Damian Downey & John E. Moore Northern Ireland Regional Adult Cystic Fibrosis Centre

1. Diagnosis and Management of Fungal Disease Damian Downey & John E. Moore Northern Ireland Regional Adult Cystic Fibrosis Centre Belfast City Hospital

2. Background/Setting the Scene - John Moore • Spectrum of fungal disease in CF - Damian Downey • Diagnostic Aspects - John Moore • Discussion - Floor • Identification of key issues: • Clinical? • Diagnostic? • Next steps & Wrap-up - Damian Downey

3. Infection in CF is very important The Reason No significant bugs = 50 years Pseudomonas aeruginosa = 30 years Burkholderia cenocepacia =19 years UK CF Trust Newsletter Aspergillus/Scedosporium = ??

4. Fungal isolates from hospital air Sporidiobolus salmonicolor Phaeococcomyces chersonesos Emericella sp. Coniosporium sp. Phoma herbarum Blumeria sp. Kondoa aeriaTrametes sp. Rhexocercosporidium sp. Sclerotinia sclerotiorum Sterem annosum Heterobasidion annosum Paecilomyces sp. Aspergillus sydowii Cryptococcus sp. Cryptococcus magnus Engyodontium album Yarrowia lipolytica Cladosporium sp. Rhodotorula sp. Aspergillus versicolor Aspergillus fumigatus Penicillium sp. Aureobasidium pullulans Fungi isolated concurrently from CF patients’ sputum Nagano Y, Elborn JS, Millar BC, Walker JM, Goldsmith CE, Rendall J, Moore JE. Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis. Med Mycol. 2010; 48:166-76.

5. 16 14 13.51 13.45 13.02 12.87 12.16 12 11.44 10.88 10 9.42 8.8 % positive for Aspergillus 8 7.31 6.18 6 4 2 0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year Increased % positive sputum cultures for Aspergillus( US CFF patient registry) Bruce Montgomery; Personal communication

6. % Positive Aspergillus in sputum Year CF Registry of Ireland (http://www.cfri.ie/)

7. ARTEFACTUAL (i). Increased reporting Efficacy/data capture (ii). Improved laboratory diagnosis - culture - NGS - MALDI-TOF - standardisation (iii). Improvements in radiological imaging REAL EFFECT (i). fungal virulence (ii). change in epidemiology (iii). increased risk factors

8. Spectrum of Fungal Disease in CF

9. Aspergillus fumigatus • Aspergillus causes significant morbidity in CF • Reported prevalence rates 6% to 58% • Wide spectrum of disease • Mean age of onset 12-14 years of age1 • Challenges over classification/diagnosis and treatments • Guidelines for ABPA2 1. Pihet et al. Med. Mycol. 2009 2. ABPA in CF – State of the Art: CFF Consensus Conference 2003

10. ABPA • Manifests as a worsening of pulmonary disease with wheezing, SOB, cough and chest pain • Frequency varies due to differences in diagnosis (1-15%)

14. ABPA • Diagnostic criteria • Clinical deterioration not attributable to another aetiology • Total IgE ˃1000 IU/ml • Aspergillus SPT or specific IgE • Precipitating antibody to Aspergillus or IgG • New or recent abnormalities on CXR/CT not cleared with antibiotics and PT

15. ABPA treatment • Variation in therapeutic approaches • Steroids are the mainstay of treatment1 • Triazole antifungals may have some steroid sparing effect • Combinations/length of treatment/monitoring 1. ABPA in CF – State of the Art: CFF Consensus Conference 2003

16. Triazole antifungals • Decrease the fungal burden and antigenic stimulation • Itraconazole1 • Drug levels/interactions/SEs • Azole resistance – 94 BAL samples2 • 4.3% azole resistance • Mixed population – sensitive/resistant • Warket al. J Allergy ClinImmunol. 2003 • Zhao et al. J Antimicrob Chemo. 2013

17. Voriconazole • Voriconazole therapy in children with CF1 • Open label retrospective • High oral bioavailability • Absorption not affected by pH • Plasma levels are variable2 • Drug interactions • Cost implications • Hilliard et al. Journal of CF. 2005 • Sprietet al. Eur J ClinMicrobiol Dis. 2011

