Lack of standardization in the procedures for mycological examination of sputum samples from CF...

Medical Mycology 2010, 48(Suppl. 1), S88–S97

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Lack of standardization in the procedures for mycological

examination of sputum samples from CF patients:

a possible cause for variations in the prevalence of

fi lamentous fungi

All authors are members of the

Respiratory Infections in Cystic F

Received 1 June 2010; Received

Accepted 24 July 2010

Correspondence: Andrew M. B

Laboratory, Health Protection Age

ries, Myrtle Road, Bristol BS2 8E

� 44 117 9226611; E-mail: Andy.B

ANDREW M. BORMAN * , MICHAEL D. PALMER * , LAURENCE DELHAES † , JACQUELINE CARR È RE ‡ ,

LO Ï C FAVENNEC § , ST É PHANE RANQUE # , JEAN-PIERRE GANGNEUX ̂ , REGINE HORR É $

& JEAN-PHILIPPE BOUCHARA ¶

* UK Mycology Reference Laboratory, Health Protection Agency, Bristol, UK, † Parasitology-Mycology Service (EA3609 - BDEEP)

Faculty of Medicine, Univ. Lille Nord de France (UDSL), University Hospital Centre & IFR-142, Institut Pasteur de Lille, France,

‡ Laboratoire de Biologie, CRCM, H ô pital Ren é e Sabran, Giens, Hy è res, France, § Laboratoire de Parasitologie-Mycologie, Centre

Hospitalier Universitaire, Rouen, France, # Laboratoire de Parasitologie-Mycologie, AP-HM Timone, Marseille, France, ^Laboratoire de

Parasitologie-Mycologie, Centre Hospitalier Universitaire de Rennes and UPRES-EA 4427, Universit é de Rennes 1, France, $ Federal

Institute for Drugs and Medical Devices, Bonn, Germany, and ¶ Groupe d ’ Etude des Interactions H ô te-Pathog è ne, UPRES-EA 3142,

Universit é d ’ Angers & Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France

© 2010 ISHAM

Filamentous fungi and yeasts are increasingly isolated from respiratory secretions of patients with cystic fi brosis (CF), and persistent fungal colonization of the airways of such patients is thought to exacerbate lung damage. While many independent studies have identifi ed Aspergillus fumigatus complex as the principal colonizing fungus in CF, increased awareness of the role of fungi in CF pathology coupled with improved mycological culture and identifi cation methods have resulted in a number of other fungi being isolated and reported from CF sputum samples, including A. terreus , mem-bers of the Pseudallescheria boydii / Scedosporium apiospermum complex, Exophiala dermatitidis , Paecilomyces and Penicillium species. However, the range of fungal path-ogens isolated and the relative prevalence of individual species vary widely between reports from different geographical CF centres, and as yet no standardized method for the mycological examination of CF sputum samples has been adopted. Here, we examine the potential contribution of the mycological methods employed to exam-ine CF respiratory secretions relative to the variability in the fungal biota reported. The role of direct microscopic examination of respiratory samples and the impact of the culture conditions used on the detection of specifi c fungal pathogens are addressed, and the potential signifi cance of isolation of yeast species from CF patient airways is discussed.

Keywords cystic fi brosis , fi lamentous fungi , yeasts , respiratory samples , culture methods

ISHAM Working group on Fungal

ibrosis.

in fi nal revised form 21 July 2010;

orman, UK Mycology Reference

ncy South-West Regional Laborato-

L, UK. Tel: � 44 117 9268683; Fax:

[email protected]

Introduction

Cystic fi brosis (CF) is the predominant genetic disease in

the Caucasian population in European countries. While

several organs may be affected, the severity of lesions

involving the respiratory tract usually dictates both patient

morbidity and eventually mortality [1]. Mutations in the

Cystic Fibrosis Transmembrane Conductance Regulator

DOI: 10.3109/13693786.2010.511287

Respiratory samples from CF patients: comparison of culture methods S89

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( CFTR ) gene result in altered mucociliary clearance, and

characteristic excessively viscous bronchial mucus

[reviewed in 2]. This viscous mucus is frequently contam-

inated with a variety of bacterial and fungal facultative

pathogens, leading to extensive colonization of the respira-

tory tract, and to recurrent exacerbations of pulmonary

disease [3 – 6].

While a variety of moulds are frequently recovered

from CF respiratory secretions, Aspergillus fumigatus

is the principal fi lamentous fungus reported to colonize

the airways of CF patients, independent of the geo-

graphic region studied or the mycological methodolo-

gies employed [6 – 8]. Prolonged colonization with

Aspergillus spp. results in sensitization and eventually

allergic bronchopulmonary aspergillosis (ABPA) in a

signifi cant proportion of CF patients, which has been

associated with accelerated deterioration of lung function

[9 – 11]. Other common fungi repeatedly cultured from

CF patient samples include Aspergillus terreus , members

of the Scedosporium apiospermum complex, Geosmithia

spp., Paecilomyces spp. and Exophiala dermatitidis

[7,8,12 – 16; for review see 6]. At least certain of these

opportunistic fungal colonizers of CF respiratory tracts

have already been shown to be capable of causing inva-

sive and disseminated infections in CF patients after lung

transplantation [see for example 17,18].

