In: Candidiasis ISBN: 978-1-62618-870-9

Editors: Felipe Contreras and Pedro Fuentes © 2013 Nova Science Publishers, Inc.

Chapter 7

FLUORESCENT STAINING FOR THE DIAGNOSIS

OF ORAL ERYTHEMATOUS CANDIDIASIS

Yoichi Nakagawa Department of Clinical Pathophysiology,

Tsurumi University School of Dental Medicine, Yokohama, Japan

ABSTRACT

The diagnosis of candidiasis is based on clinical findings, and the diagnosis is

confirmed by the identification of pseudohyphae in stained smears sampled from a lesion,

by the identification of colonies cultured on Sabouraud’s medium or by histological

examination. There are several staining techniques for the microscopic examination of

smears, including the Giemsa, Gram and periodic acid-Schiff (PAS) stains. Fungiflora Y

is a fluorescent stain. The solution binds to ß-linked polysaccharides, such as chitin and

cellulose, which are components of the fungal cell wall. The usefulness of Fungiflora Y

staining for the diagnosis of erythematous candidiasis is described in this chapter. The

sensitivity, specificity and positive and negative predictive values were calculated using

fungal culture as the gold standard, and the results were compared with the staining

results using the modified Giemsa staining system.

Keywords: Candida, candidiasis, culture, fluorescence staining, exfoliative cytology,

Fungiflora Y, quantitative analysis, dry mouth

1. INTRODUCTION

Oral candidiasis is classified into three major variants: pseudomembranous (Figure 1),

erythematous (Figure 2) and hyperplastic candidiasis [1]. The erythematous forms of the

Corresponding author: Yoichi Nakagawa, Department of Clinical Pathophysiology, Tsurumi University School of

Dental Medicine 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan. E-mail address: nakagawa-

y@tsurumi-u.ac.jp.

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Yoichi Nakagawa 180

disease occur more commonly than the pseudomembranous type [2]. Denture stomatitis,

angular cheilitis and median rhomboid glossitis are regarded to be candida-associated lesions.

The diagnosis of candidiasis is based on the clinical findings, and the diagnosis is

confirmed by the identification of blastospores and pseudohyphae in stained smears sampled

from a lesion, by the identification of colonies by culture on Sabouraud’s medium or by

histological examination [3, 4]. Smears are valuable for differentiating between the yeast and

hyphae forms, but are less sensitive than culture methods [5]. Because the number of Candida

isolates is smaller in erythematous candidiasis than in pseudomembranous candidiasis,

negative results are occasionally obtained by direct examination of erythematous candidiasis

[6]. In the diagnosis of erythematous candidiasis, examinations are reported to yield false-

negative results in 25% of culture tests and 42.5% of microscopic examinations [6]. Thus, a

more sensitive staining method for the microscopic examination has been needed. The

usefulness of fluorescent staining using Fungiflora Y for the detection of Candida in the

smear samples from erythematous candidiasis is described in this chapter.

Figure 1. Clinical manifestations of pseudomembranous candidiasis.

Figure 2. Clinical manifestations of erythematous candidiasis.

Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 181

2. EXAMINATION OF CANDIDA IN THE DIAGNOSIS

OF ORAL CANDIDIASIS

2.1. Culture Examinations

The interpretation of the results of microbial examinations is often complicated, because

Candida is present commensally in the oral cavities of up to 40% of individuals [1, 4]. There

is an overlap in the candidal counts from carriers and individuals showing infection, and there

are no established criteria to differentiate candidal commensalism from disease [1, 2]. Hence,

the isolation of Candida from the mouth is not confirmatory evidence of infection, and the

diagnosis of candidiasis is based on the combination of mycological examinations and

assessments of the clinical signs and symptoms [1, 2, 7].

The techniques available for the isolation of Candida within the oral cavity include the

use of a plain swab, an imprint culture, the collection of whole saliva, concentrated oral rinse

and oral mucosal biopsy [5]. The collection of whole saliva and concentrated oral rinse are

appropriate methods for evaluating the quantitative level of Candida in the whole mouth,

while swab and imprint culture can detect Candida from infected lesions.

For fungal cultures, the swab is directly inoculated onto an agar, such as Sabouraud’s

dextrose agar. The Candida species can be identified by the colony colors on the

CHROMagar Candida plates (Chromagar Microbiology, Paris, France; Figure 3). In our

department, the number of Candida colonies on the CHROMagar Candida plates is counted

after incubation at 37°C for 48 h and the results are expressed as the CFU/plate.

