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| ORIGINAL ARTICLE |
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| Year : 2014 | Volume
: 1
| Issue : 1 | Page : 41-46 |
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Detection of Candida species by acridine orange fluorescent dye in exfoliative smears of oral candidiasis
Kumaraswamy LR Naik1, Pushparaja Shetty2, Sarosh E Shroff2, Vimal Kumar Karnekar3, Krishna MS Prasad3, Lal P Madathil2
1 Departments of Oral Pathology and Microbiology, SJM Dental College & Hospital, Chitradurga, India
2 Department of Oral Pathology and Microbiology, A B Shetty Memorial Institute of Dental Sciences, Deralakatte, India
3 Department of Microbiology, K. S. Hegde Medical Academy, Mangalore, Karnataka, India
| Date of Web Publication | 2-Jan-2014 |
Correspondence Address:
Kumaraswamy LR Naik
Department of Oral Pathology and Microbiology, SJM Dental College & Hospital, Chitradurga - 577 501, Karnataka
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/WKMP-0056.124188
Background: Oral candidiasis continued to receive attention due to its evidenced association with its evidenced association with acquired immunodeficiency syndrome. In spite of considerable improvements in the laboratory diagnostic methods, there is still a need for more reliable, definitive and less time-consuming diagnostic techniques for Candida infection. Many of the studies were done utilizing acridine orange (AO) as a basic fluorescent dye for the demonstration of yeast in cultured Candida species and in histopathological sections. However, only very few studies report this technique to demonstrate Candida organisms in oral smears. The present study was carried out to assess the diagnostic reliability of detection of Candida by utilizing AO fluorescent dye in oral exfoliative smears of oral candidiasis.
Materials and Methods: A total of 62 clinically diagnosed cases of oral candidiasis were studied. Two smears and a swab were collected from each suspected lesion. Each slide was stained with, periodic acid Schiff (PAS) reagent and AO. PAS stained smears were then evaluated for the presence of Candida species under light microscopy. AO stained smears were observed under fluorescent microscopy. After inoculation of swab on the saborouds agar plates, the growth of Candida species was evaluated by Gram stain and germ tube test. Each technique was evaluated for sensitivity, specificity, positive predictive value and negative predictive value.
Results: It was found that the PAS stained smears were more reliable for detection of Candida species (sensitivity = 100%, specificity = 66.7%), than AO method (sensitivity = 87.5%; specificity = 0%).
Conclusion: Acridine Orange fluorescent technique even though rapid lacks specificity for Candida. Keywords: Acridine orange, Candida, candidiasis, fluorescence, oral smears, periodic acid Schiff reagent
How to cite this article:
Naik KL, Shetty P, Shroff SE, Karnekar VK, Prasad KM, Madathil LP. Detection of Candida species by acridine orange fluorescent dye in exfoliative smears of oral candidiasis. Saudi J Oral Sci 2014;1:41-6 |
How to cite this URL:
Naik KL, Shetty P, Shroff SE, Karnekar VK, Prasad KM, Madathil LP. Detection of Candida species by acridine orange fluorescent dye in exfoliative smears of oral candidiasis. Saudi J Oral Sci [serial online] 2014 [cited 2023 Mar 21];1:41-6. Available from: https://www.saudijos.org/text.asp?2014/1/1/41/124188 |
| Introduction | |  |
Oral candidiasis, caused by the yeast Candida is a relatively common, well-known opportunistic infection in man and has been recognized as a clinical entity since the time of hippocrates in 4 th century BC. [1] Examination of clinical signs and symptoms and demonstration of an organism by staining with periodic acid Schiff (PAS) or gamori methanamine silver (GMS) or Gridley stain along with culture methods are still established and accepted procedures for the diagnosis of oral candidiasis, though they are time consuming procedures. [2],[3] Furthermore the identification of fungi is easily missed on cytology as desquamated epithelial cells, inflammatory cells and necrotic debris easily mask the fungal elements. [3]
Many studies have been reported in which acridine orange (AO), a basic fluorescent dye was used in conjunction with fluorescent microscopy (FM) for the demonstration of yeast in cultured Candida species and in histopathological sections. [4],[5] However there are only few reported studies which utilize this technique to demonstrate Candida species in oral smears. [6],[7] This study was under taken to:
(a) Assess the diagnostic reliability of detection of Candida by utilizing AO fluorescent dye in oral exfoliative smears of oral candidiasis.