Numerous studies have also reported extremely elevated

isolation rates for Candida species from CF respiratory

samples, with C. albicans being by far the predominant

yeast species identifi ed [8,16,19,20]. Controversy remains

as to the potential role of Candida species colonization in

the pathology of CF, and as to whether isolation of yeasts

from sputum samples truly refl ects colonization in CF

patients, or rather results from contamination of samples

from yeasts present commensally (or introduced from

food) in the oral cavity. However, the detection of

Candida -specifi c IgE responses in ABPA patients [21]

and data indicating that serologic IgE responses against

C. albicans correlated both with ABPA and culture positiv-

ity for this yeast [22] suggest that sensitization to coloniz-

ing Candida species may be a relevant immunological

marker for ABPA development.

Although a comparison of the fungi reported from CF

patients from various studies worldwide reveals a fairly

conserved epidemiological picture of the key fungal spe-

cies involved, the relative prevalence of individual spe-

cies varies considerably. This variation may stem from

actual geographical differences in the prevalence of cer-

tain individual species, temporal changes in the relative

prevalence of certain species driven by climatic or social

factors, or from population differences in the genetic

susceptibility to certain organisms. For example it has

been suggested that the elevated isolation frequencies of

© 2010 ISHAM, Medical Mycology, 48(Suppl. 1), S88–S97

Exophiala dermatitidis in German CF patients might

result from frequenting sauna facilities, in which this

thermophilic organism is known to thrive [6,16,23,24].

However, it is also likely that the insensitivity of the

culture methods employed in some studies coupled with

the absence of a consensus approach to examining CF

respiratory secretions at least partly underpins the

apparently variable fungal biota reported from different

CF patient cohorts. Several studies describing fungal

selective media for the culture of CF secretions support

this contention. Mycological media supplemented with

a cocktail of antibiotics active principally against

Gram-negative bacteria significantly increased the sen-

sitivity of culture for both yeasts and filamentous fungi

[25]. For example, erythritol-chloramphenicol agar

and extended incubation time improved the isolation of

E. dermatitidis [16] and two different semi-selective

media (yeast extract-peptone-dextrose-agar with cyclo-

heximide [12], or SceSel � [14,26]) greatly facilitated

the detection of species belonging to the P. boydii / S. apiospermum complex.

The current paper uses data drawn from several CF

centres and mycology laboratories in the UK and France

to lend further support to the idea that the major factors

determining the prevalence of individual fungal species

reported from CF patients are indeed the mycological

approaches employed for examination and culture of

respiratory samples from such patients. We also discuss

the relative merits of direct microscopic examination

of sputum samples for the detection and enumeration of

fi lamentous fungi and yeasts, and the urgent need for

a multi-centric collaborative approach to defi ne a stan-

dardized method for examining respiratory specimens

from CF patients.

Methods

The following mycological methods were employed in the

various centres for the examination and culture of sputum

samples from CF patients:

Mycology Reference Laboratory (MRL), Bristol, UK

Sputum samples were mixed with an equal volume of

sputasol (Oxoid, Basingstoke, UK) and incubated at 37 ° C

for 15 min. Volumes of 10 ml of treated sputum were then

centrifuged at 3000 rpm (1500 g ) for 10 min in a MSE

Centaur 2 bench-top centrifuge. Supernatants were dis-

carded, leaving approximately 0.5 ml of sputum and pel-

leted sediment. Sedimented material was resuspended in

the 0.5 ml of remaining sputum, and aliquots of 0.2 ml

each were inoculated onto duplicate Sabouraud ’ s glucose

peptone agar fl asks containing chloramphenicol (Cmp;


S90 Borman et al.

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0.05 g/l) which were incubated at 30 ° C and 37 ° C for 3 – 4

weeks. Cultures were examined and reported after 48 h

and then re-incubated for a further 3 weeks and re-

examined periodically (usually weekly). Moulds were

identifi ed by examination of macroscopic and microscopic

features, according to standard descriptions. For yeasts,

Candida albicans was identifi ed by germ tube forma-

tion, with all germ tube negative isolates were reported as

Candida species.

The remaining 0.1 ml of resuspended sediment was

mixed with an equal volume of 20% KOH and a single

drop of Calcofl uor (Bactidrop, Remel), and examined

using a fl uorescence microscope with a V-2A fi lter (excita-

tion 380 – 420 nm; dichromatic mirror 430 nm; barrier fi lter

450 nm).

Bristol Royal Infi rmary, Microbiology Department, Bristol, UK

Data were collected for organisms referred to the MRL for

identifi cation. Sputum samples were mixed with an equal

volume of 0.1% dithiothreitol and incubated at 37 ° C for

15 min. After incubation, 1 μ l volumes of treated sputum

and sputum diluted 1/500 in sterile water were inoculate

in parallel onto; (a) Sabouraud ’ s glucose peptone agar

(incubation at 37 ° C for up to 6 weeks). (b) CLED agar

(incubation at 37 ° C for 48 h) (c) mannitol salt agar (incu-

bation at 37 ° C for 48 h), and (d) two Burkholderia cepacia

selective agar plates (incubation at 30 ° C for 5 days for one

plate and at 37 ° C for 48 h). All fi lamentous fungi, includ-

ing those from CF patients, were referred to the MRL for

identifi cation.