18. Cochrane Review 2012- Antifungal therapies for ABPA in people with cystic fibrosis • “At present, 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” Cochrane Database Syst Rev. 2012 Jun 13;6:CD002204

19. Nebulised amphotericin • Case series1,2 • Limited experience • Tolerability • Proesmans et al. Adv in Resp Dis. 2010 • Hayes et al. Paed Pulm 2010

20. Omalizumab therapy • Recombinant monoclonal antibody targeting Fc receptor of IgE and prevents binding of IgE to immune effector cells • Mediates inflammation and hypersensitive response • Case series1,2 • Trial 16 ABPA patients (CF excluded)3 • Exacerbations reduced. No effect on lung function • Pharma study in CF- RCT terminated early4 • Cochrane review 2013. Need for further studies 5 • Wong et al. Paed respiratory reviews 2013 • Brinkmann et al. Allergy 2009 • Tille-Leblond et al. Allergy 2011 • http://clinicaltrials.gov/show/NCT00787917 • Cochrane Database Syst Rev. 2013 Sep 17;9:CD010288

21. Aspergillus spectrum • Prevalence rates 6-58% • Detection challenges • Not all develop ABPA • How do we define this group? • Colonisation/infection/sensitisation • Does it cause clinical deterioration? • Does it affect lung function?

22. Aspergillus bronchitis • Case series – 6 patients1 • Not ABPA by criteria • But varying IgE • None received steroids • Clinical improvement with itraconazole 1.Shoseyov et al. Chest 2006

23. Aspergillus colonisation • Dutch study 20111 • 61/259 children and adults • Retrospective 2002-2007 • Defined as Aspergillus in >50% resp cultures that year • Categorized to the no. of years they met criteria above 1. Vrankrijker et al. Clinical Microbiology and Infection 2010

24. Aspergillus fumigatus colonization in cystic fibrosis: implications for lung function? • Differences in FEV1 disappeared after adjustment for confounders • Specific antibody data was not included • Colonisation with Aspergillus was not independently associated with a decline in lung fn • Aspergillus was independently associated with age and the use of inhaled antibiotics Clinical Microbiology and InfectionVolume 17, Issue 9, pages 1381-1386, 16 DEC 2010 DOI: 10.1111/j.1469-0691.2010.03429.

25. The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalisation • Retrospective paed study (1999-2006) Toronto • Did not differentiate between infection/colonisation • 230 patients, 37 (16%) Aspergillus • FEV1 (79% vs 86%) • Increased ABPA, use of neb antibiotics, steroids, Pa infection and inc trend of pulm exacerbations 1. Amin et al. Chest 2010

26. Aspergillus colonisation • Separate colonisation and sensitisation? • Toulouse Centre, 251 patients (complete data 206)1 • Retrospective 1995-2007 • ABPA (34), sensitisation group (63), persistent carriage (37) and control (72) • Groups were independently associated with FEV1 decline • Some sensitised patients had episodes of ABPA 1. Fillaux et al. Scand J Infect Dis. 2012

27. Differences • Definitions-colonisation/sensitisation/chronicity • Transient vs persistent culture • Comparing different groups • Different analyses • Retrospective • Can we define different syndromes?

28. Classification of Aspergillus syndromes? • Challenges in distinguishing colonisation, sensitisation and Aspergillus bronchitis/infection • 146 patients in Manchester1 • Assessed serologic tests, RT-PCR, sputum GM • 39 (27%) culture positive • 108 (74%) RT-PCR positive (66 GM positive) • 68 (46%) GM positive 1. Baxter et al. Amer Acad of Allergy 2013

29. Class analysis • Class 1 • ± asp in sputum, -ve GM and immunology • Class 2 • serologic ABPA with +ve GM, serology and RT-PCR • Class 3 • ± asp in sputum, IgE sensitised, -ve IgG and GM • Class 4 • -ve IgE, +ve IgG, RT-PCR and GM

30. Treatment of Aspergillus in patients with CF • Double blind, placebo RCT, 24 weeks1 • 35 Aspergillus +ve patients (non-ABPA) • Difficult recruitment • Exacerbations were the same in each group • FEV1 declined in itraconazole group • Therapeutic itraconazole levels not achieved in 43% • No benefit 1. Aaron et al. PLoS one. 2012