(b) Correlate the accuracy of this technique with special stain that is PAS and culture.
(c) Determine the relative advantages of the AO and PAS in identifying the Candida species.
| Materials and Methods | | |
The study group comprised 62 patients, who were clinically diagnosed with oral candidiasis. A thorough clinical history was recorded. A detailed general physical, extra oral and intra oral examination was done. The clinical diagnosis of oral candidiasis was made according to Lehner's (1966) [8] criteria. From each patient, samples from lesion tissue for microbial culture were obtained first and then followed by scrapings for cytosmears. The swab was then directly inoculated on to the Sabouraud's agar plate. The plates were incubated at 37°C for 48 h. Two clean glass slides were marked with the case number and the type of staining procedure to be carried out. Smears were prepared from the clinically suspected area by scraping the lesion and/or from the fitting surface of the denture (in case of chronic atrophic candidiasis) with sterile metal spatula. All the slides were fixed for ½ h in a coplin jar containing ether alcohol (1:5). One slide was then stained with AO stain [9] and the other with PAS. [10]
Three observers examined all the slides. All the slides were observed under ×40 for the presence (+) or absence (−) of organisms on morphologic basis. [8] Grading were recorded as +, ++, +++ and ++++ ; according to number, the visibility and the fluorescence of Candida in contrast with the other material such as epithelium, mucous, debris and inflammatory cells [Table 1]. The graded data were then tabulated for further analysis.
Fluorescent microscopic evaluation was performed with the help of incident light FM POLYVAR 2 Reichert-Jung.100-W HBO high-pressure mercury lamp with 220-V AC mains was used as the source. The excitation filter used was of Band Pass 450-495, in blue excitation range (VB1). The barrier filter was of Long Pass 520 with a dichoric illuminator mirror with a specification of spectral reflection and edge of transmission range in direct sequence 510. Magenta colored organisms in PAS stained smears were observed depending upon morphologic criteria. [8] AO stained smears were observed directly under FM at ×40 magnification. The fluorescing organisms were identified on the basis of morphology. After incubation of the organisms in Sabouraud's agar plates, they were examined for the presence (+) or absence (−) of yeast growth. Plates without growth at the end of 48 h were incubated [Figure 1] for up to 2 weeks before being discarded as negative.
A small suspension of the culture was then taken and stained with Gram stain. The readings were documented as positive (+) or negative (−) depending upon the presence or absence of yeast. Later yeasts were subjected to germ tube test for rapid confirmation of Candida albicans as described. [8] Prepared slides were examined at ×40 under light microscopy (LM) to see germ tube formation.
| Results | | |
A total of 62 clinically diagnosed cases of oral candidiasis were observed [Figure 2] of which 49 (79.0%) were male and 13 (21.0%) were female patients with an age range of 10-75 years. The mean age of occurrence was 48.30 years. Distributions of different clinically diagnosed patients are shown in [Table 2].
Growth of the organisms
Out of the 62 cases, 56 were positive in culture (90.30%) [Figure 1]. All positive growths were positive for Gram staining (100%). The Gram-positive organisms showed round oval budding yeast cells. There were very few hyphal/pseudohyphal forms seen in the Gram stained smears.
Germ tube test
The positive organisms showed direct extension of blastopore cytoplasm. This extension was without constriction from the cell wall to form true hyphae. Out of 56 positive cases in culture 51 showed germ tube test positivity (91.1%) [Figure 3]. | Figure 3: Photomicrograph of Candida organism showing germ tube formation. Direct light microscopy (original magnification, ×40)
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PAS - reagent technique
Out of 62 clinically suspected lesions, 58 (93.5%) demonstrated the presence of organisms [Figure 4] while 4 were negative [Table 3].
