4128. [PubMed] A multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area). Mycobacterium abscessus complex (MABSC; 50 patients) and Mycobacterium avium complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The “new” species, Mycobacterium bolletii and Mycobacterium massiliense, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).
A large survey of NTM in France showing an overall prevalence of 6.6%.

2010 Amin R, Dupuis A, Aaron SD, Ratjen F. The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalization in patients with cystic fibrosis. Chest 2010 ;137:171-176. [PubMed]
This was a retrospective cohort study of patients with CF followed at The Hospital for Sick Children Toronto from 1999 to 2006. Persistent A. fumigatus infection was defined as the presence of two or more positive sputum or bronchoalveolar cultures for A. fumigatus in a given year. The primary outcome measure was the annual number of hospitalizations for pulmonary exacerbations. Two hundred thirty patients with CF were included in the analysis. The FEV(1) of patients persistently infected with A. fumigatus was 3.61% (P< or =.0001) lower during the study period compared with uninfected patients. There was a significant interaction between A. fumigatus and Pseudomonas aeruginosa on lung function (P=.0006). Patients not infected with either organism had the highest pulmonary function. Persistent A. fumigatus infection (relative risk [RR]=1.94, P=.0002) and CF-related diabetes (RR=1.64, P=.028) were associated with an increased risk of pulmonary exacerbations requiring hospitalization, whereas there was no increased risk of pulmonary exacerbations among patients with allergic bronchopulmonary aspergillosis (RR=1.02, P=.94). When adjusted for baseline pulmonary function, none of these variables were associated with a significantly increased risk of pulmonary exacerbations, with only chronic A fumigatus infection trending toward significance (RR=1.40, P=.065).

So this study showed that persistent A. fumigatus infection is an independent risk factor for hospital admissions in patients with CF.

2010 Moss RB. Allergic bronchopulmonary aspergillosis and Aspergillus infection in cystic fibrosis. [Review] Current Opinion in Pulmonary Medicine 2010; 16:598-603. [PubMed]

 Richard Moss

Recent literature on Aspergillus fumigatus infection and allergy in cystic fibrosis have expanded our understanding of many aspects of allergic bronchopulmonary aspergillosis, and bring new attention to A. fumigatus airways infection and A. fumigatus allergy without allergic bronchopulmonary aspergillosis (ABPA).
RECENT FINDINGS: ABPA, A. fumigatus infection and A. fumigatus allergy without ABPA all likely worsen cystic fibrosis (CF) lung disease. Studies examining utility of new serologic assays for diagnosing ABPA include evaluations of standardized measurement of A. fumigatus-specific IgG, serum chemokine TARC levels, and recombinant A. fumigatus allergens; as yet, none appear ideal. Although oral glucocorticoids remain primary therapy, toxicity and incomplete control have led to an ongoing search for further safe and effective agents including itraconazole and voriconazole, intravenous pulse methylprednisolone, nebulized amphotericin B and omalizumab. Little controlled treatment data is available.
SUMMARY: Diagnosis of CF-ABPA remains difficult, but improvements in serologic assays are occurring. Treatment remains in many cases unsatisfactory, and new agents offer promise but await proper controlled trials of safety and efficacy. A. fumigatus airway infection and A. fumigatus allergy without ABPA are emerging as further complications of A. fumigatus respiratory colonization in patients with CF, but prospective studies are needed to corroborate largely retrospective findings.

Dr Richard Moss (1949- )(figure) of Stanford University is one of N. America’s leading CF clinicians and researchers. He has a major interest in the pathogenesis of chronic airways diseases of childhood particularly immunoregulation of inflammation in cystic fibrosis. In the arena of clinical research, as a member of the CFF Therapeutics Development Network his research group conducts many trials. His wide interests include aerosol therapy, gene vectors and corrrection of CFTR-dependent cell biology defects and new treatments for CF complications such as diabetes and osteoporosis, internet-based disease management, and trials of asthma drugs.

2010 Proesmans M, Vermeulen F, Vreys M, De Boeck K. Use of nebulized amphotericin B in the treatment of allergic bronchopulmonary aspergillosis in cystic fibrosis. Int J Pediatr 2010; 37:62-87.
Allergic bronchopulmonary aspergillosis (ABPA) often recurs when the corticosteroid treatment is reduced. These authors report successful steroid withdrawl without relapse over 12 months in 6 of 7 patients with difficult ABPA treated with nebulised amphotericin B.

