|Year : 2021 | Volume
| Issue : 1 | Page : 1-5
KOH wet mount versus cellophane strip without mounting medium for rapid diagnosis in superficial mycoses
Sandeep Arora1, Reetika Pal2, Devinder Kumar Suhag2, Rajeshwari Dabas3, Gulhima Arora4, Satish Chand2
1 Department of Dermatology, Army College of Medical Sciences, Base Hospital, Delhi Cantt, India
2 Department of Dermatology, Command Hospital Air Force, Bengaluru, Karnataka, India
3 Department of Dermatology, Base Hospital, Lucknow, Uttar Pradesh, India
4 Department of Dermatology, Mehektagul Derma Clinic, New Delhi, India
|Date of Submission||09-Oct-2020|
|Date of Decision||19-Dec-2020|
|Date of Acceptance||15-Feb-2021|
|Date of Web Publication||11-Aug-2021|
Department of Dermatology, Base Hospital, Delhi Cantt - 110 010, New Delhi.
Source of Support: None, Conflict of Interest: None
Context: Diagnosis of superficial mycoses in clinically doubtful cases relies on demonstration of fungal elements on a 10% potassium hydroxide (KOH) slide mount which takes 30 min to 2 h. Rapid results will lead to reduced time to diagnosis. Aims: A comparative study of conventional 10% KOH wet mount against the cellophane strip method without a mounting medium under bright-field and phase-contrast microscopy was aimed to determine the comparative efficacy of these methods in demonstrating fungal elements of superficial mycoses. Settings and Design: The study was conducted at a skin center of a tertiary care hospital between November 2019 and January 2020. Material and Methods: One hundred and fifty consecutive clinically suspect cases of superficial cutaneous mycoses were examined. Skin scrapings for 10% KOH were collected with a blunt scalpel on one slide and examined half hour later. A cellophane strip collection of squames stuck onto another slide and examined directly by bright-field and phase-contrast microscopy. Statistical Analysis Used: Descriptive and inferential statistical analysis was carried out in the present study using SPSS 22.0, and R environment version 3.2.2 was used for the analysis of the data. Chi-square/Fisher’s exact test was used to find the significance of study parameters on categorical scale between two or more groups, nonparametric setting for qualitative data analysis. Fisher’s exact test was used when samples were very small. Results: The mean percentage positivity for fungal hyphae as seen on KOH mount was 76%, for cellophane tape 97.33%, and under phase-contrast microscopy 97.33%. Pityriasis versicolor was detected by all three methods (100%). Tinea corporis had the least positive result on KOH mount. Conclusion: Cellophane strip method without a mounting medium adequately demonstrates fungal elements on light microscopy.
Keywords: Cellophane strip, dermatophytosis, potassium hydroxide mount, rapid diagnosis, superficial mycoses
|How to cite this article:|
Arora S, Pal R, Suhag DK, Dabas R, Arora G, Chand S. KOH wet mount versus cellophane strip without mounting medium for rapid diagnosis in superficial mycoses. Indian J Dermatopathol Diagn Dermatol 2021;8:1-5
|How to cite this URL:|
Arora S, Pal R, Suhag DK, Dabas R, Arora G, Chand S. KOH wet mount versus cellophane strip without mounting medium for rapid diagnosis in superficial mycoses. Indian J Dermatopathol Diagn Dermatol [serial online] 2021 [cited 2023 Apr 1];8:1-5. Available from: https://www.ijdpdd.com/text.asp?2021/8/1/1/323703
| Introduction|| |
Dermatophytosis, a widespread superficial skin infection often described as a menace of our times, is reported in high numbers in dermatology outpatient departments (OPDs). It often relapses, is recalcitrant to treatment and its mismanagement with steroid abuse often leads to atypical presentations. A wet mount with potassium hydroxide (KOH), an easy test that demonstrates fungal hyphae, is time-consuming. Cellophane strip method described in the literature also uses KOH as a mounting medium, while other methods are expensive substitutes to the simple KOH mount.,,,,, Hence, a simple bedside test/modification which could identify the fungal elements in light microscopy is desirable. A cross-sectional analytical study of suspected cases of dermatophytosis was done. Specifically, lesional scrapings were comparatively evaluated using the traditional KOH mount, direct microscopy of cellophane strip mount without KOH, and phase-contrast microscopy of cellophane strip mount without KOH. The aim was to identify which method was better or showed comparable results in identifying fungal elements; the objective was to determine the possibility of reducing result turnaround time while yielding similar results to the standard KOH mount in a busy OPD.
| Materials and Methods|| |
One hundred and fifty consecutive patients of clinically suspected cases of dermatophytosis and pityriasis versicolor above 18 years of age reporting to the dermatology OPD of a tertiary level hospital between November 2019 and January 2020 were examined for this study for which institutional ethical committee clearance and patient consent were taken. Those <18 years of age with preexisting chronic papulosquamous diseases such as psoriasis and those not consenting for study were excluded.