Centre Hospitalier R é gional Universitaire, Lille, France

Sputum samples were treated with an equal volume of a

1 � solution of 2,3-dihydroxy-1,4-dithiolbutane in sterile

water (Digest-EUR ® , Eurobio, France), followed by incu-

bation for 30 min at 37 ° C. After direct microscopic exam-

ination of either Giemsa or Ortho-Toluidin blue stained

samples, 50 ml volumes of treated sputum were inocu-

lated in parallel onto CandiSelectTM 4 (Bio-Rad; incu-

bation at 37 ° C for 3 weeks), Sabouraud ’ s glucose peptone

agar with 0.5 g/l amikacin (incubation at 25 ° C for 3

weeks) and ½ diluted Sabouraud ’ s glucose agar with 0.5

g/l amikacin (incubation at 25 ° C for 3 weeks). Moulds

were identifi ed by examination of macroscopic and

microscopic features. For isolates with atypical morphol-

ogy, identifi cation was confi rmed by amplifi cation and

sequencing of the ITS1-ITS2 region of the nuclear ribo-

somal repeat region. For yeasts, Candida albicans was

identifi ed by latex agglutination testing, tentative IDs for

non- C. albicans Candida species were obtained using

CHROMagar chromagenic media.

Laboratoire de Parasitologie-Mycologie, Centre Hospitalier

Universitaire, Angers, France

Prior to 1996, after direct examination, aliquots (10 μ l)

were plated on two yeast extract-peptone-dextrose-agar

(YPDA) plates containing 0.5 g/l chloramphenicol (Cmp),

and for one of them 0.5 g/l cycloheximide. Each sample

was also digested with an equal volume of mucolytic agent

(Digest-EUR ® , Eurobio) for 30 min at room temperature.

The digested mix was then diluted 1:5 (fi nal dilution of the

sample 1:10), and 10 μ l aliquots were inoculated onto two

YPDA plates containing Cmp and for one of them cyclo-

heximide was also included in the agar medium. All plates

were incubated at 37 ° C for 3 weeks even if some rapidly

growing fungi like A. fumigatus were detected.

From 1996, the procedure was slightly modifi ed. Before

digestion, aliquots of the sample were inoculated in paral-

lel onto CHROMAgar Candida (Becton-Dickinson, UK)

incubation at 37 ° C for 3 weeks), and YPDA with Cmp and

cycloheximide (incubation at 37 ° C for 3 weeks). After

digestion and dilution, the procedure was unchanged.

From 2006, cultures were performed exclusively after

digestion and dilution. All samples were inoculated (10 μ l

aliquots) in parallel onto (a) CHROMAgar Candida (incuba-

tion at 37 ° C for 3 weeks), (b) in-house prepared YPDA-Cmp-

cycloheximide (incubation at 37 ° C for 3 weeks), (c) in-house

prepared DRBC-benomyl (dichloran-rose bengal chloram-

phenicol agar containing 0.1 g/l Cmp and supplemented with

0,008 g/l benomyl; incubation at 37 ° C for 3 weeks), (d) in-

house prepared Erythritol agar supplemented with Cmp (incu-

bation at 37 ° C for 3 weeks), (e) Sabouraud ’ s glucose agar

containing Cmp and gentamicin (Becton-Dickinson; incuba-

tion at 20 – 25 ° C for 3 weeks), (f) in-house prepared YPDA-

Cmp-cycloheximide (incubation at 20 – 25 ° C for 3 weeks) and

(g) in-house prepared Erythritol agar � Cmp (incubation at

20 – 25 ° C for 3 weeks). Yeasts were identifi ed according to

colony colour on CHROMagar Candida (Becton-Dickinson)

and, for non-green colonies, by their carbohydrate assimila-

tion pattern using ID 32C test strips (bioM é rieux). Moulds

were identifi ed morphologically according to standard mac-

roscopic and microscopic descriptions.


Universitaire, Rouen, France

Digestion of the samples by mixing with an equal volume

of Digest-EUR (Eurobio), following which, 20- μ l aliquots

of the digested sample were inoculated in parallel onto a

commercial Sabouraud ’ s glucose peptone agar slant con-

taining 0.04g/l gentamicin (Bio-Rad, France) and a second

Sabouraud ’ s glucose peptone agar slant containing gen-

tamicin and cycloheximide (Bio-Rad France), both of

which were incubated at 30 ° C for 2 weeks. Identifi cation


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of yeasts was achieved by establishing their auxanographic

profi les using ID 32C test strips and mould isolates were

identifi ed morphologically, according to standard macro-

scopic and microscopic descriptions.

Laboratoire de Biologie, H ô pital Ren é e Sabran, Giens, France

Samples were digested by mixing with an equal volume of

Sputasol (Oxoid) and after 20-min incubation at room tem-

perature, 30- μ l aliquots of the digested sample were inoc-

ulated in parallel onto an in-house prepared DRBC-benomyl

agar plate and a gentamicin-containing Sabouraud ’ s glu-

cose peptone agar plate (bioM é rieux, France). All plates

were incubated at 37 ° C for one week. Yeasts were identi-

fi ed according to their carbohydrate assimilation pattern

using Api 20C Aux test strips (bioM é rieux) and moulds

were identifi ed morphologically according to standard

macroscopic and microscopic descriptions. For suspected

isolates of G. argillacea , the morphological identifi cation

was confi rmed by gene sequencing.


Universitaire, Rennes, France

All samples were fi rst digested by mixing with Digest-

EUR (Eurobio). After 30 min of incubation under constant

shaking, the digested samples were centrifuged, and por-

tions of the resulting pellets were inoculated in parallel on

two Sabouraud glucose agar slants with 50 mg/l Cmp (AES

Chemunex, Combourg, France), one of which was incu-

bated at 37 ° C and the other 30 ° C for 7 days. Moulds were

identifi ed by examination of macroscopic and microscopic

features, according to standard macroscopic and micro-

scopic descriptions. For yeasts, non- C. albicans Candida

species were identifi ed by a combination of chromogenic

agars (CHROMagar, Bekton Dickinson, France) and the

mini Api system (BioM é rieux, France).


