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Diagnosis and Management of Fungal Disease Cystic Fibrosis Trust Clinical Conference Damian Downey & John E. Moore Northern Ireland Regional Adult Cystic.

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Presentation on theme: "Diagnosis and Management of Fungal Disease Cystic Fibrosis Trust Clinical Conference Damian Downey & John E. Moore Northern Ireland Regional Adult Cystic."— Presentation transcript:

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

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 No significant bugs = 50 years Pseudomonas aeruginosa = 30 years Burkholderia cenocepacia =19 years UK CF Trust Newsletter The Reason Aspergillus/Scedosporium = ??

4 Fungal isolates from hospital air Cladosporium sp. Rhodotorula sp. Aspergillus versicolor Aspergillus fumigatus Penicillium sp. Aureobasidium pullulans Sporidiobolus salmonicolor Phaeococcomyces chersonesos Emericella sp. Coniosporium sp. Phoma herbarum Blumeria sp. Kondoa aeria Trametes sp. Rhexocercosporidium sp. Sclerotinia sclerotiorum Sterem annosum Heterobasidion annosum Paecilomyces sp. Aspergillus sydowii Cryptococcus sp. Cryptococcus magnus Engyodontium album Yarrowia lipolytica 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:

5 Increased % positive sputum cultures for Aspergillus ( US CFF patient registry) Year % positive for Aspergillus Bruce Montgomery; Personal communication

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

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

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 years of age 1 Challenges over classification/diagnosis and treatments Guidelines for ABPA 2 1. Pihet et al. Med. Mycol 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%)

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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 treatment 1 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 Itraconazole 1 Drug levels/interactions/SEs Azole resistance – 94 BAL samples 2 4.3% azole resistance Mixed population – sensitive/resistant 1.Wark et al. J Allergy Clin Immunol Zhao et al. J Antimicrob Chemo. 2013

17 Voriconazole Voriconazole therapy in children with CF 1 Open label retrospective High oral bioavailability Absorption not affected by pH Plasma levels are variable 2 Drug interactions Cost implications 1. Hilliard et al. Journal of CF Spriet et al. Eur J Clin Microbiol Dis. 2011

18 Cochrane Review 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.Cochrane Database Syst Rev Jun 13;6:CD002204

19 Nebulised amphotericin Case series 1,2 Limited experience Tolerability 1.Proesmans et al. Adv in Resp Dis 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 series 1,2 Trial 16 ABPA patients (CF excluded) 3 Exacerbations reduced. No effect on lung function Pharma study in CF- RCT terminated early 4 Cochrane review Need for further studies 5 1.Wong et al. Paed respiratory reviews Brinkmann et al. Allergy Tille-Leblond et al. Allergy http://clinicaltrials.gov/show/NCT http://clinicaltrials.gov/show/NCT Cochrane Database Syst Rev Sep 17;9:CD010288Cochrane Database Syst Rev.

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 patients 1 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 /259 children and adults Retrospective 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? Clinical Microbiology and Infection Volume 17, Issue 9, pages , 16 DEC 2010 DOI: /j Volume 17, Issue 9, Differences in FEV 1 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

25 The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalisation Retrospective paed study ( ) Toronto Did not differentiate between infection/colonisation 230 patients, 37 (16%) Aspergillus FEV 1 (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 ABPA (34), sensitisation group (63), persistent carriage (37) and control (72) Groups were independently associated with FEV 1 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 Manchester 1 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 weeks 1 35 Aspergillus +ve patients (non-ABPA) Difficult recruitment Exacerbations were the same in each group FEV 1 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% patients 1 14% of CF patients in a German Centre 2 Risk factors for acquisition are not clear Genotyping in a centre revealed no shared strains, mainly single strain 3 Same strains can be present for years 4 1.Cimon et al. Eur. J. Clin. Microbiol. Infect. Dis Horre et al. Respiration Defontaine et al. J Clin Microbiol Bernhardt. J Cystic Fibrosis 2013 (in press)

33 Diagnostic Aspects

34 - increasing burden of organisms + Adherence early colonisation established colonisation early infection chronic infection  Acquistion: environment patients - increasing biofilm + Phase I Phase II Phase III Phase IV Phase V 1 st PA culture f rom sputum Improved Detection Zone ANTIMICROBIAL INTERVENTION 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): ) nth fungal culture from sputum

35 - increasing burden of organisms + Adherence early colonisation established colonisation early infection chronic infection  Acquistion: environment patients - increasing biofilm + Phase I Phase II Phase III Phase IV Phase V 1 st PA culture f rom sputum Improved Detection Zone ANTIMICROBIAL INTERVENTION 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): ) nth fungal culture from sputum

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

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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 May; 28(3): ; J Clin Micro Feb; ) 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 Agar20g Yeast extract30g Peptone6.8g (per 1000ml) + Cotrimoxazole 128mg/l Chloramphenicol 50mg/l Ceftazidime 32mg/l Colistin 24mg/l

44 Ability to culture on Media B + FungiGrowth 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. fluorescens0% Morgarella morgarii 0% A. xylosoxidones 0% K. oxytoca 0% A. salmonicida 0% P. mirabilis 0% MRSA 0% S. aureus 0%

