Rezusta 2016

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mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Evaluation of incubation time for dermatophytes cultures

Antonio Rezusta,1 Sonia de la Fuente,2 Yolanda Gilaberte,3 Matxalen Vidal-Garcıa,4 Leticia


,4 Ana Lo
Alcala  pez-Calleja,4 Maria Angeles Ruiz4 and Maria Jose
 Revillo4
1
Department of Microbiology, Hospital Miguel Servet, University of Zaragoza, Zaragoza, Spain, 2Department of Dermatology, Hospital Ernest Lluch,
Calatayud, Spain, 3Department of Dermatology, Hospital San Jorge, Huesca, Spain and 4Department of Microbiolgy, Clinical Microbiology Labortory,
Hospital Miguel Servet, Zaragoza, Spain

Summary
In general, it is recommended to incubate dermatophytes cultures for a minimum of 4 weeks. Several aspects of
routine fungal cultures should be evaluated in order to implement appropriate and necessary changes. The aim of
this study was to determine the optimum incubation time for routine dermatophytes cultures, analysing the time
to find first fungal growth by visual observation. We recorded the time when the initial growth was detected for
all dermatophyte isolates during a 4-year period. A total of 5459 dermatophyte cultures were submitted to our
laboratory. From the total cultures, only 16 (1.42%) isolates were recovered over/after 17 days of incubation and
only three dermatophyte species were recovered over 17 days. Fourteen isolates belong to Trichophyton rubrum,
one isolate to Trichophyton mentagrophytes complex and one isolate to Epidermophyton floccosum. We concluded that
an incubation period of 17 days is enough to establish a microbiological diagnosis of dermatophytosis.

Key words: dermatophytes, dermatophytosis, dermatophyte carriage, onychomycosis, incubation time, mycoses.

the laboratory workload and therefore costs. In addi-


Introduction
tion, dermatophytes identification by standard proce-
Dermatophyte infections are extremely frequent world- dures, involving morphological observations and
wide and their epidemiological features vary according biochemical tests, not always provide sufficient data
to the geographical area and have changed in the last for their accurate identification. In this sense, the use
decades. Superficial mycoses are estimated to affect of the PCR-DNA sequencing methods may yield more
more than 20–25% of the world’s population and reliable results in these cases.5
their incidence is constantly increasing.1 The preva-
lence of onychomycosis infection is estimated to be at
Materials and methods
least 12% and even higher in individuals more than
65 years old.2 The aim of this study was to determine the optimum
Cultures remain critical for diagnosis of fungal infec- incubation time for routine dermatophytes cultures,
tions. There is a universal recommendation that analysing the time to find first fungal growth by visual
mycology cultures should be incubated for 4 weeks.3,4 observation. An observational, retrospective study was
Several aspects of routine fungal cultures should be performed to evaluate all positive cultures obtained at
evaluated in order to implement appropriate and nec- the Microbiological department of the University
essary changes. Microbiology laboratories should be Hospital Miguel Servet, Zaragoza (Spain) from June
more efficient and prolonged incubation times increase 2009 to May 2013. All isolates were obtained from
clinically suspected lesions of dermatophytosis from
skin, scalp or nails. Patients should have stopped any
Correspondence: Sonia de la Fuente, Department of Dermatology, Hospi-
lica s/n, 50009 Calatayud, Zaragoza,
tal Ernest Lluch, Avda/Isabel La Cato oral or antifungal treatments at least 2 weeks before
Spain. sampling except in very exceptional cases. Specimens
Tel.: +0034 616 000 377. Fax: +0034 976 565 995. were plated on potato dextrose agar, added with chlo-
E-mail: delafuente.sonia@gmail.com ramphenicol (in house) and Sabouraud dextrose agar,
with chloramphenicol (Oxoid) and dermatophyte test
Submitted for publication 8 June 2015
Revised 13 September 2015 medium (DTM) Biomerieux containing cicloheximide,
Accepted for publication 28 January 2016 and incubated at 30 °C for 4 weeks. Cultures were