Universitaire, Marseille, France

Thick mucous samples were digested with an equal volume

of Sputagest Selectavial (Mast Diagnostic, France) for 10

min at room temperature and were then processed as fl uid

samples. Direct examination of the sample was performed

using chloral-lactophenol cotton blue (VWR, France)

staining. A volume of 500 μ l of fl uid sample (or digested

mucous sample) was inoculated onto one Sabouraud dex-

trose agar slant with chloramphenicol and gentamicin

(AES Chemunex or BioM é rieux, France) and incubated

at 27 – 30 ° C for 10 days. The purity of yeast isolates was

checked by subculture on CHROMAgarTM Candida


medium. Candida albicans and Candida krusei were

identifi ed using the rapid agglutination test, i.e., Bichro-

latex albicans and Bichrolatex krusei (Fumouze Diagnos-

tics, France), respectively. Other yeast species were

identifi ed on the basis of the color of the colonies on chro-

mogenic medium, microscopic morphology on PCB

medium and carbohydrate assimilation profi les on Auxa-

color 2 (Sanofi Diagnostic, Pasteur, France). Moulds were

identifi ed on the basis of macroscopic and microscopic

morphological characteristics, according to standard

descriptions.

Results and discussion

The impact of variable culture methods on the prevalence

of fungal species reported from CF respiratory secretions

The published literature contains extensive data concerning

the range of mould species associated with respiratory

samples from CF patients. Principal among the more com-

monly encountered organisms are A. fumigatus , other

Aspergillus species (especially A. terreus and A. fl avus ),

members of the Scedosporium apiospermum complex, and

in certain CF centres Exophiala dermatitidis , Paecilomyces

spp. and Penicillium spp. (see for example [6,14]). An

examination of data collected recently from different Euro-

pean CF centres supports this general pattern (Tables 1 and

2), and confi rms the predominance of A. fumigatus in such

samples. All centres reported high prevalence rates for this

organism, both in terms of total respiratory secretions pro-

cessed and in terms of the number of CF patients sampled,

although signifi cant variation was observed in absolute

positivity rates for this organism (10.1 – 80.4% of samples;

8.6 – 88.9% of patients). Similarly, most centres also

reported the isolation of A. terreus , A. fl avus , S. apiosper-mum complex from a smaller, but signifi cant number of

patients and samples (Tables 1 and 2), in agreement with

most existing reports. Many centres also reported the isola-

tion of Penicillium spp. from patients in the various cohorts.

To date, it is unclear whether the isolation of this organism

has relevance to clinical exacerbation in CF, or rather

serves as a marker for exposure to air generally contami-

nated with fi lamentous fungi.

Repeated isolation of Aspergillus and Scedosporium

spp. from patient samples is accepted to be an indicator

of colonization with those organisms. However, an appre-

ciation of the exact burden of colonization within the CF

population as a whole is diffi cult, principally because

absolute prevalence rates vary signifi cantly between even

geographically close CF centres (compare for example

data for A. fumigatus and S. apiospermum ; Table 1; and

for A. fl avus, A. terreus and S. apiospermum reported

from the CF centres in Angers, Rouen and Lille for 2006;

S92 Borman et al.

Study Centre

Bristold Bristole Giens Hospital Rennes Hospital Marseille Hospital

Cultures S (156) P (69) S (102) P (36) S (1033) P (238) S (471) P (135) S (932) P (442)

Negative 34 (21.8) 13 (18.8) NR NR NR NR 119 (25.3) 24 (17.8) 398 (42.7) 250 (26.8) Moulds Aspergillus fumigatus 61 (39.1) 30 (43.5) 82 (80.4) 32 (88.9) 145 (14.0) 66 (27.7) 166 (35.3) 71 (52.6) 114 (12.2) 38 (8.6)

Aspergillus terreus 4 (2.6) 4 (5.8) 5 (4.9) 4 (11.1) 22 (2.1) 12 (5.0) 10 (2.1) 5 (3.7) 8 (0.9) 7 (1.6) Aspergillus fl avus 2 (1.3) 2 (2.9) 2 (2.0) 2 (5.6) 16 (1.5) 12 (5.0) 8 (1.7) 8 (5.9) 19 (2.0) 13 (2.9) Aspergillus nidulans 1 1 – – 2 (0.2) 2 (0.8) 4 (0.8) 4 (3) 4 (0.4) 4 (0.9) Aspergillus niger 1 1 – – 1 1 6 (1.3) 6 (4.5) 10 (1.1) 8 (1.8) Aspergillus spp.a – – – – – – 5 (1.1) 5 (3.7) 20 (2.1) 18 (4.1)

Scedosporium apiospermum b 1 1 12 (11.8) 3 (8.3) 23 (2.2) 7 (2.9) 2 (0.4) 2 (1.5) 8 (0.9) 2 (0.5) Scedosporium prolifi cans – – – – – – – – – – Exophiala dermatitidis – – 5 (4.9) 1 1 1 4 (0.8) 3 (2.2) – – Fusarium spp. 2 (1.3) 2 (2.9) – – – – – – – – Geosmithia argillacea – – – – 4 (0.4) 3 (1.3) – – – – Paecilomyces spp. 1 1 2 (2.0) 1 – – 2 (0.4) 2 (1.5) 1 1 Penicillium spp. 2 (1.3) 2 (2.9) 5 (4.9) 4 (11.1) 1 1 – – 69 (7.4) 56 (12.7) Rhizopus spp. 1 1 – – – – 4 (0.8) 3 (2.2) – –

