Diagnosis and Management of Fungal DiseaseCystic Fibrosis Trust Clinical Conference

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

Belfast City Hospital

Diagnosis and Management of Fungal Disease

Background/Setting the Scene - John Moore

Spectrum of fungal disease in CF - Damian DowneyDiagnostic Aspects - John Moore

Discussion - Floor Identification of key issues:

Clinical? Diagnostic?

Next steps & Wrap-up - Damian Downey

No significant bugs= 50 years

Pseudomonas aeruginosa = 30 years

Burkholderia cenocepacia=19 years

UK CF Trust Newsletter

The ReasonThe Reason

Aspergillus/Scedosporium = ??

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; 48:166-76.

Increased % positive sputum cultures for Aspergillus ( US CFF patient registry)

6.18

7.31

8.89.42

10.8811.44

12.16

12.87 13.0213.51 13.45

0

2

4

6

8

10

12

14

16

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005Year

% p

osi

tive

fo

r A

sper

gil

lus

Bruce Montgomery; Personal communication

Year

% Positive Aspergillusin sputum

CF Registry of Ireland (http://www.cfri.ie/)

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

Spectrum of Fungal Disease in CF

Aspergillus fumigatusAspergillus causes significant morbidity in CFReported prevalence rates 6% to 58%Wide spectrum of diseaseMean age of onset 12-14 years of age1

Challenges over classification/diagnosis and treatments

Guidelines for ABPA2

1. Pihet et al. Med. Mycol. 20092. ABPA in CF – State of the Art: CFF Consensus Conference 2003

ABPAManifests as a worsening of pulmonary disease with

wheezing, SOB, cough and chest painFrequency varies due to differences in diagnosis

(1-15%)

ABPADiagnostic criteriaClinical deterioration not attributable to another

aetiologyTotal IgE ˃1000 IU/mlAspergillus SPT or specific IgEPrecipitating antibody to Aspergillus or IgGNew or recent abnormalities on CXR/CT not cleared

with antibiotics and PT

ABPA treatmentVariation in therapeutic approachesSteroids 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

Triazole antifungalsDecrease the fungal burden and antigenic

stimulationItraconazole1

Drug levels/interactions/SEsAzole resistance – 94 BAL samples2

4.3% azole resistance Mixed population – sensitive/resistant

1.Wark et al. J Allergy Clin Immunol. 20032.Zhao et al. J Antimicrob Chemo. 2013

VoriconazoleVoriconazole therapy in children with CF1

Open label retrospectiveHigh oral bioavailabilityAbsorption not affected by pHPlasma levels are variable2

Drug interactionsCost implications

1. Hilliard et al. Journal of CF. 20052.Spriet et al. Eur J Clin Microbiol Dis. 2011

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

Nebulised amphotericinCase series1,2

Limited experienceTolerability

1. Proesmans et al. Adv in Resp Dis. 20102. Hayes et al. Paed Pulm 2010

Omalizumab therapyRecombinant monoclonal antibody targeting Fc receptor of

IgE and prevents binding of IgE to immune effector cellsMediates inflammation and hypersensitive responseCase 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

1. Wong et al. Paed respiratory reviews 20132. Brinkmann et al. Allergy 2009

3. Tille-Leblond et al. Allergy 20114. http://clinicaltrials.gov/show/NCT00787917

5. Cochrane Database Syst Rev. 2013 Sep 17;9:CD010288

Aspergillus spectrumPrevalence rates 6-58%Detection challengesNot all develop ABPAHow do we define this group?Colonisation/infection/sensitisationDoes it cause clinical deterioration?Does it affect lung function?

Aspergillus bronchitisCase series – 6 patients1

Not ABPA by criteriaBut varying IgENone received steroidsClinical improvement with itraconazole

1.Shoseyov et al. Chest 2006

Aspergillus colonisationDutch study 20111

61/259 children and adultsRetrospective 2002-2007Defined as Aspergillus in >50% resp cultures that

yearCategorized to the no. of years they met criteria

above

1. Vrankrijker et al. Clinical Microbiology and Infection 2010

Aspergillus fumigatus colonization in cystic fibrosis: implications for lung function?