Lesions were photographed, cleansed with surgical spirit, and subjected to scraping at the edge followed by cellophane strip for collection of skin scrape specimens. A blunt preflamed scalpel was used to scrape the edge of the lesions, collected onto the first slide, 2 drops of 10% KOH (NICE Chemical Pvt. Ltd, Kochi, India) added to slide and covered with coverslip for 30 min. A 5 cm × 2.5 cm cellophane strip (Wonder 555 tape, Wonder Polymers Pvt. Ltd, New Delhi, India) with sticky side down was applied on to the same edge, firmly stroked, stripped off in one motion, and stuck onto the second slide.
For light microscopy, we used a trinocular microscope (Olympus biological microscope CX43, Tokyo, Japan) with bright field and phase contrast. Image capture was done on an attached computer using coupled Magcam DC-10, 10-megapixel Universal Serial Bus camera, at ×400 with ToupLite software (MacOS Version 1.0).
Slides were initially examined under lower magnification (×100) and low light intensity followed by higher magnification (×400), with higher light intensity for better illumination. KOH slides were examined for hyphae, arthroconidia, and yeasts after 30 min, and during this period, the second slide was examined for fungal elements directly after sticking the cellophane tape, without KOH, first with conventional bright light followed by phase contrast, and images were captured [Figure 1] and [Figure 2].
|Figure 1: Panel showing slide mounts of dermatophytosis: (a) Hyphae crossing over squames (10% potassium hydroxide ×400), (b) Branched and broken hyphae over a background of squames (cellophane tape ×400), (c) Phase-contrast view of the same slide (phase contrast using polarizing filter 400nm ×400)|
Click here to view
|Figure 2: Panel showing slide mounts of pityriasis versicolor: (a) Malassezia spp. with short mycelial forms and clusters of yeast forms (10% potassium hydroxide ×400), (b) Bright-field view of the case without mounting medium (cellophane tape ×400), (c) Phase-contrast view of the same case (phase contrast using polarizing filter 400nm ×400)|
Click here to view
Descriptive and inferential statistical analysis was carried out in the present study using SPSS 22.0, and R environment version 3.2.2 was used for the analysis of the data and Chi-square/Fisher’s exact test was used to find the significance of study parameters on categorical scale between two or more groups, nonparametric setting for qualitative data analysis. Fisher’s exact test was used when cell samples were very small.
| Results|| |
The patient population consisted of 122 males (81.3%) and 28 females. The mean age of the cohort was 34.58 years. Occupation of males included students 34, general duty soldiers 30, drivers 19, with desk job 16, retired with no active occupation 10, sportsmen 7, and farmers 6, while among women, 11 had a desk job, 8 were homemakers, 6 students, and 3 farmers. Four males and one female with tinea corporis had well-controlled diabetes mellitus. The rest had no comorbidities. Thirty-eight cases (27 males and 11 females) had a history of self-medication with topical steroids in the past before presenting to our center.
There was no statistical significance in the pattern of fungal infection with respect to age or gender (P > 0.05). The mean percentage positivity for fungal hyphae as seen in KOH mount was 76%, for cellophane tape 97.33%, and under phase-contrast microscopy 97.33% [Table 1].
|Table 1: Diagnosis distribution in relation to potassium hydroxide, cellophane tape, and phase contrast of patients studied|
Click here to view
The positive yield was highest in pityriasis versicolor (P < 0.05) followed by tinea cruris and tinea corporis with standard KOH mount. The likelihood of a negative KOH mount (P < 0.05) was maximum in tinea corporis patients compared to other patterns, while there was no difference in rate of demonstration of fungal elements by cellophane strip and phase-contrast method.