Yeasts Candida albicans 74 (47.4) 33 (47.8) NR NR NR NR 210 (44.6) 76 (56.3) 279 (29.9) 143 (32.4) Candida spp.c 7 (4.5) 5 (7.2) NR NR NR NR 32 (6.8) 21 (15.5) 63 (6.8) 49 (11.1) Trichosporon spp. – – – – – – – – – – Cryptococcus neoformans – – – – – – – – 1 1

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Table 2). Particularly with reference to the data from Bris-

tol (Table 1), these variations cannot be explained by

geographic variations in organism prevalence or by differ-

ent CF populations under study. Effectively, both Bristol

centres received respiratory samples from the same patients

within the same CF cohort, although at different times

within the same year samples from a single patient were

referred to one or the other centre, or both. It is possible

that some of the variations in relative organism prevalence

rates might stem from bias introduced by the repeated sam-

pling of a few patients that are chronically colonized by

less common moulds. For example, S. apiospermum was

cultured from statistically more CF samples at the Bristol

Royal infi rmary than at the MRL but no statistical differ-

ence was seem in the absolute numbers of CF patients from

which this organism was isolated at these two centres

(compare data for Bristol d and e; Table 1).

However, it is also highly probable that many of the

variations in prevalence rates result from different myco-

logical methods employed in the various centres. This is

underscored by the sequential data collected at the CF

centre in Angers (Table 2). Although the numbers of CF

patients followed in Angers rose steadily and continually

throughout the period 1992 (46 samples from 32 patients)

to 2006 (251 samples from 78 patients), the emergence

of A. fl avus , A. terreus , S. apiospermum and Penicillium

spp. as signifi cant CF pathogens coincided principally

with major changes introduced in the culture methods

employed in 2006. These included the introduction of

a number of selective media and incubation at several

temperatures (see Methods). Indeed, a direct comparison

of the use of selective versus non-selective media in

Angers suggested that the use of semi-selective culture

media greatly facilitated the detection of Scedosporium

apiospermum , probably at least in part due to the regular

association between of Aspergillus fumigatus and Sce-dosporium apiospermum , and to the faster and more

extensive growth of Aspergillus fumigatus (Table 3).

However, it can not be excluded that a small proportion

of the observed increase in the detection of A. fl avus , A. terreus , S. apiospermum and Penicillium spp. over the

period 1992 – 2006 also resulted from the increased cumu-

lative risk of colonization with the advancing age of the

patients in the Angers CF cohort.

Several recent reports have also suggested that Exo-phiala dermatitidis is frequently present in CF respiratory

samples from German and Dutch CF patients, with preva-

lence rates ranging from 1.8% to 15.6% depending on the

particular study (see for example [6,14]). In most cases,

isolation rates were signifi cantly improved by use of selec-

tive media and prolonged incubation times [14]. Exophiala

dermatitidis was sporadically isolated from CF sputum

samples in a restricted number of the centres involved in

the current study, albeit with low relative prevalence (see

Table 1 Moulds and yeasts isolated from CF respiratory samples (S) and CF patients (P) in 2008, according to laboratory.

Numbers in parentheses indicate the percentage of total isolates or patients. aAll other Aspergillus species; bincludes all members of the P.boydii / S. apiospermum complex; cnon-albicans Candida species; dMRL, Bristol; eMicrobiology Department, Bristol Royal Infi rmary. NR: not registered.




Tabl

e 2

Mo

uld

s an

d y

east

s is

ola

ted f

rom

CF

res

pir

atory

sam

ple

s (S

) an

d C

F p

atie

nts

(P

) in

lab

ora

tori

es i

n t

hre

e U

niv

ersi

ty h

osp

ital

s fr

om

Fra

nce

, ac

cord

ing t

o l

abora

tory

. N

um

ber

s in

par

enth

eses

indic

ate

the

per

centa

ge

of

tota

l is

ola

tes

or

pat

ients

.

Anger

s 1992

Anger

s 1996

Anger

s 2000

Anger

s 2

006

Lil

le 2

006

Rouen

2006

S (

46)

P (

32)

S (

143)

P (

44)

S (

148)

P (

52)

S (

251)

P (

78)

S (

333)

P (

76)

S (

646)

P (

137)

Yea

sts

Can

dida

alb

ican

s 26 (

56.5

)17 (

53.1

)94 (

65.7

)29 (

65.9

)87 (

58.8

)27 (

51.9

)132 (

52.3

)41 (

52.6

)80 (

24.0

)55 (

72.4

)233 (

36.1

)78 (

56.9

) C

andi

da d

ubli

nien

sis

––

––

––

––

2 (

0.6

)2 (

2.6

)–

– C

andi

da fa

mat

a –

––

––

––

––

–1

1 C

andi

da g

labr

ata

––

12 (

8.4

)3 (

6.8

)11 (

7.4

)2 (

3.9

)15 (

6.0

)4 (

5.1

)8 (

2.4

)5 (

6.6

) 2 (

0.3

)1

Can

dida

inte

rmed

ia

–

–

–

–

–

–

–

–

–

–

11

Can

dida

kef

yr

––

––

––

2 (

0.8

)2 (

2.6

)1

1–

– C

andi

da p

arap

silo

sis

––

4 (

2.8

)4 (

9.1

)6 (

4.1

)4 (

7.7

) 32 (

12.8

) 9 (

11.5

)2 (

0.6

)2 (

2.6

) 5 (

0.8

)2 (

1.5

) C

andi

da li

poly

tica

–

––

––

–1

1–

– 3 (

0.4

)3 (

2.2

) C

andi

da in

cons

picu

a –

––

–1

1–

––

––

– C

andi

da s

phae

rica

–

––

––

–1

1–

––

– C

andi

da tr

opic

alis

–

––

–1

11

1–

–1

1 C

andi

da s

pp.

