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Mycopathologia (2015) 180:373377

DOI 10.1007/s11046-015-9933-y

Direct Microscopy: A Useful Tool to Diagnose Oral


Candidiasis in Children and Adolescents
Mathieu Marty . Emmanuelle Bourrat .
Frederic Vaysse . Mark Bonner .
Isabelle Bailleul-Forestier

Received: 11 March 2015 / Accepted: 24 August 2015 / Published online: 2 September 2015
Springer Science+Business Media Dordrecht 2015

Abstract
Introduction Oral candidiasis is one of the most
common opportunistic fungal infections of the oral
cavity in human. Among children, this condition
represents one of the most frequent affecting the
mucosa. Although most diagnoses are made based on
clinical signs and features, a microbiological analysis
is sometimes necessary. We performed a literature
review on the diagnosis of oral candidiasis to identify
the techniques most commonly employed in routine
clinical practice.
Materials and Methods A MedlinePubMed search
covering the last 10 years was performed.
Results Microbiological techniques were used in
cases requiring confirmation of the clinical diagnosis.
In such cases, direct microscopy was the method most
commonly used for diagnosing candidiasis.
Conclusion Direct microscopy appears as the
method of choice for confirming clinical diagnosis
and could become a routine chair-side technique.

M. Marty (&)  F. Vaysse  I. Bailleul-Forestier


Department of Pediatric Dentistry, Toulouse Dental
School, University of Toulouse III, Toulouse, France
e-mail: martymat@hotmail.fr
E. Bourrat
St-Louis Lariboisie`re University Hospital, Paris, France
M. Bonner
International Institute of Periodontology, Nice, France

Keywords Oral candidiasis  Children  Direct


microscopy  Diagnosis

Introduction
The last decade had seen a gradual increase in the
incidence of fungal infections [1]. Oral candidiasis is
one of the most common opportunistic fungal infections of the oral cavity in human. Its clinical expression is largely varied, and it has been classified into
different subtypes, as presented in Table 1 [2].
Although most fungal species live as saprophytes,
under certain conditions they can become pathogenic
and cause infectious diseases, known in animals as
mycoses. Fungi of the genus Candida cause oral
candidiasis. One hundred and fifty species of this
genus have been isolated from the oral cavity,
although 80 % of the isolates corresponded to Candida albicans [3]. All types of oral candidiasis are
found in the pediatric population where they represent
one of the most frequent mucosal disorders. Some
predisposing factors have been associated with an
increased incidence of infection in children, such as
prolonged antibiotic therapy, malnutrition, diabetes,
HIV infection and poor oral hygiene. An oral
candidiasis encountered in a young child must lead
clinician to search for an immune deficiency. Denturerelated stomatitis may also be encountered in young
patients during long-term orthodontic treatment using

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Mycopathologia (2015) 180:373377

Table 1 Clinical classification of oral candidiasis


Type
Acute

Subtypes
Pseudomembranous
Erythematous

Chronic

Pseudomembranous
Erythematous
Hyperplasic

Others

Angular cheilitis
Denture-related median
rhomboid glossitis

a removable material [4, 5]. In the adult population,


wearing of prosthetic denture is also a predisposing
factor, as well as smoking and xerostomia. While this
pathology occurs more rarely among children and
teenagers, methods of diagnosis and treatment have
been proposed [6, 7]. The diagnosis of oral candidiasis
is often done with clinical symptoms [7], mostly for
acute pseudomembranous cases (Fig. 1). However,
due to its clinical variability, microbiological techniques are often necessary to confirm the diagnosis
and establish a differential diagnosis with other
diseases, such as oral lichen planus or leucoplasia,
especially in acute erythematous candidiasis (Fig. 2),
or in cases characterized by resistance to antifungal
drugs [8]. Several approaches are used to isolate and
identify Candida species, including direct microscopy
of smears, stains and cultures, as well as PCR [9].
Recent advances in optical devices offered dental
practitioners the opportunity to equip their surgeries
with powerful optical microscopes for direct

Fig. 2 Erythematous tongue candidiasis in a child

microscopic examination. The procedure involves


taking a representative sample from the infected site
by exfoliative cytology, which is transferred on glass
slide for microscopic examination. Ideally, it is treated
with potassium hydroxide (KOH), Gram stain or
periodic acidSchiff (PAS) stain. The KOH clears
organic material from the background, making the
fungi stand out, as clear blastoconidia, hyphae or
pseudohyphae in the case of Candida infection. With
Gram staining, hyphae and yeasts will appear dark
blue, whereas they will be red to purple on PASstained specimens [10].
We aimed to study the recent literature and
international policies regarding the diagnosis and
monitoring of oral candidiasis in children and adolescents using direct microscopy, in order to define the
interest for a pediatric dentist to use these technics.

Materials and Methods

Fig. 1 Acute pseudomembranous candidiasis in a 7-year-old


boy

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A PubMed search was performed using the following


key words: oral AND candidiasis AND diagnosis.
This review respected the Prisma checklist. The key
words were validated by the Medical Subject Heading
(MeSH) dictionary, and the Boolean operator AND
was used to relate them. All systematic reviews,
clinical trials and meta-analyses were considered in
this review.
We identified a total of 63 articles from which we
selected 11 after analysis, all of which were reviews.
The excluded publications fell outside the scope of the
present study.