–   Just as corticosteroids, antibiotics, rhDNase are effective via the nebulised route it is not surprising that so is nebulised amphotericin. We have found this treatment to be useful in a child with a mixed fungal infection of the airways and its use seems logical in troublesome Aspergillus infection to eradicate the antigen from the airways. Also Aspergillus causing bronchitis, as distinct from allergic bronchopulmonary aspergillosis, is now well described (Shoseyov D et al. Chest 2006; 130:222-226.

2011 Zalar P, Novak M, de Hoog GS, Gunde-Cimerman N. Dishwashers – a man-made ecological niche accommodating human opportunistic fungal pathogens. Fungal Biology 2011; 115:997-1007. [PubMed]
Enrichment of fungi that may require specific environmental conditions was observed in dishwashers, 189 of which were sampled in private homes of 101 towns or communities. One-hundred-two were sampled from various localities in Slovenia; 42 from other European countries; 13 and 3 from North and South America, respectively; 5 from Israel; 10 from South Africa; 7 from Far East Asia; and 7 from Australia. Isolation was performed on samples incubated at 37C. Species belonging to genera Aspergillus, Candida, Magnusiomyces, Fusarium, Penicillium and Rhodotorula were found occasionally, while the black yeasts Exophiala dermatitidis and Exophiala phaeomuriformis (Chaetothyriales) were persistently and most frequently isolated. Sixty-two percent of the dishwashers were positive for fungi, and 56% of these accommodated Exophiala. Both Exophiala species are known to be able to cause systemic disease in humans and frequently colonize the lungs of patients with cystic fibrosis. The authors conclude that high temperature, high moisture and alkaline pH values typically occurring in dishwashers can provide an alternative habitat for species also known to be pathogenic to humans.

This is a very detailed paper the full version of which is available without cost on the internet. In view of the increasing importance of fungal infections such as Aspergillus in people with CF, this is useful information although the presence of these organisms seem to represent a potential more than definite threat at this stage.

2011 Massam J, Bitnun A, Solomon M, Somers GR, Guerguerian AM, van Wylick R, Waters V.  Invasive aspergillosis in cystic fibrosis: a fatal case in an adolescent and review of the literature. Infect Dis J 2011; 30:178-180. [PubMed]
Invasive aspergillosis is a rare complication of cystic fibrosis. In this article, the authors describe a case of an adolescent with cystic fibrosis, which was well-controlled previously, colonized with Aspergillus fumigatus. The patient developed fatal disseminated aspergillosis in the absence of any preexisting risk factors after a short course of intravenous corticosteroid treatment.

2012 Aaron SD, Vandemheen KL, Freitag A, Pedder L, Cameron W, Lavoie A, Paterson N, Wilcox P, Rabin H, Tullis E, Morrison N, Ratjen F. Treatment of Aspergillus fumigatus in patients with cystic fibrosis: a randomized, placebo-controlled pilot study. PLos One 2012;7:e36077.[PubMed]
The objective of this study was to determine whether treatment directed against Aspergillus fumigatus improves pulmonary function and clinical outcomes in patients with cystic fibrosis (CF).  A double-blind randomized placebo-controlled pilot clinical trial involving 35 patients with CF whose sputum cultures were chronically positive for A. fumigatus. Participants were centrally randomized to receive either oral itraconazole 5 mg/kg/d (N=18) or placebo (N=17) for 24 weeks. The primary outcome was the proportion of patients who experienced a respiratory exacerbation requiring intravenous antibiotics over the 24 week treatment period. Secondary outcomes included changes in FEV(1) and quality of life.

Four of 18 (22%) patients randomized to itraconazole experienced a respiratory exacerbation requiring intravenous antibiotics, compared to 5 of 16 (31%) placebo treated patients, P=0.70. FEV(1) declined by 4.62% over 24 weeks in the patients randomized to itraconazole, compared to a 0.32% improvement in the placebo group (between group difference=-4.94%, 95% CI: -15.33 to 5.45, P=0.34). Quality of life did not differ between the 2 treatment groups throughout the study. Therapeutic itraconazole blood levels were not achieved in 43% of patients randomized to itraconazole.