Figure 3. Candida colonies on the CHROMagar Candida plates. The swab was directly inoculated onto

the agar, the number and color of the colonies was then observed after incubation at 37°C for 48 h. A:

Candida albicans, B: Several species are seen in the agar.

2.2. Microscopic Examinations

Samples for exfoliative cytology are obtained by scraping the organisms, along with the

superficial epithelial cells. The samples are then smeared onto microscope slides. Several

Yoichi Nakagawa 182

staining methods are used for visualization of the organisms. The presence of pseudohyphae

is considered to be a positive finding for candidiasis.

2.2.1. Conventional Staining

For microscopic examinations, a smear of a sample that was taken from a lesion site is

fixed onto a microscope slide and then stained either by Gram staining or by the periodic acid

Schiff (PAS) technique [5]. A rapid staining test, the CytoQuick staining system (Muto Pure

Chemicals Co., Ltd., Tokyo, Japan), which is a modification of the Giemsa stain, is also

useful for the diagnosis of candidiasis [6, 8].

The CytoQuick staining system (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) is

composed of solution A (eosin) and solution B (methylene blue). The slides are placed into

solution A for 5 s, rinsed with tap water, placed into solution B for 15 s, and then examined.

Representative examples of the microscopic findings are shown in Figure 4, where

CytoQuick staining dyed the fungus body dark blue, while the cell walls remained colorless.

The exfoliated epithelial cells and bacteria were stained dark blue.

Figure 4. Modified Giemsa staining in exfoliative cytology. CytoQuick staining dyed the fungus body

dark blue, while the cell walls remained colorless. The exfoliated epithelial cells and bacteria were

stained dark blue.

2.2.2. Fluorescent Staining

Several fluorescent dyes that are specific for fungal cell wall polysaccharides have been

reported to be effective in the screening of clinical specimens for fungi [7, 9]. These include

Calcofluor white, Blankophor and Fungiqual [7, 9-11]. It has been recognized that fluorescent

techniques have significant advantages over traditional staining methods with respect to

rapidity, cost and the absence of interference with permanent fungal stains [12]. The

usefulness of fluorescent dyes has therefore been recognized as an effective tool not only in

histopathology, but also in cytopathology [13].

Fungiflora Y (Biomate Co., Ltd., Tokyo, Japan) is a nonspecific fluorescent stain for

fungi in cytological specimens. The solution binds to ß-linked polysaccharides, such as chitin

Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 183

and cellulose, which are components of the fungal cell wall [14, 15]. Fungiflora Y staining

offers a method for the rapid screening of cytological specimens for fungi and Acanthamoeba

because of its speed and technical simplicity [16-19]. However, Fungiflora Y has not been

used clinically for the diagnosis of oral candidiasis, and the application of the fluorescent dye

has so far only been performed in animal studies [14, 20]. We applied this staining method to

detect oral Candida and examined its usefulness.

2.2.3. The Staining Method Using Fungiflora Y

To stain samples using Fungiflora Y, a smear taken from the lesional site is fixed onto

microscopic slides, and Fungiflora Y is placed onto the slide for one minute. A cotton swab

cannot be used for sampling, because Fungiflora Y stains cotton fibers. The slide is then

observed under a fluorescent microscope at a wavelength of 365 nm using a CyScope®

(Partec GmbH, Münster, Germany). A CyScope® is a portable fluorescence microscope,

which enables it to be used at the bedside or at the side of a dental chair. A darkroom is not

necessary to observe stained slides, because the fluorescent intensity of the Fungiflora Y stain

is stable, and its fluorescent attenuation is smaller than that of the other fluorescence staining

methods [16]. A BZ−9000 fluorescent microscope (Keyence Corporation, Tokyo, Japan) was

used to obtain the images for this manuscript.

Fungiflora Y staining clearly shows fungal walls, because the compound specifically

attaches to polysaccharides, such as cellulose or chitin, which are present in the fungal cell

walls. Under fluorescent microscopy, typical hyphae and yeast of the Candida species display

brilliant green fluorescence, which readily differentiates them from exfoliated cells (Figure

5). Although Fungiflora Y nonspecifically binds to epithelial cells and to bacteria, the binding

is very weak. please remove ¶

Figure 5. Fluorescent staining in exfoliative cytology. Fungiflora Y staining clearly showed the fungal

walls, because the fluorescent molecules specifically attached to polysaccharides, such as cellulose or

chitin, which are present in fungal walls. Under fluorescent microscopy, typical hyphae and yeast of the

Candida species display brilliant green fluorescence, which readily differentiates them from exfoliated

cells. This specimen is from a case of erythematous candidiasis.