––

––

––

––

11

11

Geo

tric

hum

spp.

––

––

––

6 (

2.4

)5 (

6.4

)1

1 6 (

0.9

)6 (

4.4

) P

ichi

a et

sche

llsi

i –

––

––

–1

1–

––

– Sa

ccha

rom

yces

cer

evis

iae

––

––

––

2 (

0.8

)2 (

2.6

)–

–1

1 M

ould

s A

sper

gill

us fu

mig

atus

14 (

30.4

) 9

(28.1

)49 (

34.3

)17 (

38.6

)66 (

44.6

)22 (

42.3

) 93 (

37.1

)27 (

34.6

)55 (

16.5

)28 (

36.8

) 65 (

10.1

)39 (

28.5

) A

sper

gill

us fl

avus

–

––

–1

1 26 (

10.4

)10 (

12.8

)2 (

0.6

)2 (

2.6

) 4 (

0.6

)4 (

2.9

) A

sper

gill

us n

idul

ans

11

––

11

2 (

0.8

)1

––

––

Asp

ergi

llus

nig

er

––

11

––

––

––

2 (

0.3

)2 (

1.5

) A

sper

gill

us o

chra

ceus

–

––

––

–1

1–

––

– A

sper

gill

us te

rreu

s 1

12 (

1.4

)2 (

4.5

)7 (

4.7

)4 (

7.7

)22 (

8.8

) 8 (

10.3

)–

– 5 (

0.8

)3 (

2.2

) A

sper

gill

us v

ersi

colo

r –

––

––

– 4 (

1.6

)4 (

5.1

)–

– 3 (

0.5

)3 (

2.2

) Sc

edos

pori

um a

pios

perm

um

11

––

11

48 (

19.1

)10 (

12.9

)17 (

5.1

)4 (

5.2

) 5 (

0.8

)2 (

1.5

) Sc

edos

pori

um p

roli

fi can

s –

––

––

– 4 (

1.6

)1

––

––

Alt

erna

ria

spp.

––

––

––

2 (

0.8

)2 (

2.6

)–

– 2 (

0.3

)2 (

1.5

) C

lado

spor

ium

spp.

11

––

––

3 (

1.2

)3 (

3.9

)–

– 3 (

0.5

)2 (

1.5

) B

eauv

eria

spp.

––

––

––

11

––

––

Cer

atoc

ysti

s sp

p.

––

––

––

11

––

––

Fus

ariu

m s

pp.

––

––

––

––

––

11

Geo

smit

hia

argi

llac

ea

––

––

––

11

––

––

Paec

ilom

yces

var

iott

ii

11

––

––

11

––

––

Peni

cill

ium

spp.

11

4 (

2.8

)3 (

6.8

)–

– 26 (

10.4

)20 (

25.7

)1

112 (

1.9

)9 (

6.5

) Pa

ecil

omyc

es s

pp.

––

––

––

––

––

11

Tric

hode

rma

spp.

––

––

––

11

––

––

Muc

or s

pp.

––

11

––

2 (

0.8

)2 (

2.6

)–

––

– E

xoph

iala

der

mat

itid

is

––

––

––

––

––

––

Ste

rile

mould

s–

–1

1–

–14 (

5.6

)10 (

12.8

)–

– 2 (

0.3

)2 (

1.5

)

Med

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S94 Borman et al.

2006 2007

Total number of samples (patients) 51 (78) 253 (84) S. apiospermum positive cultures:

Non-selective culture media 23 (7) 15 (7)

YPDA � cycloheximide 35 (8) 24 (7)

DRBC-benomyl 41 (8) 35 (10)

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Tables 1 and 2). However, this organism was not detected

in samples by the majority of centres involved in this inves-

tigation. While this may again refl ect variations in the cul-

ture techniques and media employed, it is worth noting that

a least one of the centres that failed to detect signifi cant

numbers of E. dermatitidis isolates has employed appropri-

ate (ECA) selective media since 2006 (see data for Angers,

2006 ; Table 2). Conversely, no specifi c media likely to

improve isolation rates of Exophiala spp. were used for

samples analyzed in Bristol (Table 1), yet fi ve sequential

respiratory samples from a single patient were positive for

this organism in 2008.

An analysis of the methods employed by the various CF

centres participating in the current study also reveals a lack

of standardization in the absolute volumes of respiratory

samples that are subjected to examination, and especially

culture. Whilst all laboratories currently treat sputum sam-

ples with an equal volume of an appropriate mucolytic

agent, the volumes of treated sample that are then cultured

range from as little as 0.001 and 0.5 μ l (Bristol Royal Infi r-

mary), through 10 μ l (Angers), 20 μ l (Rouen) , 30 μ l

(Giens), 50 μ l (Lille) to 500 μ l (Marseille). At the MRL,

Bristol, and in Rennes, sputum is concentrated by cen-

trifugation and the pellet of the centrifuged, treated sputum

is cultured. It is clear that such variations in methodologies

are likely to signifi cantly infl uence the numbers and variety

of organisms isolated from a sample, and the subsequent

relative prevalence of the various fungal species. Con-

trolled studies are required to determine whether increas-

ing the sample volumes that are cultured will signifi cantly

increase isolation rates, or whether culture of excessive

volumes of respiratory secretions might actually inhibit the

growth of some or all fungal species (for example via com-

petition for nutrients or due to the presence of elevated

concentrations of inhibitory substances/bacteria). Impor-

tant variations also exist in both the nature and concentra-

tions of antibiotics employed in the mycological media

used by the various centres that have contributed to the

current study (see Materials and Methods). Studies per-

formed in Angers demonstrated a marked decrease in the

recovery of moulds from respiratory samples (presumably

due to overgrowth of bacteria) when the cycloheximide

concentration in media was reduced from 0.5 g/l to 0.05

g/l (data not shown). These fi ndings are in keeping with

suggestions that the selectivity of SceSel � culture media

is related in part to the mix of antibiotics used to inhibit

bacterial growth [25,26], and serve to highlight a further

aspect of culture media which requires standardization.