–   So the authors did not identify clinical benefit from itraconazole treatment for CF patients whose sputum was chronically colonized/infected with A. fumigatus. The obvious limitations of this pilot study were its small sample size, and failure to achieve therapeutic levels of itraconazole in many patients. Also the decline of FEV1% (4.62% over 24 weeks) in the itraconazole group seems to be excessive.

2013 Baxter CG, Moore CB, Jones AM, Webb AK, Denning DW. E-mediated immune responses and airway detection of Aspergillus and Candida in adult cystic fibrosis. Chest 2013; 143:1351-7. [PubMed]
The recovery of Aspergillus and Candida from the respiratory secretions of patients with cystic fibrosis (CF) is common. Their relationship to the development of allergic sensitization and effect on lung function has not been established. Improved techniques to detect these organisms are needed to increase knowledge of these effects.    A 2-year prospective observational cohort study was performed. Fifty-five adult patients with CF had sputum monitored for Aspergillus by culture and real-time polymerase chain reaction and Candida by CHROMagar and carbon assimilation profile (API/ID 32C). Skin prick tests and ImmunoCAP IgEs to a panel of common and fungal allergens were performed. Lung function and pulmonary exacerbation rates were monitored over 2 years.

The authors concluded alllergic sensitization is not associated with recovery of Candida or Aspergillus from the sputum of patients with CF. Aspergillus but not Candida sensitization is associated with greater lung function decline and pulmonary exacerbations.

2013 Rundfeldt C. Steckel H. Scherliess H. Wyska E. Wlaz P. Inhalable highly concentrated itraconazole nanosuspension for the treatment of bronchopulmonary aspergillosis.  Eur J Pharm Biopharm 2013; 83:44-53.[PubMed]
57% of CF patients are colonized by Aspergillus species and 10-20% of colonized patients develop symptoms of allergic bronchopulmonary aspergillosis (ABPA).    The aim of this study was to describe an aqueous nanosuspension of ITRA and to characterize the pharmacokinetics after single dose inhalation. Using wet-milling with organic milling beads, a stable nanosuspension with particle size in the range of 200nm and an ITRA concentration of 20% (v/w) could be obtained, using polysorbate 80 at a concentration of 14% relative to ITRA. The suspension was stable if stored at 8degreeC for 3 months without particle growth and could be nebulized using standard nebulizer technologies including mesh technology and pressured air nebulisers. A 10% suspension was well tolerated upon repeated dose inhalation once daily for 7 days at a predicted dose of 45mg/kg in rats. A single dose inhalation at a predicted dose of 22.5mg/kg resulted in maximum lung tissue concentration of 21.4mug/g tissue with a terminal half-life of 25.4h. Serum concentrations were lower, with a maximum concentration of 104ng/ml at 4h after dosing and a terminal half-life of 10.5h.

The data indicate that ITRA nanosuspension represents an interesting formulation for inhaled administration in CF patients suffering from ABPA. High and long lasting lung tissue concentrations well above the minimal inhibitory concentration of Aspergillus species enable once daily.

Lehmann S. Pfannenstiel C. Frmiedrichs F. Kroger K. Wagner N. Tenbrock K. Omalizu aspergillosis in patients with cystic fibrosis. Ther Adv Respir Dis 2014; 8:141-9. [PubMed]
A retrospective study of six patients (four female, two male, age 4-33 years old) with CF and ABPA treated with omalizumab within an observation period of 7.5 years. All patients showed clinical and laboratory stability or even an improvement within the treatment and post-treatment observation period, although omalizumab therapy was less effective in patients with progressed lung disease and long-term ABPA. Side effects of systemic steroids were reduced.

– Omalizumab has the potential to be an additional and solitary treatment option in patients with CF and ABPA. Early onset treatment may be beneficial and patients with early stage of lung disease seem to benefit more.