Mycopathologia (2015) 180:373377

Discussion
The occurrence of candidiasis among children is quite
high, especially in those who are medically compromised. An accurate diagnosis is essential to ensure
selection of the most suitable treatment option. In most
cases, the clinical appearance of lesions enables a
pediatric dentist to make the right diagnosis. However,
mistakes can be made differentiating candidiasis from
other clinical conditions such as bacterial gum disease
or keratosis. In such cases, further microbiological
investigation is needed. The number of recent publications on this subject reflects the interest of the

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scientific community (Table 2). Yet, only one review


specifically concerned the diagnosis of pediatric
candidiasis [17]. All studies considered microscopy
less selective than culture and genomics techniques
[1020]. However, some did conclude that direct
microscopy is an interesting technique, allowing
differentiation between yeast and hyphal forms, which
is interesting because of the greatest pathogenicity of
hyphal forms [11, 12, 15, 18]. For use as a first
intention test, it can orientate toward the diagnosis.
Considering the simplicity of the procedure, some
reviews conclude that direct microscopy could help
for a valuable chair-side diagnosis [13, 16, 17, 19].

Table 2 Usefulness of biological techniques for diagnosing oral candidiasis


References

Recommendation

Fixation type

Authors comments

Farah et al. [18]

Yes, only in the


pseudomembranous subtype of
oral candidiasis, using cytology
smears

Periodic acid
Schiff stain

In other oral Candidal lesions, cytology smears


usually fail to show any hyphal elements

Saint-Jean et al. [17]

Yes

No data

Thompson et al. [19]

Yes, with a mucosal scraping

Potassium
hydroxide

Valuable in differentiating between yeasts and


hyphal forms
Diagnosis of oral candidiasis can be confirmed
by direct microscopic examination after
clearing with 10 % potassium hydroxide

Periodic acid
Schiff stain
Giannini et al. [8]

Yes, with samples for exfoliative


cytology

Air dry

Laurent et al. [15]

No, culture recommended

No data

Culture if resistance to treatment

Smitha Byadarahally
et al. [16]

Yes, direct microscopic


examination

Potassium
hydroxide

Valuable in differentiating between yeast and


hyphal forms, but less sensitive than cultures

Krishnan [13]

Yes, direct examination of smears

Potassium
hydroxide

A chair-side diagnostic procedure that can be


performed by stain depositing a drop of 10 %
potassium hydroxide (KOH) onto the fixed
slide

Alcohol fixative

Gram stain
Periodic acid
Schiff

A chair-side diagnostic procedure can be


performed by direct microscopic examination
after clearing with a drop of 10 % potassium
hydroxide deposited onto the slide. Less
valuable than biopsy

Kumaraswamy et al.
[14]

Yes, with smears, swabs and oral


rinse samples

95 % ethyl
alcohol

Common for diagnosing candidiasis

Coronado-Castellote
et al. [12]

Yes, direct microscopic


examination

No data

First intention test

Muzyka and Epifanio


[10]

Yes, direct microscopy

Potassium
hydroxide

Less selective than culture

Stoopler et al. [11]

Yes, as an adjunctive diagnostic


technique, with samples for
exfoliative cytology

Potassium
hydroxide

Valuable in differentiating between yeasts and


hyphal forms

Gram stain
Periodic acid
Schiff stain

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Anyways in cases of resistance to conventional


therapy, a culture should be done for isolation of the
causative agent and determination of its susceptibility
to antifungals [1, 14].
While the general agreement is that direct microscopy is useful in diagnosing candidiasis, the sample
preparation method is still a matter of debate. Periodic
acidschiff (PAS) appears to give the best results [18].
However, most authors agree that clearing in a drop of
10 % KOH is the most simple and reproducible
method available, allowing a quick and accurate
diagnosis [10, 11, 16, 18, 19]. An alternative method
used in the field of oral parasitology is to spread the
sample directly onto the glass slide with physiological
serum [20] (Fig. 3). While this certainly represents the
quickest way to observe oral flora, it is less reliable

Fig. 3 Candida species at direct microscopy (wet mount in


physiological serum 91000)

Mycopathologia (2015) 180:373377

than other methods for detecting Candida yeasts.


Whatever the method chosen for sample preparation,
the sampling method allowing the best results is the
scraping of the affected area with a spatula and the
subsequent preparation of thin smears.

Conclusion
The diagnosis of oral candidiasis is mostly based on
clinical examination. However, in some situations,
further investigations are needed to obtain a more
targeted treatment. The simplicity of the sampling
method is particularly well adapted to young patients.
The care of these patients is specific with regard to the
need to involve their parents in the process. A full
explanation of the etiology is key to obtaining parents
cooperation and finally compliance with medication.
In this respect, direct microscopic examination of the
clinical sample obtained by scraping of the affected
area with a spatula as a chair-side diagnostic method
could improve pediatric candidiasis management
(Fig. 4). Clearly, the main disadvantage of this
technique is its incapacity to differentiate between
species. Also, it should be performed by an operator
trained in microbiology, with proper material and in
close relationship with the microbiology laboratory.
Nevertheless, it remains a simple, cost-effective and
noninvasive procedure which, when used as a complementary tool alongside clinical observation, could
help to establish a correct diagnosis and guide the
clinician toward a more appropriate treatment.
Compliance with Ethical Standards
Conflict of interest
interest.

The authors declare having no conflict of

Ethical Statement All procedures performed in the study


involving human participants were in accordance with the ethical standards of Toulouse University and with the 1964 Helsinki Declaration and its amendments.
Informed Consent Informed consent was obtained from all
patients and their parents.

References

Fig. 4 Normalization of oral flora after treatment at direct


microscopy (wet mount in physiological serum, 91000)

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