The number of organisms and the delineation in PAS stained smears were analyzed by Chi-square test and the association was found to be very highly significant (χ2 = 99.39: P = 0.001). | Figure 4: Photomicrograph of periodic acid Schiff reagent stained smear observed under light microscope (original magnification, ×40)
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AO-FM
Candida species fluoresced in varying intensities of red, green and orange yellow colors [Figure 5], [Figure 6], [Figure 7]. The results were shown in [Table 4], [Table 5], [Table 6] & [Table 7]. | Figure 5: Orange yellow colored fluorescing Candida hyphae/pseudohyphae with fluorescing epithelial cells (original magnification, ×40)
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 | Figure 6: Orange yellow colored fluorescing clusters of Candida hyphae/ pseudohyphae with fluorescing epithelial cells (original magnification, ×40)
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 | Figure 7: Orange yellow colored fluorescing candidal hyphae/pseudohyphae budding out from spore form with fluorescing epithelial cells (original magnification, 40×)
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 | Table 4: Comparison of AO fluorescent microscopy and culture techniques
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 | Table 5: Comparison of the number of Candida species seen in each slide stained with PAS and AO
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 | Table 6: Comparison of delineation of Candida species seen in each slide stained with different techniques
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The number and the delineation of organisms were compared with Chi-square test and was very highly significant (χ2 = 120.5; P = 0.001). Comparison of fluorescence and delineation of organisms was done using Chi-square test. It was very highly significant (χ2 = 139.63; P = 0.001). Comparison of fluorescence and number of organisms was done using Chi-square test. It was very highly significant (χ2 = 111.528; P = 0.001).{Figure 5}
Comparative analysis of visibility of organisms in each technique (PAS-LM and AO-FM)
The results were obtained for a number of organisms seen, in each slide stained by PAS observed under LM and AO observed under FM, were analyzed by Chi-square test and found to be very highly significant [Table 5]: P = 0.001].
The results obtained for delineation of Candida species seen, in each slide were analyzed by Chi-square test and found to be very highly significant [Table 6]: P = 0.001].
| Discussion | | |
Microbial evaluation
In 62 clinically diagnosed cases of oral candidiasis, 6 were negative in a culture which included 4 cases of median rhomboid glossitis and 1 each case of acute and chronic atrophic candidiasis. Though the original classification by Lenher (1966) was considered, 13 cases of median rhomboid glossitis were included in the present study. This was because it is reported in literature that a significant number of cases were associated with Candida species. [11] Out of 56 growths in culture, 4 were germ tube negative and this could be due to the growth of other yeast species other than C. albicans that inhabit the oral cavity. It is also reported that only 95% of C. albicans show germ tube formation. [12]
Cytology
The value of a cytological diagnosis of Candida compared with other investigations must be addressed. Besides cytopathology, the main techniques used to search for the fungus were Potassium hydroxide (KOH) preparations and Gram stained smears. KOH preparations although rapid, have inherent artifacts caused by protein denaturation. [13] In routinely used Papanicolaou stained smears under LM, Candida was poorly stained and at times it was difficult to demonstrate the organisms. [14] Because of such problems, PAS and GMS stains are recommended for all KOH negative preparations though they are more time consuming procedures. While some rapid silver stains have been introduced, they are comparatively slow, require frozen sections and have an unacceptably high incidence of technical failure. The organisms can also be cultured on Sabouraud's medium, although with this procedure it often takes days to confirm fungal growth. While C. albicans may be suspected in routine sections of formalin fixed, paraffin embedded tissue stained with hematoxylin and eosin, the organisms are often obscure and the diagnosis must be confirmed with time consuming special staining techniques, such as PAS or GMS. [13] Each technique has its own advantages and disadvantages, such as cost, ease of technique, induction of artifacts, sensitivity, ease of diagnosis, rapidity of the procedure and necessary equipment. Not all clinical forms of candidiasis are advised for biopsy. [2]
Periodic Acid Schiff (PAS) reagent
Many authors consider the demonstration of Candida species (especially numerous hyphal form) by PAS stained smears as one of the diagnostic methods. [12] In this study, 2 false positive results were seen accounting for sensitivity = 100% and specificity = 66.7%. In a few smears, PAS also stained mucin in a magenta color, causing interference in identification of organisms, but Candida species could be well-delineated by morphology.