2014  Zicari AM. Celani C. De Castro G. Valerio De Biase R. Duse M. Anti IgE antibody as treatment of allergic bronchopulmonary aspergillosis in a patient with cystic fibrosis. Eur Rev Med Pharmaco 2014; 18(13):1839-41. [PubMed]
A girl with CF who experienced clinical and functional improvement over 12-months treatment with omalizumab. At 12 years, she presented with a worsening respiratory condition, asthma symptoms and reduced lung function (FEV1 of 78%). Blood tests showed an increased concentration of plasma total IgE and positive specific IgE antibodies to Aspergillus fumigatus; allergic skin tests were also positive for A. fumigatus. The patient started steroid therapy but had impaired glucose tolerance due to long-term steroid use. Subcutaneous omalizumab 300 mg every two weeks was initiated and after 14 weeks she had improved respiratory symptoms (FEV1 99%) and a marked reduction in the use of systemic antibiotic and corticosteroid therapies. No side effects were reported. This case shows that therapy with omalizumab for a prolonged period can resolve symptoms of asthma.

– Omalizumab certainly appears to be a useful addition to the treatment available for variety of conditions including asthma and chronic urticaria. There are now reports of success in treatment ABPA in people with cystic fibrosis. A previous report suggests early treatment may be more effective.

2015  Beam KT, Coop CA. Steroid sparing effect of omalizumab in seropositive allergic bronchopulmonary aspergillosis. Allergy Rhinol (Providence). 2015 Jan;6(2):143-5. doi: 10.2500/ar.2015.6.0128.  Free PMC Article [PubMed]
A patient with CF with serologic ABPA who did not tolerate therapy with antifungals was able to significantly reduce (by nearly 80%) her average daily steroid use while receiving anti-IgE therapy with omalizumab added to her other respiratory medications
Omalizumab may reduce corticosteroid dependence in patients with allergic bronchopulmonary aspergillosis for patients unable to tolerate antifungals, though the authors warn the use may be limited by cost.

– Omalizumab is a recombinant DNA-derived humanized IgG1k monoclonal antibody that specifically binds to free human immunoglobulin E (IgE) in the blood and interstitial fluid and to membrane-bound form of IgE (mIgE) on the surface of mIgE-expressing B lymphocytes (Schulman ES. Am J Respir Crit Care Med 2001; 164(8 Pt 2):s6-11)

2014 Casciaro R, Naselli A, Cresta F, Ros M, Castagnola E, Minicucci L. Role of nebulized amphotericin B in the management of allergic bronchopulmonary aspergillosis in cystic fibrosis: Case report and review of literature.  J Chemother. 2015 Oct;27(5):307-11. doi: 10.1179/1973947814Y.0000000194. Epub 2014 May 14.    [PubMed] The authors used nebulized liposomal amphotericin B (L-AMB) in a patient affected by CF, complicated by ABPA. The previous combined treatment with oral steroids and azoles had no respiratory benefit and caused relevant side effects. Amphotericin B has always been well tolerated and permitted a slight steroid tapering. They also observed benefits in pulmonary function and laboratory tests.
Few data are available in literature about the use of nebulized AMB in CF and there are no RCTs evaluating antifungals in CF-ABPA. In the authors’ opinion, the reported case suggests that nebulized L-AMB could represent a possible strategy in ABPA management in CF patients.

– There is a later RCT of nebulised budesonide +/- amphotericin in 21 asthmatic patients with ABPA. Over a year 66.7% of the controls but only 8.3% of the treated patients had exacerbations of their ABPA (Ram et al.2016)

2016 Demir SO; Atici S; Akkoc G; Yakut N; Ikizoglu NB; Eralp EE; Soysal A; Bakir M. Neurologic Adverse Events Associated with Voriconazole Therapy: Report of Two Pediatric Cases. Case Reports Infectious Diseases. 2016:3989070, 2016. [PubMed]
The current report presents two cases, one a 9-year-old boy and the other a 17-year-old girl, who experienced neurologic side effects associated with voriconazole therapy. The first case was a 9-year-old boy with cystic fibrosis and invasive aspergillosis that developed photophobia, altered colour sensation, and fearful visual hallucination. The second case was a 17-year-old girl with cystic fibrosis and allergic bronchopulmonary aspergillosis, and she experienced photophobia, fatigue, impaired concentration, and insomnia, when the dose of voriconazole therapy was increased from 12mg/kg/day to 16mg/kg/day. The complaints of the two patients disappeared after discontinuation of voriconazole therapy. In addition, although serum voriconazole concentration was not measured in the present cases, therapeutic drug monitoring for voriconazole seems to be critically important in preventing neurologic side effects in pediatric patients.

