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Mycopathologia

https://doi.org/10.1007/s11046-018-0290-5

ORIGINAL PAPER

Diagnosis of Superficial Mycoses by a Rapid and


Effective PCR Method from Samples of Scales,
Nails and Hair
Irene A´lvarez-Mosquera . Silvia Herna´ez . Juan Sa´nchez . Maria Dolores
Sua´rez .
Ramo´n Cisterna

Received: 1 February 2017 / Accepted: 28 July 2018


© Springer Nature B.V. 2018

Abstract Superficial mycoses are the most fre- subsequent sequencing of amplified samples. A total
quently diagnosed affections of the stratum corneum of 211 samples (34%) resulted in positive diagnosis
of the skin, nails and hair. It is generally caused by the with at least one of the two applied methods: culture
presence of yeasts and dermatophytes. Onychomyco- (21%) and PCR (22%). Despite the low percentage of
sis is the most common infection with an incidence of identification achieved by the sequencing technique
80–90% in Europe generally produced by Trichophy- (40%), the value contributed by the amplification of
ton rubrum. The aim of this study is to compare the the 18S region of the rRNA was considered important
traditional diagnostic techniques of superficial in the identification as it showed a high predictive
mycoses with a homemade and wide-spectrum fungal values for both positive and negative diagnoses
polymerase chain reaction (PCR) technique that (90.9% and 94.6%, respectively). The proposed PCR
amplifies a specific region of the 18S ribosomal RNA method has been confirmed as a complementary,
(rRNA) directly from samples of scales, nails and rapid, and effective method in the diagnosis of
hair. A total of 626 clinical samples (obtained in the superficial mycoses. Additionally, it reduces the time
Basurto University Hospital, Bilbao, Spain) were to obtain satisfactory results from 4 weeks to 7 h.
analysed by traditional culture, microscopy and PCR.
DNA extraction was carried out by using an extraction Keywords Onychomycosis · Dermatophyte
buffer and bovine serum, and amplification of samples ·
and performance of the PCR were checked by Diagnosis · PCR method · Rapid and effective
conventional agarose gel electrophoresis with

Introductio
Handling editor: Jesu´s Guinea n
Superficial mycoses (SM) or dermatomycoses include
S. Herna´ez · J. Sa´nchez · M. D. Sua´rez · R. Cisterna all the cutaneous and cutaneo-mucous fungal infec-
Clinical Microbiology and Infection Control Department, tions. The causative fungi are divided into two large
Basurto University Hospital, Avda.Montevideo 18, 48013
Bilbao, Bizkaia, Spain groups: (1) yeasts (genera Candida, Trichosporon and
Malassezia) and (2) filamentous fungi, basically
I. A´lvarez-Mosquera ( & ) · R. Cisterna dermatophytes that include the genera Microsporum,
Immunology, Microbiology and Parasitology Department, Trichophyton and Epidermophyton [1].
School of Medicine, University of Basque Country (UPV/
EHU), Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain e- Within the SM, onychomycosis (Tinea unguium)
mail: irealvarez15@gmail.com stands out for its greater prevalence in our