Finally, consideration should be given to whether the iden-

tifi cation methods used by the various centres might also

result in some variability in the organisms reported from

CF respiratory secretions. While this is certainly a possibil-

ity for yeast isolates, where the different participating cen-

tres employ a wide range of chromogenic and biochemical

approaches (see Materials and Methods), it is much less

likely for fi lamentous fungi (moulds), since most centres

that participated in the current study identifi ed isolates of

mould by macro-and microscopic examination using com-

mon and well established methodologies (see Materials

and Methods).

Yeast species isolated from CF sputum samples

Although Candida species, and principally C. albicans ,

are regularly recovered from samples of CF patients

[19,27,28], uncertainty remains as to whether this refl ects

transient contamination of the respiratory tract by com-

mensal fl ora or persistent colonization. Based primarily

on the poor predictive value of the isolation of Candida

species from respiratory secretions for invasive diseases

in non-neutropenic (non-CF) patients and on the rarity of

Candida pneumonia despite the extremely common

recovery of Candida from respiratory samples [29,30],

recent updates to IDSA guidelines concluded that anti-

fungal therapy should not be considered on the basis of

the isolation of Candida from the respiratory tract alone

for neutropenic or non-neutropenic patients, but did not

specifi cally address the CF population [31]. However,

both signifi cant long-term persistence and strain mainte-

nance of Candida species has been shown in CF respira-

tory tracts [19], and specifi c anti- Candida IgE responses

in CF patients appear to correlate with repeated isolation

of the yeast [22].

In agreement with all previous reports, C. albicans was

the predominant yeast isolated in the CF centres participat-

ing in the current study. Candida albicans accounted for

greater than 60% of all Candida species recovered (range

68.4 – 100%; Tables 1 and 2), and had been isolated at least

once in approximately 50% of the patients, irrespective of

the particular study centre (range 47.8 – 72.4%). The other

most frequent Candida species found in respiratory sam-

ples from CF patients were C. glabrata and C. parapsilosis

complex, with prevalence rates in the order of 2 – 10%

depending on the species and the CF centre (Table 2).

Indeed, the variations in prevalence rates amongst the var-

ious Candida species were less than those observed with

Table 3 Comparison of the use of non-selective versus semi-selective media

for the isolation of S. apiospermum complex in Angers, 2006 – 2007.



Microscopy result

Organism isolated N

Negative

(%)

Yeast

(%)

Filamentous

fungus (%)

A. fumigatus alone 30 10 (33.3) 1 (3.3) 19 (63.3)

A. fumigatus � C. albicans 27 2 (7.4) 8 (29.6) 17 (63.0)

A. fumigatus � Other mould 4 0 0 4 (100)

C. albicans alone 47 26 (55.3) 17 (36.2) 4 (8.5)Other Aspergillus spp. 8 2 (25) 3 (37.5) 3 (37.5)Other Candida spp. 7 4 (57.1) 3 (42.9) 0

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fi lamentous fungi reported from the same CF centres for

the same time periods, and agreed fairly closely with previ-

ous reports [14,27]. It is possible that this lack of variation

refl ects the effi cacy of the standard mycological media

commonly employed in most CF centres for the isolation

of yeasts from clinical samples. Candida dubliniensis was

also reported from several CF centres participating in the

present study, but never approached the 11% colonization

rate reported by Peltroche-Llacsahuanga et al . [28].

However, since at least several of the centres in the current

study did not attempt the complete identifi cation of non-

C. albicans Candida species, or reported all germ tube

positive yeasts as C. albicans , it is possible that the true

prevalence rate of this organism might indeed be higher in

our CF patient cohorts.

Direct microscopic examination of CF respiratory samples

Respiratory samples that are submitted to certain CF cen-

tres are examined microscopically for the presence of

fungal elements (see Methods). At the MRL, Bristol, all

samples are subjected to direct microscopic examination

using Calcofl uor fl uorescent brightener (see Methods).

An analysis of the data correlating microscopic fi ndings

with culture results allows a crude evaluation of the util-

ity of this approach for the detection of potential coloni-

zation of patients with moulds and yeasts (Tables 4 and

5). Forty-one of 45 samples from 45 different CF patients

that were microscopy-positive for fi lamentous fungus

yielded at least one species of mould upon culture, and

all 12 samples in which moderate or large amounts of

fi lamentous fungi was reported proved to be culture pos-

itive for moulds (Table 4), demonstrating the potential

utility of direct microscopy examination as a marker for

mould isolation/colonization in CF respiratory samples.