2016 Durham SH; Garza KB; Eiland LS.  Relationship between vancomycin dosage and serum trough vancomycin concentrations in pediatric patients with cystic fibrosis. American Journal of Health-System Pharmacy. 73(13):969-74, 2016 Jul 1. [PubMed]
The association between vancomycin dosage and serum trough vancomycin concentrations in pediatric patients with cystic fibrosis (CF) was examined. A study of hospital admissions involving pediatric patients with CF revealed no significant correlation, when all admissions were considered, between the initial or final vancomycin dosage regimen and the corresponding serum trough vancomycin concentration. Both correlations were significant for the subgroup of admissions representing patients who were at least 12 years old.

2016 Emiralioglu N, Dogru D Tugcu GD, Yalcin E, Kiper N, Ozcelik U. Omalizumab Treatment for Allergic Bronchopulmonary Aspergillosis in Cystic Fibrosis. Ann Pharmacother. 2016 Mar;50(3):188-93. doi: 10.1177/1060028015624204. Epub 2015 Dec 23 [PubMed]
A review of 6 CF patients with ABPA receiving omalizumab. All patients were treated with oral prednisolone and itraconazole. Omalizumab was started if the patient was not responding to steroid treatment, which was determined according to serum IgE levels and/or clinical findings or depending on if there were side effects caused by steroid treatment.The mean age of patients at the beginning of omalizumab treatment was 16.1 years. The mean duration of ABPA by the time that treatment with omalizumab started was 13 ± 12.4 months (range = 2-29 months). With omalizumab treatment, IgE levels were decreased in all patients, and Aspergillus-specific IgE levels were decreased in 4 patients; however, FEV1(% predicted) improved only in 2 patients who had mild disease. Corticosteroids were reduced in the first, second, and third months of omalizumab treatment in 2, 1, and 3 patients, respectively. In 2 patients, steroid treatment was stopped. None of the patients suffered from side effects of omalizumab. The mean duration of omalizumab treatment was 12.5 months (range = 6-18 months).

– This study showed steroid-sparing effect, decreasing IgE levels, and improvement in respiratory symptoms in 6 CF patients with omalizumab treatment.

2016 Ram B; Aggarwal AN; Dhooria S; Sehgal IS; Garg M; Behera D; Chakrabarti A; Agarwal R. A pilot randomized trial of nebulized amphotericin in patients with allergic bronchopulmonary aspergillosis. J Asthma 2016; 53(5):517-24. [PubMed]
Consecutive subjects of ABPA with recurrent exacerbations were randomised to receive nebulised amphotericin plus nebulized budesonide (NEB) or NEB alone.  At one year, the numbers of patients experiencing exacerbation was significantly lower in the NAB arm (1/12 [8.3%] vs. 6/9 [66.7%]; p=0.016). The other secondary end points were not different between the two groups. There were no major adverse events leading to discontinuation of any of the study drugs. Three patients experienced bronchospasm after first dose of NAB; however, the subsequent doses were well tolerated.                The authors concluded nebulised amphotericin seems to be beneficial in decreasing the frequency of exacerbations in patients with ABPA complicating asthma. The suggest larger trials are required to confirm their study results.

– These patients had asthma, not CF, complicated by ABPA but the findings are helpful and not unexpected.

Brandt CRoehmel JRickerts VMelichar VNiemann NSchwarz C. Aspergillus Bronchitis in Patients with Cystic Fibrosis. Mycopathologia. 2018 Feb;183(1):61-69. doi: 10.1007/s11046-017-0190-0. Epub 2017 Aug 17     [Pubmed] 

Claudia Brandt

Aspergillus fumigatus frequently colonizes the airways of patients with cystic fibrosis (CF) and may cause various    severe

Carsten Schwarz

infections, such as bronchitis.     Serological data, sputum dependent markers and longitudinal data of treated cases of Aspergillus bronchitis were evaluated for further description of this infection. This study, which comprises three substudies, aimed to analyze epidemiological data of Aspergillus in CF and the entity of Aspergillus bronchitis. A retrospective data analysis of 10 treated cases revealed the clinical course of Aspergillus bronchitis, including repeated positive sputum culture findings for A. fumigatus, no  antibiotic treatment response, total serum IgE levels <200 kU/l, no observation of new pulmonary infiltrates and appropriate antifungal treatment response. Antifungal treatment durations of 4 ± 1.6 (2-6) weeks significantly reduced cough (P = 0.0067), sputum production (P < 0.0001) and lung function measures (P = 0.0358) but not physical capacity (P = 0.0794)From this retrospective study, a prevalence of 1.6% was calculated. In addition, two cases of Aspergillus bronchitis were identified in the prospective cohort study according to immunological, molecular and microbiological parameters. A prevalence of 9% was assessed. Aspergillus bronchitis appears to occur in a minority of colonized CF patients. Antifungal treatment may reduce respiratory symptoms and restore lung function.