Clinical Microbiology and InfectionVolume 17, Issue 9, pages 1381-1386, 16 DEC 2010 DOI: 10.1111/j.1469-0691.2010.03429.

•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

The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalisation

Retrospective paed study (1999-2006) Toronto Did not differentiate between infection/colonisation230 patients, 37 (16%) AspergillusFEV1 (79% vs 86%)

Increased ABPA, use of neb antibiotics, steroids, Pa infection and inc trend of pulm exacerbations

1. Amin et al. Chest 2010

Aspergillus colonisation

Separate colonisation and sensitisation?Toulouse Centre, 251 patients (complete data 206)1

Retrospective 1995-2007ABPA (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

DifferencesDefinitions-colonisation/sensitisation/chronicityTransient vs persistent cultureComparing different groupsDifferent analysesRetrospective Can we define different syndromes?

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 positive108 (74%) RT-PCR positive (66 GM positive)68 (46%) GM positive

1. Baxter et al. Amer Acad of Allergy 2013

Class analysisClass 1

± asp in sputum, -ve GM and immunologyClass 2

serologic ABPA with +ve GM, serology and RT-PCRClass 3

± asp in sputum, IgE sensitised, -ve IgG and GMClass 4

-ve IgE, +ve IgG, RT-PCR and GM

Treatment of Aspergillus in patients with CFDouble blind, placebo RCT, 24 weeks1

35 Aspergillus +ve patients (non-ABPA)Difficult recruitmentExacerbations were the same in each groupFEV1 declined in itraconazole group

Therapeutic itraconazole levels not achieved in 43%No benefit

1. Aaron et al. PLoS one. 2012

AspergillusMany challengesDiagnosisDefinition of groupsEffect of treatmentsRelationship to bacteria/viruses

Scedosporium apiospermumFrench 5 yr prospective study – 8.6% patients1

14% of CF patients in a German Centre2

Risk factors for acquisition are not clearGenotyping in a centre revealed no shared strains, mainly single strain3

Same strains can be present for years4

1. Cimon et al. Eur. J. Clin. Microbiol. Infect. Dis. 20002. Horre et al. Respiration 20093. Defontaine et al. J Clin Microbiol 20024. Bernhardt. J Cystic Fibrosis 2013 (in press)

Diagnostic Aspects

- increasing burden of organisms +

Adherence early colonisation established colonisation early infection chronic infectionAdherence early colonisation established colonisation early infection chronic infection Acquistion:

environmentpatients

- increasing biofilm +

Phase I Phase II Phase III Phase IV Phase V

1st PA culture from 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):2706-11 )

nth fungal culture from sputum

- increasing burden of organisms +

Adherence early colonisation established colonisation early infection chronic infectionAdherence early colonisation established colonisation early infection chronic infection Acquistion:

environmentpatients

- increasing biofilm +

Phase I Phase II Phase III Phase IV Phase V

1st PA culture from 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):2706-11 )

nth fungal culture from sputum

133,317 fungal pyrosequences30 species or genera, including 24

micromycetes and 6 basidiomycetous macroscopic fungi.

• variation in the initial processing with lytic

• agent, • volume of sputa used to inoculate

plates, • type of media,• length of incubation• temperature,

Laboratory diagnosis of fungi from CF patientsCulture-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

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

Media B +

Glucose 16.7gAgar 20gYeast extract 30gPeptone 6.8g

(per 1000ml)

+

Cotrimoxazole 128mg/l

Chloramphenicol 50mg/l

Ceftazidime 32mg/l

Colistin 24mg/l

Ability to culture on Media B +Fungi GrowthYeasts 100%Filamentous fungi 100%

BacteriaP. 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%

Media B

SDA

Media B with antibiotics

SDA

Media B

Media B with antibiotics

SDA

Media BMedia B with

antibiotics

Selectivity and sensitivity: Medium B+ antibiotics > SDA > Medium B

Sensitivity

Fungal Selective Medium

Specificity Yeasts Filamentous fungi

Combined fungi

SDA 46.4% 76.0% 83.0% 84.6%

SDA+ 89.2% 84.0% 33.0% 84.6%

Medium B+ 85.7% 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):566-72.