Yoichi Nakagawa 184

Therefore, the fungi appear to stand out against the background. This allows for detection

even at low magnifications. Furthermore, the margin of the fungal body emits fluorescence

that is brighter than the inner portion because the dye binds to the fungal wall, thereby

showing the characteristic fungal form [14]. This makes it easy to discriminate fungi from

nonspecific foreign bodies.

For samples of suspected erythematous candidiasis, a 60–70 µL drop of sterile water was

dropped onto the surface of the lesion, usually the dorsum of the tongue, and was swabbed

using a dental mirror.

3. DIFFERENCES IN THE CYTOLOGICAL FINDINGS BETWEEN

PSUEDOMEMBRANOUS AND ERYTHEMATOUS CANDIDIASIS

One helpful clinical feature of the pseudomembranous type of candidiasis (Figure 1) is

the ability to scrape off the superficial white plaques. In these white plaques, there are

numerous superficial Candida hyphae, and these are more likely to result in a positive smear

test (Figure 6). On the other hand, the cytological findings of erythematous candidiasis are

completely different from the findings of pseudomembranous candidiasis (Figure 5).

Negative results are occasionally obtained by fungal examinations for this type of infection,

because the number of Candida isolates is smaller in atrophic candidiasis than in

pseudomembranous candidiasis [6]. Based on this background, we have developed

fluorescent staining methods for microscopic examinations.

The studies performed in our department were as follows; comparison of Fungiflora Y

and modified Giemsa staining of erythematous candidiasis, and quantitative evaluation of

Candida by microscopy in erythematous candidiasis. The outline of these studies will be

described in the following sections.

Figure 6. Fungiflora Y staining in a case of pseudomembranous candidiasis.

Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 185

4. COMPARISON OF FUNGIFLORA Y WITH MODIFIED GIEMSA

STAINING IN ERYTHEMATOUS CANDIASIS

The usefulness of Fungiflora Y staining for the diagnosis of erythematous candidiasis

was investigated. The study subjects were recruited from consecutive patients who had

subjective dry mouth and visited the Dry Mouth Clinic at Tsurumi University Dental

Hospital. Microscopic and cultural examinations were performed in the cases that were

clinically diagnosed with erythematous candidiasis, which was based on findings such as the

redness of the oral mucosa, an atrophic or smooth tongue, as well as soreness or pain of the

tongue [21]. A total of 48 patients [nine males and 39 females; age ranging from 26 to 96

years; mean ± SD = 68.0 ± 12.0] who were clinically diagnosed with oral erythematous

candidiasis were enrolled in this study. Of the 48 patients, 37 (77.1%) complained of pain in

their mouths. All of the patients had soreness of their tongue.

The sensitivity, specificity and positive and negative predictive values were calculated

using fungal culture as the gold standard, and the results were compared with the staining

results obtained using the CytoQuick system. The accuracy was calculated, and the

differences between CytoQuick and Fungiflora Y groups were examined using contingency

tables and the chi square test.

4.1. Comparison of the Reliability of Staining with Fungiflora Y

and Modified Giemsa Staining

The inter-observer agreement was assessed to compare the reliability of each staining

method. The microscopic findings of the presence (positive) or absence (negative) of

pseudohyphae on the smear specimens were evaluated by an oral surgeon (Dr. S. Yamachika)

and a general dentist (Dr. M.R. Okamoto). Dr. Yamachika had 30 years of experience in the

oral surgery field and Dr. Okamoto had one year of experience in general dental practice.

Kappa values were calculated in order to determine the inter-observer agreement.

The inter-observer agreement was poor for the CytoQuick staining and fair for the

Fungiflora Y staining; the kappa values for the assessment of the presence of pseudohyphae

or yeast were 0.47 and 0.61, respectively. A kappa value less than 0.40 indicates poor

agreement, 0.40–0.59 indicates fair agreement, 0.60–0.74 indicates good agreement and 0.75–

1.00 indicates excellent agreement. Thus, the results suggested that the Fungiflora Y staining

was a superior method for the microscopic examination of erythematous candidiasis

compared to the CytoQuick staining method.