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Mycopathologia

environment. This fungal infection of the nail is Until now, some authors have already mentioned
mainly caused by dermatophytes, yeast, and to a lesser the utility of molecular techniques for the diagnosis of
degree by other non-dermatophyte fungi [2]. It is superficial mycoses when considering dermatophyte
distributed worldwide and represents up to 50% of nail strains or cultures [9, 16]. However, fewer studies
disorders [3] with Trichophyton rubrum being the have shown its applicability on the direct detection
most common species in Europe with an incidence of from clinical samples [17]. In this light, the main
80–90% [4, 5]. On the other hand, the presence of non- objective of this work is to compare the traditional
dermatophyte fungi, i.e. Fusarium spp., Aspergillus diagnostic techniques of superficial mycoses with a
spp. and Scopulariopsis spp., in affected nails is homemade and wide-spectrum fungal PCR technique
considered rare [5]. that analyses a specific region of the 18S ribosomal
Due to the fact that onychomycoses present clinical RNA (rRNA) [18] from samples of scales, nails and
characteristics similar to other nail disorders of non- hair in order to demonstrate its applicability in the
infectious origin, such as psoriasis, lichen planus and daily routine.
non-inflammatory onychodystrophy, it is important to
conduct a differential diagnosis in order to identify the
causative fungi and provide the most appropriate and Materials and
effective treatment in each case [6]. The current Methods
diagnosis methods are based on the detection of fungal Clinical Sampling
elements by direct microscopy of clinical samples
(with KOH solution) followed by in vitro culture and A total of 626 clinical samples of scales (70 scales
morphological identification of the fungus [7]. How- from feet; 20 scales from hands), nails (440 toenails;
ever, these analytical procedures present two main 83 fingernails) and hair (13) were obtained in the
drawbacks. The former is that the microscopic exam- Mycology Specialist’s office of the Basurto University
ination of dermal scale, hair and nail samples presents Hospital (Bilbao, Spain). Unselected samples were
low sensitivity and specificity, and therefore, it may obtained from patients that were diagnosed by a
lead to false positive or negative results. On the other dermatologist or a general practitioner depending on
hand, culture of samples allows recognition at species the case with skin, nail or hair injuries to test the
level, but the slow growth of the causative fungi may prevalence or absence of fungal infection as the
delay the diagnosis up to 4 weeks. Additionally, this causative agent. Scales samples were obtained by
procedure could diagnose false negatives [8]. scraping off the skin of the patients with a sterile
On the contrary, the current molecular methods scalpel. In the case of nails, biological samples were
based on polymerase chain reaction (PCR) for the collected from the subungual portion. Regarding
detection of dermatophytes in skin, hair and nails are gender, the majority of the diagnosed patients were
more reliable, rapid and present higher sensitivity and women (68%) with an average age of
specificity values if they are compared with the most 52.23 ± 17.48 years, whereas in men (32%) it was
traditional procedures, i.e. microscopic techniques and of 50.43 ± 20.15 years. Of them, all the samples
fungi culture [9, 10]. To date several molecular included in this study were obtained from volunteer
methods have been described for the detection of donors under informed consent, following the ethical
fungal species causing dermatomycoses, such as the standards of Helsinki Declaration.
study of restriction fragment length polymorphisms
(RFLPs) [11] or the analysis of different regions Traditional Diagnosis
throughout the genome based on the polymerase chain
reaction (PCR), i.e. 18S ribosomal RNA (rRNA) [12], All the samples considered herein were firstly
28S region of the rRNA [13] and the chitin synthase 1 observed through microscope with a 20% KOH
region [14]. In this context, there is consensus on the solution (20 gr of KOH ? 40 ml of DSMO ? 60
selection of the internal transcribed spacer (ITS) ml of Milli-Q water) by an expert technician.
region as the most suitable target to identify the bulk Moreover, they were incubated at 27 °C for 4 weeks
of fungal species [15]. by spreading them over Sabouraud Dextrose agar
with chloram- phenicol and Sabouraud with
chloramphenicol and
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Mycopathologia

cycloheximide (Becton–Dickinson Microbiology Sys- fungal species investigated displayed values higher
tems, Sparks, Maryland, USA). In the case of nails, than 95% sequence identity.
they were crushed and finally, the nail powder was
incubated. Diagnosis

Molecular Diagnosis Diagnosis of each sample was carried out by combin-


ing the results obtained from the above-mentioned
DNA extraction was carried out by incubating the techniques: (1) microscopic observation with KOH
samples in 100 l L of an extraction buffer composed solution, (2) macro- and microscopic characteristics of
by (60 mM NaHCO3, 250 mM KCl and 50 mM Tris the culture, and (3) study of the 18S region of the
pH = 9.5) during 10 min at 95 °C and subsequently rRNA.
adding 100 l L of an anti-inhibition buffer (2% bovine
serum), the tube was vortex briefly and the sample was
ready to analyse [19]. The genomic DNA was directly Result
amplified by polymerase chain reaction (PCR). The s
following primers were used for the amplification of A total of 626 samples including 440 toenails, 83
the 18S region of the rRNA: 18S 1 50-ATTGGAGGG- fingernails, 70 scales from feet, 20 scales from hands
CAAGTCTGGTG-30 and 18S 2 50-CCGATCCC- and 13 hair samples were analysed by the three
TAGTCGGCATAG-30 [18, 20]. methods described in the previous section.
The PCR was performed in a total reaction volume Of all samples, 211 (34%) resulted in positive
of 50 l l composed by 1 9 reaction buffer (Roche diagnosis through any of the performed methods.
Diagnostics, Mannheim, Germany), 200 l M of each Within the positive samples, 47 of 211 (22.3%)
dNTP, 0.5 l M of each primer, 2 U of Taq polymerase displayed concordance among the three identifying
(Roche Diagnostics) and Milli-Q water. It was carried methods performed herein. The rest of the samples,
out in a 9800 thermal cycler from Applied Biosystems i.e., 164 of 211, (77.7%), did not showed concordance
(Thermofisher Scientific, Waltham, MA, USA). The among the three different methods carried out in the
PCR protocol consisted of an initial denaturing step of present work (Table 1).
2 min at 95 °C followed by 35 cycles of1 min at 95 We found that the greater number of positive results
°C, 1 min at 55 °C, and 2 min at 72 °C. After that, a with the three identifying methods corresponded to
final elongation step at 72 °C for 7 min was toenail samples (33/47); however, fingernails only
performed in order to avoid adenylation. represented 10/47 of the positive samples in the three
Amplification of samples and performance of the methods performed.
PCR were checked by conventional agarose gel (2%)
electrophoresis (50 min at 85 V), taking as a reference
the DNA molecular weight marker VIII (Roche Table 1 Results for each analysed methods
Diagnostics, Mannheim, Germany). Positive and N KOH Culture PCR-G Sequencin
g
negative controls were included in each case.
Finally, those samples that displayed good PCR 47 ? ? ? 18
amplification in agarose gel were purified by using the 56 - ? ? 18
MoBio kit and sequenced. Sequencing was conducted 26 ? - ? 12
on an ABIPRISM3130 Genetic Analyzer (Ther- 9 - - ? 8
mofisher Scientific, Waltman, MA, USA) by using 12 ? ? -
the Big Dye Terminator v3.1 Cycle sequencing kit 15 - ? -
(Applied Biosystems, Foster City, CA, USA). The 46 ? - -
obtained DNA sequences of the analysed region of 415 - - -
interest were analysed and used to identify the fungus ? , positive result; - , negative result; N, number of samples;
at specie level by using the Basic Local Alignment KOH, microscopy technique; PCR-G, visualization of the
Search Tool (BLAST) [21]. PCR productsof all the amplicon in the agarose gel