45 Media B SDA Media B with antibiotics

46 SDA Media B Media B with antibiotics

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

48 Sensitivity Fungal Selective Medium SpecificityYeastsFilamentous fungi Combined fungi SDA46.4%76.0%83.0%84.6% SDA %84.0%33.0%84.6% Medium B %92.0%83.0% 92.3% 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):

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51 Moore JE, Murphy A, Millar BC, Loughrey A, Rooney PJ, Elborn JS, Goldsmith CE. Improved cultural selectivity of medically significant fungi by suppression of contaminating bacterial flora employing gallium (III) nitrate J Microbiol Methods ;76(2):

52 Selective isolation techniques were superior in detecting non-Aspergillus hyphomycetes compared with conventional methods. Although liquid media detected fewer strains of Exophiala, Pseudallescheria and Scedosporium species, additional hyphomycete species not detected by other methods were isolated. Current conventional methods are insufficient to detect non-Aspergillus hyphomycetes, especially Exophiala, Pseudallescheria and Scedosporium species, in sputum samples of cystic fibrosis patients.

53 Conclusions: These data suggest that standard microbiological media and procedures are not sufficient to detect colonization of the respiratory tract by Pseudallescheria/Scedosporium in CF patients. By use of Sce-Sel+ agar, fungi belonging to this complex were isolated more frequently. Therefore, this semiselective mycological isolation medium should be used for the detection of these fungi in the respiratory tract of CF patients, especially in patients in whom a fungal infection is assumed or who are scheduled for lung transplantation.

54 n=77 adult patients attend Regional Adult CF Centre, BCH Median age:28.5 (18-59 years) Male: 48% Female: 52% Plate onto SDA Media B 22 ˚C 2-3 weeks DNA extraction PCR (ITS1-ITS4) Direct sequencing Identification Fresh CF Sputum ( post physiotherapy) Plate onto SDA 30 ˚C 1week Resend to mycology specialist laboratory Identification Add 1:1 Sputalysin DNA extraction PCR (ITS1-ITS4) Nested PCR (ITS3-ITS4) One band more than one band Direct sequencing Cloning Sequencing Identification 1. Employing conventional mycological culture 2. Mycological culture with CF-derived fungal selective culture medium 3. Direct DNA extraction from sputum (non-cultured) approach + Colistin Ceftazidime Cotrimoxazole Chloramphenicol 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:

55 18S rRNA gene5.8S rRNA gene28S rRNA gene ITS1 regionITS2 region ITS 1ITS 4 5’-TCC GTA GGT GAA CCT GCG G-3’5’-TCC TCC GCT TAT TGA TAT GC-3’ ITS3ITS 4 5’-GCA TCG ATG AAG AAC GCA GC-3’ Nested PCR Primarily PCR Millar BC, Xu J, Earle JA, Evans J, Moore JE. Comparison of four rDNA primer sets (18S, 28S, ITS1, ITS2) for the molecular identification of yeasts and filamentous fungi of medical importance. Br J Biomed Sci. 2007;64(2):84-9.

56 Prevalence rate of fungi in CF patients 97 % 58 % 39 % 9.1 % 6.5 % 5.2 % 3.9 % 2.6 % 1.3 % Candida spp. Candia albicans Candia dubliniensis Candida parapsilosis Aspergillus spp. Penicilium sp. Saccharomyces cerevisiae Candida glabrata Aspergillus fumigatus Exophiala dermatitidis Scedosporium apiospermum Trichospororn sp. Malassezia sp. Rhodotorula sp. Aureobasidium pullulans Fuscoporia ferrea Fusarium culmorum Acremonium strictum Thanatephorus cucumeris Cladosporium sp. Yeasts and filamentous fungi % patients positive Yeast=6 genera Filamentous fungi=9 genera

57 The number of fungi detected in CF patients n=77 1 fungi=46 2 fungi=18 3 fungi=12 4 fungi=0 5 fungi=1

58 Method 1=14 (18%) [NHS standard] Method 2=60 (78%) [Selective agar + molecular ID] Method 3=77 (100%) [Full molecular workup] Yeasts and filamentous fungi organisms % positive (n=77)

59 I very significant fungi related to CF III potentially significant fungi related to CF IV not reported as human pathogens Aspergillus fumigatus Aspergillus versicolor Aspergillus sydowii Aspergillus spp. Candida albicans Candida dubliniensis Candida parapsilosis Candida glabrata Exophiala dermatitidis Scedosporium apiospermum Trichosporon sp. Malassezia sp. Penicillium sp. Rhodotorula sp. Saccharomyces cerevisiae Aureobasidium pullulans Acremonium strictum Cladosporium sp. Cryptococcus sp. Fuscoporia ferrea Fusarium culmorum Thanatephorus cucumeris Sporidiobolus salmonicolor Phaeococcomyces chersonesos Emericella sp. Blumeria sp. Phoma herbarum trametes sp. Coniosporium sp. Kondoa aeria Rhexocercosporidium sp. Sclerotinia sclerotiorum Sterem annosum Heterobasidion annosum Paecilomyces sp. Engyodontium album Yarrowia lipolytica The classification of fungi related to CF II relatively significant fungi related to CF Decreasing clinical significance in CF patients

60 Fungal Workshop – What are the diagnostic and clinical challenges?

61 How do we define Aspergillus groups with the tools currently available? Should there be a nominated specialist laboratory to support fungal workup in CF? What RCTs should be carried out?

62 cysticfibrosis.org.uk Thank you


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