© 2016 Blackwell Verlag GmbH


doi:10.1111/myc.12484 Mycoses, 2016, 59, 416–418
Evaluation of 4-week incubation

read twice a week recording the number of days until Table 1 Distribution of incubation time for days when dermato-
dermatophyte growth was observed. On the other phytes were isolated.
hand, they were considered clinically non-relevant if 0–7 days 0–14 days 0–17 days >17 days
they were recovered from patients who had the same
organism isolated within the first 17 days of incuba- 820 1095 1112 1128
72.69% 97.07% 98.758% 100%
tion from the other specimen source.
Dermatophytes were identified by their macro and
micromorphologic, phenotypic characteristics accord-
six isolates we could not justify the slow growth or the
ing with Summerbell and a sequence analysis of the
delay in the identification process (Fig. 1).
internal transcribed spacer (ITS) of the ribosomal DNA
Only three dermatophyte species were recovered
was performed for strains with no reliable identifica-
over 17 days. Fourteen isolates (87.5%) belong to Tri-
tion by phenotypic methods. For this purpose, a DNA
chophyton rubrum, one isolate (6.25%) to Trichophyton
fragment of about 1000 bp was amplified by poly-
mentagrophytes complex and one isolate (6.25%) to Epi-
merase chain reaction (PCR) using universal fungal
dermophyton floccosum. Regarding to the location
primers (ITS4 and ITS5).6 The sequences of the total
where the samples were obtained, 14 (87.5%) isolates
ITS region were subjected to a BLASTn search (com-
recovered over 17 days were from nails and only two
pares nucleotide similarities) of the National Center for
(12.5%) from the skin. Dermatophyte species recov-
Biotechnology database.
ered over 17 days and their location are summarised
Considering that some factors could influence the
in Table 2.
recovery and identification of dermatophytes, we anal-
ysed the used of previous antifungal drugs by the
patients or possible failures based on untrained person- Discussion
nel in the laboratory.
We conducted a critical evaluation of the protocol for
dermatophytes isolation. This study shows that incu-
Results bation times shorter than 17 days are enough time for
dermatophytes to grow from clinical isolates. Although
A total of 5459 dermatophyte cultures were submitted
dermatophyte differentiation could be ready some days
to our laboratory. One thousand one hundred and
twenty-eight (20.66%) of these were positive for der-
matophytes. Eight hundred and twenty (72.69%) iso-
lates were recovered during the first 7 days, 275
isolates (24%) between 7 and 14 days, and 17 isolates
(1.51%) 15 and 17 days. Only 16 (1.42%) isolates
were recovered over/after 17 days of incubation
(Table 1). The proportion of dermatophytes recovered
before 17 days was significantly higher than those
recovered over 17 days. (P < 0.001, X2 test). Consid-
ering only isolates recovered over 17 days, in four
cases were non-relevant due to the same organism
had already been isolated from other of their samples
during the first 17 days. In addition, seven of the iso-
lates recovered over 17 days, had positive direct vision
with KOH. Therefore, in these patients no change in
the therapeutic approach was implemented because
specific antifungal treatment had been prescribed
based on direct vision findings.
From the 12 isolates diagnosed after 17 days con-
sidered clinically relevant, four isolates belong to
patients under antifungal treatment on the specimen
collection date. In other two cases, the mycology
expert was not present during the identification pro- Figure 1 Summary of the culture results and clinical relevance
cess, which can justify the delay. Therefore, only in or possible justification of growth that occur over 17 days.

© 2016 Blackwell Verlag GmbH


Mycoses, 2016, 59, 416–418 417
A. Rezusta et al.

Table 2 Distribution and location of dermatophytes species perform the procedure. The reduction of the incuba-
recovered over 17 days. tion time to 17 days will make the technique more
No. % Location effective, because the microbiological diagnose is estab-
lished sooner, and more efficient, because it allows to
Trichophyton rubrum 14 87.50 Nail save money.
Trichophyton mentagrophytes complex 1 6.25 Skin
On the basis of these results, we conclude that
Epidermophyton floccosum 1 6.25 Skin
16 100 mycological cultures positive after 17 days are rarely
relevant for patient care, and in most cases, the incu-
bation time could be reduced. Validation of our pro-
later, we emphasise that to shorten the incubation posal in different settings should be done, especially in
time until visual growth is very convenient. Previous fungal reference laboratories, before being established
studies indicate that fungal cultures for the detection as a general recommendation.
of dermatophytes fungi have to be incubated for
4 weeks. The reduction of the incubation time to Conflicts of interest
17 days will make the technique more effective and
more efficient. The authors have no conflict of interest to declare.
Traditional recommendations are based on the slow
growth of some fungal species, notably dimorphic References
fungi.7,8 However, there is no recent evidence support-
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418 Mycoses, 2016, 59, 416–418

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