Claudia Brandt is a scientist working at Charite Universitatsmedizin Berlin.    Carsten Schwarz is Director of the Adult Cystic Fibrosis Unit at Charite Universitatsmedizin Berlin

Harun SNWainwright CEGrimwood KHennig SAustralasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) study group.Collaborators (24)Aspergillus and progression of lung disease in children with cystic fibrosis.  Thorax. 2018 Oct 1. pii: thoraxjnl-2018-211550. doi: 10.1136/thoraxjnl-2018-211550. [Epub ahead of print]  [Pubmed] 
The impact of Aspergillus on lung disease in young children with cystic fibrosis is uncertain. Study to determine if positive respiratory cultures of Aspergillus species are associated with: (1) increased structural lung injury at age 5 years; (2) accelerated lung function decline between ages 5 years and 14 years and (3) to identify explanatory variables.

A cross-sectional analysis of association between Aspergillus positive bronchoalveolar lavage (BAL) cultures and chest high-resolution CT (HRCT) scan findings at age 5 years in subjects from the Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) study was performed. A non-linear mixed-effects disease progression model was developed using FEV1% predicted measurements at age 5 years from the ACFBAL study and at ages 6-14 years for these subjects from the Australian Cystic Fibrosis Data Registry.

Results. Positive Aspergillus BAL cultures at age 5 years were significantly associated with increased HRCT scores for air trapping (OR 5.53, 95% CI 2.35 to 10.82). However, positive Aspergillus cultures were not associated with either FEV1% predicted at age 5 years or FEV1% predicted by age following adjustment for body mass index z-score and hospitalisation secondary to pulmonary exacerbations. Lung function demonstrated a non-linear decline in this population.

The authors concluded in children with cystic fibrosis, positive Aspergillus BAL cultures at age 5 years were associated contemporaneously with air trapping but not bronchiectasis. However, no association was observed between positive Aspergillus BAL cultures on FEV1% predicted at age 5 years or with lung function decline between ages 5 years and 14 years.

Préville-Ratelle SCoriati AMénard ABourdeau ITremblay FBerthiaume Y. Adrenal Insufficiency in Cystic Fibrosis: A Rare Phenomenon?   Can Respir J. 2018 Mar 13;2018:3629031. doi: 10.1155/2018/3629031. eCollection  2018. [Pubmed]  Free article
The prevalence of adrenal insufficiency (AI) in cystic fibrosis (CF) is unknown. The frequent use of glucocorticoids (inhaled or systemic) may induce the long-term suppression of the hypothalamic-pituitary-adrenal axis. The authors reviewed the results of adrenocorticotropic hormone (ACTH) stimulation tests done over a 10-year period to evaluate adrenal function in 69 CF patients of the CHUM CF clinic. Clinical characteristics of AI patients were compared to adrenal-sufficient (AS) patients.

Adrenal insufficiency was confirmed in 33 of the 69 CF patients. A higher rate of dysglycemia (P=0.022) and of Aspergillus positive culture (P=0.006) was observed in AI patients compared to AS patients. Weight, CFTR genotype, and pulmonary function were comparable between AI and AS patients. The use of systemic corticosteroids (SC) prior to the diagnosis of AI was observed in 42.4% of patients, but none of the AS patients. Inhaled corticosteroids in 67% of the AS and 94% of the AI patients. Compared to AI patients without SC, SC-treated AI patients were older and had a higher rate of allergic bronchopulmonary aspergillosis.

This study is the first to systematically examine the presence of AI in the largest cohort of CF patients studied to date with a prevalence of 8%. Patients treated with corticosteroids and those colonized with Aspergillus have a greater risk of AI.

Corresponding author: Professor Yves Berthiaume.  Institut de recherches cliniques de Montréal (IRCM),and Clinique de fibrose kystique, Centre hospitalier de l’Université de Montréal (CHUM), Canada H2X.