31. Aspergillus • Many challenges • Diagnosis • Definition of groups • Effect of treatments • Relationship to bacteria/viruses

32. Scedosporium apiospermum • French 5 yr prospective study – 8.6% patients1 • 14% of CF patients in a German Centre2 • Risk factors for acquisition are not clear • Genotyping in a centre revealed no shared strains, mainly single strain3 • Same strains can be present for years4 • Cimon et al. Eur. J. Clin. Microbiol. Infect. Dis. 2000 • Horre et al. Respiration 2009 • Defontaine et al. J Clin Microbiol 2002 • Bernhardt. J Cystic Fibrosis 2013 (in press)

33. Diagnostic Aspects

34. - increasing burden of organisms + - increasing biofilm + Phase I Phase II Phase III Phase IV Phase V ANTIMICROBIAL INTERVENTION Acquistion: environment patients  Adherence early colonisation established colonisation early infection chronic infection Improved Detection Zone 1st PA culture f rom sputum Improvements: Radiological imaging (chest x—ray; HRCT; MRI;) selective culture/selective enrichment (agars) direct molecular detection (PCR, RT-PCR, qPCR) serological/antibody detection (galactomannan) Biomarkers via fluorescence correlation spectroscopy (FCS) (Sahahzad et al.J Cell Mol Med. 2011; 15(12):2706-11 ) nth fungal culture from sputum

35. - increasing burden of organisms + - increasing biofilm + Phase I Phase II Phase III Phase IV Phase V ANTIMICROBIAL INTERVENTION Acquistion: environment patients  Adherence early colonisation established colonisation early infection chronic infection Improved Detection Zone 1st PA culture f rom sputum Improvements: Radiological imaging (chest x—ray; HRCT; MRI;) selective culture/selective enrichment (agars) direct molecular detection (PCR, RT-PCR, qPCR) serological/antibody detection (galactomannan) Biomarkers via fluorescence correlation spectroscopy (FCS) (Sahahzad et al.J Cell Mol Med. 2011; 15(12):2706-11 ) nth fungal culture from sputum

36. 133,317 fungal pyrosequences 30 species or genera, including 24 micromycetes and 6 basidiomycetous macroscopic fungi.

40. variation in the initial processing with lytic • agent, • volume of sputa used to inoculate plates, • type of media, • length of incubation • temperature,

41. Laboratory diagnosis of fungi from CF patients • Culture-based • Most CF labs performing fungal cultures • cheap • Non-culture-based • PCR (specific & broad range), • RT-PCR, • qPCR, NGS, Sanger Sequencing • Highly specialised • Reference/Specialist laboratory involvement

42. Problems associated with fungal isolation on agar 1. Previously, the inhibition of fungal growth by P.aeruginosa and B. cepacia complex was reported (J.R. Kerr, J infect. 1994 May; 28(3): 305-10; J Clin Micro. 1994 Feb; 525-527) 2. Overgrowth by rapidly growing bacterial organisms Objectives of novel medium 1. Promote selectivity and sensitivity of yeasts and filamentous fungi, whilst inhibiting co-flora (i.e. pan-resistant P. aeruginosa and B. cepacia complex 2. Develop medium that could be used for quantitative of fungi

43. Media B + Glucose 16.7g Agar 20g Yeast extract 30g Peptone 6.8g (per 1000ml) Cotrimoxazole 128mg/l Chloramphenicol 50mg/l Ceftazidime 32mg/l Colistin 24mg/l +

44. Ability to culture on Media B + Fungi Growth Yeasts 100% Filamentous fungi 100% Bacteria P. aeruginosa 0% B. cepacia complex 67% (poor growth) S. maltophilia 0% E. coli 0% H. influenzae 0% P. fluorescens 0% Morgarella morgarii 0% A. xylosoxidones 0% K. oxytoca 0% A. salmonicida 0% P. mirabilis 0% MRSA 0% S. aureus 0%

45. SDA Media B with antibiotics Media B

46. SDA Media B with antibiotics Media B

47. SDA Media B with antibiotics Media B Selectivity and sensitivity: Medium B+ antibiotics > SDA > Medium B

48. Sensitivity Nagano Y, Millar BC, Goldsmith CE, Walker JM, Elborn JS, Rendall J, Moore JE. Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF). J Cyst Fibros. 2008; 7(6):566-72.