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) nitrateJ Microbiol Methods. 2009 ;76(2):201-203.

• 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.

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.

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; 48:166-76.

18S rRNA gene 5.8S rRNA gene 28S rRNA gene

ITS1 region ITS2 region

ITS 1 ITS 4

5’-TCC GTA GGT GAA CCT GCG G-3’ 5’-TCC TCC GCT TAT TGA TAT GC-3’

ITS3 ITS 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.

Prevalence rate of fungi in CF patients

97 ％

58 ％

39 ％

9.1 ％ 9.1 ％ 9.1 ％6.5 ％

5.2 ％ 5.2 ％ 3.9 ％ 3.9 ％ 2.6 ％ 2.6 ％1.3 ％ 1.3 ％ 1.3 ％ 1.3 ％ 1.3 ％ 1.3 ％ 1.3 ％

0

10

20

30

40

50

60

70

80

90

100

Candid

a sp

p.

Candia

albi

cans

Candia

dub

linien

sis

Candid

a pa

raps

ilosis

Asper

gillus

spp.

Penici

lium

sp.

Sacch

arom

yces

cere

visiae

Candid

a gla

brat

a

Asper

gillus

fum

igatu

s

Exoph

iala

derm

atitid

is

Scedo

spor

ium a

piosp

erm

um

Tricho

spor

orn

sp.

Mala

ssez

ia sp

.

Rhodo

toru

la sp

.

Aureo

basid

ium p

ullula

ns

Fusco

poria

ferre

a

Fusar

ium cu

lmor

um

Acrem

onium

stric

tum

Thana

teph

orus

cucu

mer

is

Clados

poriu

m sp

.

Yeasts and filamentous fungi

% p

ati

en

ts p

os

itiv

e

Yeast=6 genera

Filamentous fungi=9 genera

5 fungi1%

3 fungi16%

2 fungi23%

1 fungi60%

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

Candid

a sp

p.

Candia

albica

ns

Candia

dub

linien

sis

Candid

a pa

rapsil

osis

Candid

a gla

brat

a

Exoph

iala

dermatiti

dis

Rhodo

toru

la sp

.

Sacch

arom

yces

cere

visiae

Tricho

spor

on sp

.

Asper

gillus

spp.

Asper

gillus

fumiga

tus

Penici

lium sp

.

Scedo

spor

ium a

piosper

mum

Aureo

basidium

pull

ulans

Fusco

poria

ferre

a

Fusar

ium cu

lmor

um

Acremon

ium stric

tum

Malass

ezia

sp.

Thana

teph

orus

cucu

meris

Clados

porium

sp.

0

10

20

30

40

50

60

70

80

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)

Ivery significant fungi

related to CF

IIIpotentially significant

fungi related to CF

IVnot 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 ferreaFusarium culmorum

Thanatephorus cucumerisSporidiobolus salmonicolor

Phaeococcomyces chersonesos

Emericella sp. Blumeria sp.

Phoma herbarum trametes sp.

Coniosporium sp.Kondoa aeria

Rhexocercosporidium sp.Sclerotinia sclerotiorum

Sterem annosumHeterobasidion annosum

Paecilomyces sp.Engyodontium album Yarrowia lipolytica

The classification of fungi related to CF

IIrelatively significant fungi

related to CF

Decreasing clinical significance in CF patients

Fungal Workshop –

What are the diagnostic and clinical challenges?

Fungal Workshop –

What are the diagnostic and clinical challenges?

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?

cysticfibrosis.org.uk

Thank you