4.2. Comparison of the Accuracy of Fungiflora Y with Modified Giemsa

Staining

Positive microscopic or cultural examinations confirmed the diagnosis of candidiasis in

38 (80.9%) of the 48 patients. The rest of the patients were thought to be false negatives or to

have other oral diseases, such as burning mouth syndrome. The CytoQuick staining

Yoichi Nakagawa 186

examination detected pseudohyphae with occasional yeast in 20 cases (41.7%). In contrast, 31

cases (64.6%) were positive on the microscopic examination using Fungiflora Y staining.

The sensitivity and specificity of the CytoQuick staining was 0.51 and 0.91, respectively;

the positive predictive value was 0.95, and the negative predictive value was 0.36 (Table 1).

The positive likelihood ratio was 5.65, and the negative likelihood ratio was 1.87. The

accuracy was (19 + 10)/48 = 0.60.

The sensitivity and specificity of the Fungiflora Y staining were 0.84 and 1.0,

respectively; the positive predictive value was 1.00, and the negative predictive value was

0.65 (Table 2). The positive likelihood ratio was more than 10 (infinity). The accuracy was

(31 + 11)/48 = 0.88, which was superior to that of CytoQuick (p = 0.0052). These results

suggest that the detection of fungal elements from erythematous candidiasis was more

accurate using Fungiflora Y staining compared to traditional staining.

Although it is not clear which method is the most accurate among the various fluorescent

staining methods, Fungiflora Y seems to have an advantage, because Fungiflora Y only

involves a one-step process, whereas the Calcofluor method involves two steps [11].

Moreover, one minute is adequate for the Fungiflora Y staining. Therefore, the microscopic

examination of a smear specimen using Fungiflora Y staining was useful for the diagnosis of

oral erythematous candidiasis.

Table 1. Contingency table created from the results of the CytoQuick

staining and culture

Culture

Positive Negative Total PPV NPV

CytoQuick Positive 19 1 20 19/20 = 0.95

Negative 18 10 28 10/28 = 0.36

Total 37 11 48

Sensitivity 19/37 = 0.51

Specificity 10/11 = 0.91

Positive likelihood ratio 5.65

PPV, positive predictive value; NPV, negative predictive value.

Table 2. A contingency table created from the results of the Fungiflora Y

staining and culture

Culture

Positive Negative Total PPV NPV

Fungiflora Y Positive 31 0 31 31/31 = 1.0

Negative 6 11 17 11/17 = 0.65

Total 37 11 48

Sensitivity 31/37 = 0.84

Specificity 11/11 = 1.0

Positive likelihood ratio >10

PPV, positive predictive value; NPV, negative predictive value.

Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 187

5. QUANTITATIVE EVALUATION OF CANDIDA

BY MICROSCOPY IN ERYTHEMATOUS CANDIDIASIS

Light microscopy has been used for a differential diagnosis of fungal infections,

especially in the dermatological field, because it is quick.. However, the quantitative

evaluation of fungus in terms of cytology has not been studied. The Gaffky’s scale is widely

used for quantitative evaluation of Mycobacterium tuberculosis in sputum smears. The

presumption of the number of bacteria with quantitative relevance by Gram staining and

bacterial culture has also been studied. In a textbook on urinary tract infections, it is stated

that a number of investigators have reported that staining methods correlate with quantitative

culture in about 80 to 90% of cases [22]. Based on this background, the quantitative

relationship between Candida detected in microscopic and cultural examinations was

investigated to evaluate the usefulness of exfoliative cytology. This study may also elucidate

why negative results are occasionally obtained by direct examination of erythematous

candidiasis [6].

The subjects in this study were recruited from consecutive patients who had subjective

dry mouth and visited the Dry Mouth Clinic at Tsurumi University Dental Hospital.