Harun SN, Wainwright CE, Grimwood K, Hennig S; Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) study group. Aspergillus and progression of lung disease in children with cystic fibrosis. Thorax. 2019;74(2):125–131. doi:10.1136/thoraxjnl-2018-211550  [Pubmed]

Sabariah Noor Harun

The impact of Aspergillus on lung disease in young children with cystic fibrosis is uncertain.  This study was to determine if positive respiratory cultures of Aspergillus species are associated with: (1) increased structural lung injury at age 5 years; (2)  lung function decline between ages 5 years and 14 years and (3) to identify explanatory variables.                                                                                                                                                                                              A cross-sectional analysis of the association between Aspergillus positive bronchoalveolar lavage (BAL) cultures and chest high-resolution CT (HRCT) scan findings at age 5 years in subjects from the Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) study was performed. A non-linear mixed-effects disease progression model was developed using FEV1% predicted measurements at age 5 years from the ACFBAL study and at ages 6-14 years for these subjects from the Australian Cystic Fibrosis Data Registry.Positive Aspergillus BAL cultures at age 5 years were significantly associated with increased HRCT scores for air trapping (OR 5.53, 95% CI 2.35 to 10.82). However, positive Aspergillus cultures were not associated with either FEV1% predicted at age 5 years or FEV1% predicted by age following adjustment for body mass index z-score and hospitalisation secondary to pulmonary exacerbations. Lung function demonstrated a non-linear decline in this population.

The authors concluded that In children with cystic fibrosis, positive Aspergillus BAL cultures at age 5 years were associated contemporaneously with air trapping but not bronchiectasis. However, no association was observed between positive Aspergillus BAL cultures on FEV1% predicted at age 5 years or with lung function decline between ages 5 years and 14 years.

Dr Sabariah Noor Harun is a lecturer in the School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia and School of Pharmacy, University of Queensland, Brisbane, Queensland, Australia.

Engel TGPErren EVanden Driessche KSJMelchers WJGReijers MHMerkus PVerweij PE.  Aerosol Transmission of Aspergillus fumigatus in Cystic Fibrosis Patients in the Netherlands.  Emerg Infect Dis. 2019 Apr;25(4):797-799. doi: 10.3201/eid2504.181110. [Pubmed]  Free PMC Article

     Tobias Engel

The authors collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.

Tobias G.P. Engel is currently in a residency with a PhD programme at Radboud University Medical Center, Department of Medical Microbiology, Route 777, Geert Grooteplein Zuid 10, 6500 HB Nijmegen, the Netherlands;

Chevalier B, Hinzpeter A. The influence of CFTR complex alleles on precision therapy of cystic fibrosis.  J Cyst Fibros. 2019 Dec 25. pii: S1569-1993(19)30988-9. doi: 10.1016/j.jcf.2019.12.008. [Epub ahead of print] [Pubmed]

CFTR is an extensively studied gene and multiple sequence variants have been identified, many of which still need to be defined as neutral or disease causing. Complex alleles are defined when at least two variants are identified on the same allele. Each pathogenic variant can affect distinct steps of the CFTR biogenesis. As CFTR modulators are being developed to alleviate specific defects, pathogenic variants need to be characterized to propose adequate treatments. Conversely, cis-variants can affect treatment response when defects are additive or if they alter the binding or efficacy of the modulator. Hence, complex alleles increase the complexity of CFTR variant classification and need to be assigned as neutral, disease causing or modulating treatment efficacy. This review was based on a symposium session presented at the 16th ECFS Basic Science Conference, Dubrovnik, Croatia, 27 to 30 March, 2019.

Dr Benoit Chevalier is at INSERM U1151, Institut Necker Enfants Malades, Paris, France; Université Paris Descartes, Paris, France.

Eickmeier OZissler UMWittschorek JUnger FSchmitt-Grohé SSchubert RHerrmann EZielen S. Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.  Clin Exp Allergy. 2019 Dec 30. doi: 10.1111/cea.13557. [Epub ahead of print] [Pubmed]

        Olaf Eickmeier

The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A. fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease.     The authors aimed to ascertain whether allergen exposure to A. fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease.