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Mycopathologia

In the cases of variants among the three identifying Table 2 Type of samples analysed and species identified by
methods, toenails were also the majority in the cases of culture
a negative microscopic observation and positive Type of sample CULTURE ?
culture and PCR (30/56) and negative culture and
N % Species
positive microscopy and PCR (19/26). On the other
hand, we found more samples of scales from feet in the Toenail 65 77.4 Trichophyton rubrum
cases of positive PCR and negative traditional diag- 6 7.1 Candida spp.
nosis (5/9) and fingernails samples in the cases of 6 7.1 Scopulariopsis brevicaulis
negative PCR and positive results with traditional 3 3.5 Candida albicans
techniques (6/12). 2 2.4 Candida parapsilosis
2 2.4 Trichophyton interdigitale
Traditional Diagnosis Overall 84 100
Fingernail 7 36.8 Candida albicans
In total, 130 of 211 samples (84 toenails, 19 finger- 5 26.3 Trichophyton rubrum
nails, 19 scales from feet, 2 scales from hands and 6 4 21 Candida spp.
hair samples) were diagnosed as positive for superfi- 1 5.3 Candida parapsilosis
cial mycoses by microscopic observation and deter- 1 5.3 Candida tropicalis
mination of the macro- and microscopic 1 5.3 Trichophyton tonsurans
characteristics of the culture methods. In this study, Overall 19 100
the traditional diagnosis resulted in 81false negatives. Scale from foot 15 80 Trichophyton rubrum
Trichophyton rubrum was the most frequently 2 10 Candida parapsilosis
identified dermatophyte species in the samples of
2 10 Trichophyton interdigitale
scales (15/19 or 80.0%) and toenails (65/84 or 77.4%).
Overall 19 100
Microsporum audouinii, which is a highly contagious
Scale from hand 2 100 Trichophyton rubrum
anthropophilic dermatophyte which has experienced a
Overall 2 100
renaissance in recent years, especially for families
Hair 1 16.6 Trichophyton violaceum
who migrate from Africa, was the most frequent
5 83.3 Microsporum audouinii
species found in hair samples [22]. On the other hand,
Overall 6 100
non-dermatophyte fungi, such as Candida parapsilo-
sis and Candida albicans, were isolated in samples of N, number of samples
fingernails (7/19) and to a lesser degree in scales from
foot (2/19).
Other dermatophyte species, such as Trichophyton There was a concordance between the species
interdigitale and Trichophyton tonsurans were also found in culture and PCR; however, it did not
identified. T. interdigitale, tend to cause tinea unguium happened in the case of Candida species in which we
and tinea pedis, whereas T. tonsurans is the most could see differences at subspecies level. We also
common pathogen causing tinea capitis [23]. found cases of negative culture and positive molecular
Moreover, we also found non-dermatophyte diagnosis where we could detect species of non-
moulds as Aspergillus, Fusarium (in the cases of dermatophyte moulds (Table 3).
negative culture and positive PCR) and Scopulariopsis Despite the percentage of identification achieved
found in toenail samples among other species detailed through sequencing, this technique may be important
in Table 2. in diagnosis of the onychomycosis as it showed a
positive predictive value (PPV) of 90.9% and a
Molecular Diagnosis negative (NPV) value of 94.6%. If this results are
compared with those obtained through the microscopy
Only the 40.5% (56/138) of the samples that showed technique (71.4% and 87.0%, respectively), the study
positive results in the agarose gel could be identified at of the 18S region of the rRNA has shown to be a more
species level through sequencing of the PCR
amplicons.