| Discussion|| |
Porto in 1953 initially described the use of cellophane tape in diagnosis of pityriasis versicolor. A number of subsequent reports have demonstrated that 10% KOH addition after obtaining skin specimens with strip method had equal efficacy to standard scrape and KOH mount [Table 2].,,,
|Table 2: Studies on cellophane tape method for demonstration of superficial mycosis|
Click here to view
The likelihood rate of detecting fungal elements by KOH method is reported at 2.86 versus 3.28 for culture. Using KOH in addition to a strip method for harvesting material adds up to processing time of the slide from 30 min to 2 h. Reported advantage of KOH in lower concentrations (10%–30%) is that the fungal cell wall can resist its ability to clear the examining field by digesting keratin and lysing excess protein. However, detection rates with KOH are reported variably., It fails to offer a contrasting color field, may have artifacts, crystalizes over time, and is time-consuming. Other methods used to improve detection rates are either on light microscopy using contrast stains such as Parker and blue-black ink, chlorazol black E, cotton blue C4B, calcofluor white, and even molecular diagnostic tests such as polymerase chain reaction.,, The former are limited by expense and are time-consuming, while the latter suffer from inadequate representation of various species in reference libraries of different commercial systems, their cost, and the delay in processing. Hence, either of these cannot be used as rapid tests. Phase-contrast microscopy, although expensive allows minute specimen detail observation, has a high positive observation rate without the need for special stains or reagents., Fungal mounts from culture colonies have used the cellophane strip method too but with a mounting medium.
In this study, we used the standard method of collecting skin scrapings for the KOH mount. Using the strip method at the same site prevented the collection of thicker scales which could interfere with observation in the absence of a mounting medium. Using the KOH method in a similar way after initial scraping was not feasible as no further scales could be collected for the mounting medium.
Our study had a KOH positivity for fungal elements of 76% which is comparable to what’s reported in the literature.,,, No studies for strip method without a mounting medium have so far been reported for a comparison with this study. Time duration for keeping the squames in KOH has been mentioned as 30 min to 2 h in the literature, although in practice, they may be assessed earlier. Our method involved no wait time.
The demographic profile and pattern of infection were consistent with the reported literature.,
Fungal culture from patients of superficial mycoses may yield a positive result, but they are often negative and hence have a poor negative predictive value. This study was structured to compare the methods of detecting fungal elements on microscopy and not to determine a gold standard in their diagnosis which possibly still is a combination of a positive microscopy, positive culture, and clinical confirmation of superficial mycoses. Prior or present treatment was not an exclusion criterion as all cases were symptomatic, and this was a comparative study where treatment would have affected all three diagnostic methods.
KOH mount may need a level of expertise from those examining it. In our case, at least three experienced authors examined them independently with the same results, hence minimizing observer bias. Limitations of direct light microscopy which are perceived in detection of fungal elements, is that margins of normal epidermal cells may mimic fungal structure. Dissolution of keratin and cell wall with KOH may help; however, the absence of KOH in our study did not hinder their identification.
Advantages of using the cellophane strip as in this study are its ease of use, direct examination without waiting time, and storage of slides with preservation of specimen for later review [Figure 3].
|Figure 3: Bright-field view of the slide of a case of dermatophytosis with cellophane tape method at 6 months showing preservation of hyphae (cellophane tape ×400)|
Click here to view
However, the cellophane strip while stripping does traumatize the fungal hyphae, resulting in sometimes broken hyphae or altering their morphology. Furthermore, the learning curve may be steeper as compared to KOH mount for a novice and observer dependence on interpretation of observation may result in false negatives just as in KOH mounts. Pityriasis versicolor does not have this issue and their morphology is well maintained. Phase-contrast microscopy yields greater contrast of the microscopy field and allows easier and instant detection of fungal elements. However, it is an added expense and yielded no added advantage in our study. Other limitations of this study include artifactual changes induced by glue of the cellophane strip and possibility of false positives and negatives just as in a KOH mount.