A total of 35 patients, aged 14.8±8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n=18): specific (s)IgE negative, and group 2 (n=17): sIgE positive (≥ 0.7 KU/L) for A. fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n=13) underwent BAP with A. fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured.                        Results: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After bronchial allergen provocation, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells.

The authors conclude their study demonstrated the clinical relevance of A. fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A. fumigatus exposure.

Dr Olaf Eickmeier,  Department for Children and Adolescents, Division of Allergology, Pulmonology and Cystic fibrosis, Goethe University, Frankfurt.

Patel D, Popple SClaydon AModha DEGaillard EA. Posaconazole therapy in children with cystic fibrosis and Aspergillus-related lung disease  Med Mycol. 2020 Jan 1;58(1):11-21. doi: 10.1093/mmy/myz0 [Pubmed]

      Erol Gaillard

A prospective study over a fifty-three month period evaluating the safety, tolerability, and efficacy of posaconazole in pediatric CF. Fourteen children (seven males, median age 13 years, range 3-17 years) received a total of twenty-three courses of posaconazole (13 oral suspension and 10 tablet formulation). Of these patient episodes, nine received posaconazole for emerging or active allergic bronchopulmonary aspergillosis (ABPA) and two required a combination of posaconazole and systemic corticosteroids for difficult-to-treat ABPA. A subgroup of patients (n = 12) with persistent isolates of Aspergillus fumigatus, in the absence of serological markers of ABPA, received posaconazole monotherapy for pulmonary exacerbations not responding to conventional broad-spectrum antibiotic treatment. Posaconazole levels, full blood count, electrolytes, and liver function were monitored on day 7 of treatment and then monthly. Posaconazole was well tolerated in all but three patients. Therapeutic plasma levels >1 mg/l were achieved in all receiving the tablet formulation in comparison to 60% on the liquid preparation. There was a modest but significant improvement in FEV1 (% predicted) demonstrated for the cohort as a whole (p = 0.015) following posaconazole therapy.

Posaconazole is well tolerated in children as young as six years old, improvements in lung function are observed, and therapeutic plasma levels are readily achieved in patients taking the tablet formulation and in adherent patients taking the liquid formulation.

Dr Erol Gaillard, Senior Lecturer in Child Heath and Honorary Consultant in Paediatric Respiratory Medicine.                           Department of Respiratory Sciences, NIHR Biomedical Research Centre (Respiratory theme) and Institute for Lung Health, University of Leicester, Leicester, United Kingdom and the Department of Paediatric Respiratory Medicine, University Hospitals of Leicester NHS Trust, Leicester, United Kingdom.

Mahasin Al ShakirchiKimmo SorjonenLena KlingsporPeter BergmanLena HjelteIsabelle de Monestrol .  The Effects of Aspergillus fumigatus Colonization on Lung Function in Patients with Cystic FibrosisJ Fungi (Basel) [Pubmed] Nov 9;7(11):944.doi: 10.3390/jof7110944   Free PMC article
Aspergillus fumigatus is commonly isolated from CF airways. However, the impact on CF lung progression is not completely understood. In this study, using a 16-year retrospective observational cohort study (2000-2015) that included 132 patients, we determined the annual lung function, measured as percent predicted forced expiratory volume in the first second (ppFEV1), decline before and after the first colonization with A. fumigatus. Further, in the same individual, the ratios of lung function when patients were colonized with A. fumigatus and when they were not were calculated. The impact of eradication, with antifungal treatment or spontaneously, was assessed.
The annual ppFEV1 was significantly lower after the first colonization with A. fumigatus. Furthermore, within the same individual, colonization with A. fumigatus for two and three years in a row was associated with 4.3% and 7.9% lower ppFEV1, respectively, compared to when not colonized.
Finally, patients who eradicated A. fumigatus the following two years after colonization exhibited 9.9% and 14.5% higher ppFEV1 compared to patients who continued to produce cultures with A. fumigatus for two and three years. Our study demonstrated that A. fumigatus colonization was associated with a negative impact on lung function in the long term and eradication, spontaneously or with treatment, was associated with a better pulmonary outcome.

Mahasin Al Shakirch is at the Stockholm Cystic Fibrosis Centre, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. and the Department of Clinical Science, Intervention and Technology, Division of Pediatrics, Karolinska Institutet, 171 77 Stockholm, Sweden.