Diagnosis and Management of Fungal Disease Cystic Fibrosis Trust Clinical Conference Damian Downey & John E. Moore Northern Ireland Regional Adult Cystic Fibrosi Centre Belfast City Hospital Diagnosis and Management of Fungal Disease
Dec 15, 2015
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