Indeed, more than 65% of isolates (40/61) of A. fumigatus

were cultured from samples which had been found to


Culture result

Microscopy N

Culture

negative

(%)

Mould

isolated

(%)

Yeast

isolated

(%)

Mould �

Yeast

isolated (%)

Negative 76 28 (36.8) 15 (19.7) 30 (39.5) 3 (3.9)Yeast detected 33 3 (9.1) 2 (6.1) 17 (51.5) 11 (33.3)Small amounts of

fi lamentous fungus

33 3 (9.1) 13 (39.4) 1 (3.0) 16 (48.5)

Moderate amounts of

fi lamentous fungus

6 0 6 (100) 0 0

Large amounts of

fi lamentous fungus

6 0 3 (50) 0 3 (50)

be microscopy positive for fi lamentous fungal hyphae/

hyphal fragments (Table 5). Interestingly, A.fumigatus

grew but was not recorded microscopically from nearly

30% of samples that were culture and microscopy posi-

tive for yeasts, as compared to only 3% of samples that

were microscopy positive but culture negative for yeast

(Table 5). This suggests that high concentrations of

Candida albicans blastospores/hyphae in certain CF res-

piratory secretions might hinder the microscopic detec-

tion of mould hyphae. It will be interesting to determine

if samples that are microscopy-negative but culture posi-

tive for A. fumigatus contain recently ‘ acquired ’ , non-

germinated fungal conidia as opposed to growing fungal

hyphae, and if so, whether such conidia are an indicative

precursor of future colonization.

For yeasts, the value of direct microscopy examination

of sputum samples appears somewhat less clear. Yeasts

were recovered from almost 40% of microscopy negative

samples (Table 4) and only 33.8% (25/74) of the C. albi-cans isolates were from samples in which yeasts had been

seen by direct microscopy (Table 5), suggesting that micro-

scopic examination is less sensitive for the detection of

yeasts, and/or that a high proportion of samples contain

very low numbers of yeast cells. Data from the MRL con-

cerning the yeasts isolated from sputum samples from non-

CF patients, and those isolated from mucosal sites and sites

indicative of systemic Candida infections (blood, tissue

etc) reveals a similar range of organisms and relative prev-

alences to those seen in CF populations (data not shown).

Thus, the signifi cance of isolation of a particular yeast

from CF secretions cannot be assessed on the basis of the

identity of the organism alone. It remains to be determined

whether those samples that are microscopy-negative but

culture-positive for yeasts correspond to samples that have

been ‘ contaminated ’ by low numbers of food yeasts/com-

mensal organisms from the oropharynx. Finally, any dis-

cussion on the potential utility of direct examination of

respiratory secretions should include an assessment of the

likelihood that the results would impact favourably on

Table 4 Correlation between results of direct microscopic examination

and culture of CF respiratory secretions, data from CF secretions

examined at the MRL in 2008.

Table 5 Fungi isolated from CF respiratory secretions ( N � 123) at

the MRL in 2008, as a function of the results of direct microscopic

examination.

S96 Borman et al.

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patient management, especially since any correlations

between the result of direct examination and culture and

clinical condition are likely to be considerably complicated

in patients receiving antifungal therapy.

Conclusions

A considerable volume of data now supports the conten-

tion that a number of key fungal species regularly and

persistently colonize the respiratory tract of patients with

CF. However, a true picture of the prevalence of different

fungal species in CF lungs, and of the signifi cance of their

isolation is hindered by the absence of a standardized

approach to examining such samples. While several stud-

ies have identifi ed semi-selective media that improve the

isolation of certain of the fungal pathogens associated

with CF, these media are not employed routinely in many

laboratories, and the number of different media that would

be required to effectively capture the complete fungal

biota remains unknown. It is becoming clear that once a

standardized approach has been implemented, it will be

possible to accurately analyze the role of some of the

‘ rarer ’ fi lamentous fungi in the exacerbation of the

clinical picture in CF, and the possibility that there are

regional or population-driven variations in the fungi that

colonize CF patients. In this respect, it is worth noting

that several recent taxonomic studies have identifi ed

cryptic species within key clinical morpho-species of

both moulds and yeasts [32 – 36], including Aspergillus fumigatus species complex, S. apiospermum complex and

C. parapsilosis complex. To date, correct identifi cation

of members of these species complexes necessitates

molecular approaches, including sequencing of various

conserved fungal target genes [36], and as such is not

routinely available to many laboratories. It remains to

be determined whether discrimination of such cryptic

species will provide signifi cant clinical benefi t or epide-

miological impact in the context of CF.

We would suggest that the development of a standardized

approach to the culture of respiratory samples from CF

patients will require a two stage process. The fi rst stage

would involve a limited number of key CF centres and be

aimed at developing a defi nitive and easily implementable

approach to the processing and culture of respiratory speci-

mens. This could be achieved in a step-wise fashion (perhaps

using selected CF respiratory samples that are distributed

across these pilot centres), in which fi rst the optimal sample

volume/processing method is determined, followed by a par-

allel comparison of appropriateness of the available semi-

selective media for the isolation of the key fungal pathogens

currently implicated in colonization of CF patients. The sec-

ond stage would then involve adoption of this standardized

methodology in CF centres across the globe, and rigorous

comparison of the epidemiological data on CF biota collected

in different geographic locations. Irrespective of the exact

approach that is chosen, it is undeniable that there is a press-

ing requirement for a concerted effort towards defi ning the

optimal methods for the mycological analysis of the fungal

component of CF microbiology.

Acknowledgments

We wish to thank all of the members of the laboratories

that have contributed data to this study.

Declaration of interest: The authors report no confl icts of

interest. The authors alone are responsible for the content

and writing of this paper.

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Lack of standardization in the procedures for mycological examination of sputum samples from CF...

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