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Mycopathologia

Table 3 Comparison of the species identified by culture and procedure which could delay the diagnosis from days
sequencing to weeks in realistic routine conditions. In this way, a
N Culture Positive and sequenced PCR rapid diagnosis could increase the effectiveness of the
treatment and therefore reduce the cost per patient. In
13 Trichophyton rubrum Trichophyton rubrum our study, molecular diagnosis was carried out in a day
1 Trichophyton rubrum Trichophyton tonsurans routine, approximately consuming 7 h.
7 Trichophyton rubrum Trichophyton spp. The region amplified in this study was the 18S
1 Trichophyton rubrum Candida albicans rRNA due the satisfactory results obtained as it
2 Candida albicans Candida parapsilosis specifically amplify the dermatophyte DNA. There-
2 Candida parapsilosis Candida parapsilosis fore, the dermatophyte primer is specific, and it is
3 Candida spp. Candida spp. accurate in the direct identification of dermatophytosis
2 Candida spp. Candida parapsilosis from clinical material [25].
2 Scopulariopsis brevicaulis Scopulariopsis brevicaulis In our study, the PCR worked better with nail
1 Trichophyton interdigitale Trichophyton spp. samples in which the amount of sample was higher.
1 Trichophyton tonsurans Trichophyton tonsurans A great part of our positive results correspond with
1 Microsporum audouinii Microsporum audouinii those samples of toenails, being T. rubrum the main
Trichophyton rubrum species causing onychomycosis in feet, as expected
8 Negative
5 Negative
Trichophyton spp. since it is the most common dermatophyte species
1 Negative
Scopulariopsis brevicaulis with the major incidence in Europe. It rarely affects
Fusarium spp. hair and scalps but is usually found in nails [4, 22, 26].
1 Negative
Candida parapsilosis In the same way, the detected species are in concor-
4 Negative
Aspergillus spp. dance with the bibliography, being the genera Candida
1 Negative
the most frequent responsible of onychomycosis in
N, number of samples
hands. Candida onychomycosis affects fingernails
more often than toenails being Candida albicans the
most common cause of candidal onychomycosis (80%
efficient method for the diagnosis of the of such infections) [27, 28]. On the other hand, M.
onychomycosis.
audouinii was found in hair samples (Table 2).
Although onychomycoses are mainly caused by
dermatophytes, various non-dermatophyte filamen-
Discussio
tous fungi (NDF) have also been detected in our study
n
such as Scopulariopsis brevicaulis or Fusarium spp.
The usual protocols for detecting fungi in SM consider
isolated from abnormal nails [13] (Tables 2, 3).
both the direct microscopy and the culture of frag-
Our results showed that a great number of false
ments of nails, hairs and dermal scales, followed by
positives and negatives would be diagnosed by using
the identification of species using microscopy tech-
only the direct visualization of the sample through
niques and/or determination of biochemical charac-
microscopy despite being a rapid diagnosis method.
teristics. However, these methods present several
This may be due to the subjectivity of the technique,
disadvantages, such as low efficiency of the cultures,
artefacts, environmental contaminants, or in some
delay in the diagnosis, subjectivity of the microscopy
cases, the low degree of parasitization of the sample
for the identification, possibility of contamination of
[17]. On the other hand, some cases that showed
the culture, and possibility of bias of the culture in the
positive diagnosis through the microscopy and culture
case of mixed infections [24].
techniques could not be confirmed by molecular
Amplification of regions of interest through the
methods. This could be due to the fact that the direct
genome of the causative agents has proved to be an
analysis of the samples did not provide the minimum
interesting and valuable complement to the traditional
quantity of DNA to obtain satisfactory results after the
diagnosis methods of SM. The detection by direct PCR
amplification of the DNA available. Other factors
of the causative agents in the collected samples can
affecting the performance of this technique could be a
produce reliable results faster than the traditional
non-homogeneous distribution within the material,

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Mycopathologia

degradation of the genetic material or the inhibition of Referenc


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