5.1. Subjects and Methods

5.1.1. Subjects

Samples were obtained from a total of 54 patients (three males and 51 females; mean age

± SD = 68.4 ± 12.5) at the Dry Mouth Clinic, Tsurumi University Dental Hospital. The

patients were clinically diagnosed to have erythematous candidiasis and underwent both

microscopy of a smear specimen stained with a fluorescent dye, Fungiflora Y, and fungal

cultural examination. Of the 54 patients, 34 (77.1%) had redness of the tongue, 36 atrophy of

the tongue papillae, 15 angular cheillitis, six redness of the lips, 14 redness of the palate and

11 had redness of the buccal mucosa. All complained of pain in their mouths, and all had

soreness of their tongue. Of the 54 patients, Candida were detected in 51 (94.4%) of the

cultures, so that these cases were diagnosed to have oral candidiasis. In the cytological

examination, candidal hyphae were detected in 46/51 of these cases (90.2%).

5.1.2. Methods for Microscopic Examination

The specimens were obtained from surface of the dorsum of the tongue by swabbing. The

number of Candida was expressed as the number of fungi/field of vision (FOV) by

microscopy, and colony-forming units (CFU) in the culture. For microscopic examination of

the Fungiflora Y staining of the exfoliative cytology, a specimen was observed under

magnification of 200-power and the number of the fungi was counted. At first, the

examination with the fluorescent microscope was performed for more than four FOV in

succession in the top and bottom directions. Then, the observation was continued for more

than four FOV in the right and left direction. When no cell bodies were found, the number of

the FOV was further increased. As a result, more than 18 FOV were observed, for an average

of 51 FOV, in each specimen.

Yoichi Nakagawa 188

5.2. Relationship between the Microscopic and Cultural Examinations

of Candida

The results of the microscopic and cultural examinations demonstrated that there were 0 to

3.7 fungi/FVO and 0 to 1992 CFU/plate, respectively. The correlation coefficient of the

examinations was 0.569 (p<0.001); the regression equation was [CFU/plate] = 330.4x

[number of fungi/FOV] + 124.6 (Figure 7). please remove ¶Because this result demonstrated

that there was a quantitative correlation between the microscopic and cultural examination,

the possibility of quantitative evaluation of Candida by cytology was suggested.

Since numerous factors are involved in the establishment of candidiasis, such as the

number of organisms, the enzyme produced from Candida and the impairment of the host

defense, a diagnosis of oral candidiasis cannot be made based only on the amount of detected

Candida [23-25]. However, a decrease in the number of Candida is important for the

management of oral candidiasis, as well as elimination of the risk factors for further infection.

Quantitative culture of saliva has been thought to be helpful in the diagnosis of oral

candidiasis. This is because patients with candidiasis have more than 400 CFU/ ml of saliva,

whereas carriers of Candida albicans have less than 400 CFU/ ml [26]. The establishment of

a cut-off point will be helpful for the daily oral care of dry mouth patients in order to prevent

the risk of erythematous candidiasis [27]. Since it is recognized that there is a relationship

between the amount of Candida and the clinical signs, the quantitative analyses are useful for

managing oral candidiasis. Rapid microscopic examinations will give helpful information for

the treatment and prevention of oral candidiasis.

Because the number of Candida isolates is smaller in cases of erythematous candidiasis

than pseudomembranous candidiasis, negative results are occasionally obtained by direct

examination of erythematous candidiasis [6]. In our present study, the intercept of the

regression equation was 124.6 for erythematous candidiasis (Figure 8).

Figure 7. The correlation between the numbers of Candida detected in microscopic and cultural

examinations.

Microscope (Number of fungi / FOV)

Cultu

re (

CF

U / p

late

)

Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 189

This result suggested that the sensitivity of exfoliative cytology was relatively low.

However, even when only one mycelium is recognized from the lesion by microscopy, the

lesion is regarded to be due to oral candidiasis. This means that the microscopic examination

has high specificity.

CONCLUSION

Exfoliative cytology has been recognized to be of value in differentiating between the

yeast and hyphal forms of infection, and is advantageous due to the rapid examination.

Additionally, our study suggested that the direct examination of a smear was helpful, even for

quantitative evaluations, because a positive correlation was recognized between the number

of Candida detected by microscopic and cultural examinations. Although the number of

Candida isolates is smaller in erythematous candidiasis than in pseudomembranous

candidiasis, and negative results are sometimes obtained, an examination of smears from the

lesion is still useful, because a sensitive fluorescence staining option is available. Fungiflora

Y staining clearly shows the presence of fungal walls, because the fluorescent molecule

specifically binds polysaccharides, such as cellulose or chitin, which are present in fungal

walls. Under fluorescent microscopy, typical hyphae and yeast of the Candida species display

brilliant green fluorescence, which readily differentiates them from exfoliated cells.

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