| Conclusion|| |
Cellophane strip method can serve as a rapid bedside test and diagnostic tool for diagnosis of superficial fungal infections. It scores over the conventional KOH mount by eliminating the need for a mounting medium and reduces time. Its sensitivity although proved higher than standard KOH mount in our study shall need corroboration with a larger cohort. Phase-contrast microscopy although very sensitive equals the strip method and has no added advantage. The main limitation of the strip method is that species identification may be difficult as stripping occasionally results in broken hyphae and their true morphology may not be retained. However, its purpose as a screening tool and a rapid test is still served. The high sensitivity we observed shall need reconfirmation with larger studies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dabas R, Janney M, Subramaniyan R, Arora S, Lal VS, Donaparthi N. Use of over-the-counter topical medications in dermatophytosis: A cross-sectional, single-center, pilot study from a tertiary care hospital. Indian J Drugs Dermatol 2018;4:13-7. [Full text]
Dogra S, Narang T. Emerging atypical and unusual presentations of dermatophytosis in India. Clin Dermatol Rev 2017;1:12-8. [Full text]
PORTO JA. The use of cellophane tape in the diagnosis of tinea versicolor. J Invest Dermatol 1953;21:229-31.
Raghukumar S, Ravikumar BC. Potassium hydroxide mount with cellophane adhesive tape: a method for direct diagnosis of dermatophyte skin infections. Clin Exp Dermatol 2018;43: 895-8.
Thirumurthy M, Sethuraman G, Srinivas CR. KOH mount for superficial fungal infections using cellophane tape: comparison with standard technique. Indian J Dermatol Venereol Leprol 2002;68:136.
Miranda MF, Silva AJ. New uses of vinyl tape for reliable collection and diagnosis of common superficial mycoses. Skinmed 2003;2:156-8.
Hassab-El-Naby HM, Salem AS, Abdo HM, Hassan HM, Ch MB. Comparative study for the reliability of cellophane tape and standard KOH mount in diagnosis of pityriasis versicolor. Gulf J Dermatol Venerol 2010;17:29-34.
Garg J, Tilak R, Garg A, Prakash P, Gulati AK, Nath G. Rapid detection of dermatophytes from skin and hair. BMC Res Notes 2009;2:60.
Ponka D, Baddar F. Microscopic potassium hydroxide preparation. Can Fam Physician 2014;60:57.
Levitt JO, Levitt BH, Akhavan A, Yanofsky H. The sensitivity and specificity of potassium hydroxide smear and fungal culture relative to clinical assessment in the evaluation of tinea pedis: a pooled analysis. Dermatol Res Pract 2010;2010:764843.
Fonseka S, Lim CS, Bandara UN, Dissanayake M. New contrast stain for the rapid diagnosis of dermatophytosis and pityriasis versicolor. Lab Med 2011;42:649-52.
Avram A, Grupper C. [Tridimensional aspects of fungi studied by interference contrast]. Ann Dermatol Syphiligr (Paris) 1974;101:277-80.
Bergmans AM, van der Ent M, Klaassen A, Böhm N, Andriesse GI, Wintermans RG. Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material. Clin Microbiol Infect 2010;16:704-10.
Haufe U, Haufe F. [Demonstration of fungus elements in unstained histological preparations by phase contrast microscopy]. Dermatol Wochenschr 1958;138:1217-20.
Roberts GD. Detection of fungi in clinical specimens by phase-contrast microscopy. J Clin Microbiol 1975;2:261-5.
Harris JL. Safe, low-distortion tape touch method for fungal slide mounts. J Clin Microbiol 2000;38:4683-4.
Kurade SM, Amladi SA, Miskeen AK. Skin scraping and a potassium hydroxide mount. Indian J Dermatol Venereol Leprol 2006;72:238-41.
] [Full text]
Patro N, Panda M, Jena AK. The menace of superficial dermatophytosis on the quality of life of patients attending referral hospital in eastern india: A cross-sectional observational study. Indian Dermatol Online J 2019;10:262-6.
] [Full text]
Noronha TM, Tophakhane RS, Nadiger S. Clinico-microbiological study of dermatophytosis in a tertiary-care hospital in north karnataka. Indian Dermatol Online J 2016;7:264-71.
] [Full text]
Lorch Dauk KC, Comrov E, Blumer JL, O’Riordan MA, Furman LM. Tinea capitis: predictive value of symptoms and time to cure with griseofulvin treatment. Clin Pediatr (Phila) 2010;49:280-6.
Das S, De A, Saha R, Sharma N, Khemka M, Singh S, et al
. The current indian epidemic of dermatophytosis: A study on causative agents and sensitivity patterns. Indian J Dermatol 2020;65:118-22.
] [Full text]
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]