Acridine Orange (AO)
Many authors utilized the modifications of originally described AO technique. [7],[9],[15] They found that the advantage were minimal by their modifications. We utilized staining method described by Riva et al. due to the ease of preparation of solution and a minimal number of staining containers required. [9] However the less specific results were a serious disadvantage (specificity = 0%). Along with the organisms, the epithelial cells, inflammatory cells, mucin and debris fluoresced variably in colors of bright green, orange, red and yellow. Small bacteria like cells also fluoresced but Candida were delineated by their larger size. Other than AO fluorescent, dyes like Calcofluor white (CFW) and Fungiqual have also been used to demonstrate Candida though they have been shown to have non-specific fluorescence and other artifacts. [3],[13],[14] Many of the authors stated that the screening of AO smear is as laborious and time consuming as the conventional cytological diagnosis. [7] In our experience, we felt that because of differential intensities of fluorescence by Candida species it is easy to screen the AO smears and the fungi can be easily picked up. In few instances, it was agreed that the background fluorescence may interfere with the proper delineation of Candida. [5] If the organisms were less in the smears, we agree that the exposure of human eyes to brilliant fluorescent colors overtime may cause more eyestrain than working with ordinary microscopy. [7]
AO fluorescence of fungi is due to nuclear binding of the organism. [9],[16] AO binds to both DNA and RNA with an excitation maximum of approximately 470 nm. The distribution of dead, inactive but living or living cells can be noted as DNA retains its staining properties even in non-viable cells. AO stains single stranded nucleic acids and emit orange red fluorescence, whereas those that are double stranded tend to fluorescence green. [17] The intensity of color depends upon the concentrations of nucleic acids. [18] Fluorescence of Candida could also be due to the acid mucopolysaccharides in the wall of fungi that reacts with basic AO. [16] As the AO is a basic dye it would be possible to give the background fluorescence for oral acid mucin ropes causing confusions, but this could be ruled out on morphologic grounds of hyphae and spores. [9] It was advised to use hematoxylin to quench the background fluorescence. We did not try this method as it might prolong the technique time. A study done by Mirrett et al. found that AO was more sensitive (44-62%) than Grams staining in a series of clinical specimens for detection of bacteria. [19] The overall sensitivity of AO was less, when compared to our study (87.5%). It was claimed that the other fluorescent dyes such, as CFW staining does not interfere with subsequent GMS or PAS staining. [13] We did not use the AO stained smears to stain subsequently with PAS. On comparison of the sensitivity and specificity of AO stained smears under fluorescence microscopy and with PAS under LM, it was found that the PAS is still more reliable (sensitivity of 100% and specificity 66.7%) than AO-FM (sensitivity of 87.5% and specificity 0.0%). However PAS requires additional staining, time and cost. On comparison of number, visibility and delineation of Candida species seen in each slide it was found that PAS showed better results than by AO-FM.
It is possible that the variation in the number, visibility and fluorescence grading of organisms detected by these methods depend upon the color stained or the fluorescence of the organisms. It is also possible that the error in the sample collection and distribution of the sample over each slide is a factor for variance. Furthermore as each observer recorded the scores independently, there are chances of missing the most representative field areas where the organisms were more aggregated. Differences in estimates between observers are likely to depend on individual interpretations of what actually constitutes a countable Candida. Because of differences in which particular objects are recognized as stained/fluorescing Candida, the operator variation may be large. [17] To reduce these variability three different observers were selected for the study. In the present study, negative controls were not included as the general population harbors the Candida species as normal commensal, in the oral cavity and Sabouraud's agar culture technique is highly sensitive. [2]
| Conclusion | | |
AO fluorescent technique even though rapid, lacks specificity. The fluorescence is not specific for Candida and hence close attention to the morphology of the fluorescing organisms is mandatory. Although PAS unquestionably remains the mainstay of identification of fungus in routine cytological preparations, AO fluorescence preparations offer several advantages in routine cytological smears. The AO fluorescence of Candida is brilliant, but occasional misdiagnosis occurs due to artifacts. These artifacts are not considered to be sufficiently numerous so as to be a serious handicap to using this technique. When the greater number of organisms is present in the smear, the artifact is not considered to be important.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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