Clinico-mycological study and comparison of efficacy of three different techniques of sample collection from skin lesions for potassium hydroxide mount preparation in dermatophytoses

INTRODUCTION

Dermatophytoses refers to superficial fungal infection of keratinized tissues caused by dermatophytes.¹ The dermatophytes belong to 3 genera i.e. Trichophyton, Microsporum and Epidermophyton.²

The prevalence of superficial mycotic infections varies from 20-25%, making these one of the most frequent forms of infection worldwide. It is common in tropics and may present as epidemic proportions in areas with high rate of humidity, over population and poor hygienic conditions.³ Worldwide the most common species causing dermatophytoses is Trichophyton rubrum.^3-7

Direct microscopic examination in KOH solution is a reliable rapid test for demonstrating dermatophytes for Out Patient Department (OPD) procedure. Various studies are done on alternative methods for collecting sample for KOH preparation and fungal culture, but sensitivity and specificity of these methods have been lacking.^8-12

Thus this study was taken up to define current clinical pattern and the ethological prevalence in our locality, to compare the efficacy of three different sampling techniques from skin lesions and to correlate KOH mount results with the culture results.

METHODS

Clinically suspected cases for dermatophytoses attending Dermatology Out Patient Department of a hospital in Mysore over a period of two year (September 2015 to October 2017) were included.

Patient who are on topical and/or systemic antifungal agents in the past 6 weeks or who were on topical steroids and / or systemic steroid in the past 8 weeks or patient on immunosuppressive agents, other than steroids in the past 6 months were excluded

After taking consent, detailed clinical history and through clinical examination was conducted, specimens were collected by following methods,

3 methods of sample collection from skin lesions:

• • Standard scraping method: The active border was scraped with a glass slide.
• • The cellophane tape method: cellophane tape was stuck on the active border & it was peeled off, which was then stuck on a glass slide.
• • Surface skin biopsy (SSB): was done by taking a drop of instant adhesive (fevikwik) on a slide and then the slide is placed on lesion, after few seconds the slide is removed.

Infected hairs from the lesion were plucked and were put on slide. Nails were thoroughly cleaned with 70% alcohol or spirit, and then clipped using scissors or scraping of the nail was obtained from the nail bed and underside (ventral side) of the nail plate from the advancing edge, most proximal to the cuticle.

The above collected samples were subjected to direct microscope examination by KOH mount and culture study. On microscopic examination, long branching septate hyphae or arthrospores were looked in skin and nail scraping. Fungi in infected hair specimens were seen as hyphae or chains or arthrospores and the nature of invasion of hair and spore formation was studied.

Culture examination: For primary isolations, Sabouraud’s dextrose agar (SDA) slope was used as selective media. One set was incubated at room temperature for one month. Growth was usually seen by 2 weeks. Growth was identified based on macroscopic appearance and microscopic features in lactophenol cotton blue mount. If no growth was observed at the end of 4^th week, the culture was labelled as negative.

Statistical method

All the statistical calculations were done through SPSS for windows (v 16.0) - The Chi-Square Test procedure tabulates was done.

RESULTS

In this study of 210 patients, dermatophytoses was more common in males (111) than in females (99), (M: F= 1.2:1). Maximum number of cases were encountered in the age group of <10 years of age (74 cases) (Table 1). The most common clinical pattern of dermatophytic infection observed was tinea corporis in 83 cases (40.5%) followed by tinea capitis 74 cases (35.2%), (Table 1).

Overall positivity by culture was in 188 patients and direct microscopy was in 170 cases (Table 2). Fungal element from the skin samples was detected by three methods (standard-97%, SSB-98%, Cellophane tape method-96.2%), Scraping method, cellophane tape method, SSB method of sample collection was found to be almost equally effective (Table 3).

Overall positivity by culture was in 188 patients and direct microscopy was in 170 cases (Table 2). Fungal element from the skin samples was detected by three methods (standard-97%, SSB-98%, Cellophane tape method-96.2%), Scraping method, cellophane tape method, SSB method of sample collection was found to be almost equally effective (Table 3).

Out of 74 hair clipping samples, 53 were positive for fungal element by direct microscopy and 71 were positive by culture. Out of 31 samples of nail clipping 16 (51.6%) were positive for fungal element by direct microscopy and 14 (45%) were positive by culture.

All the 3 genera of dermatophytes i.e. Trichophyton, Epidermophyton and Microsporum have been isolated as the causative agent in this study (Table 4, Figure 4 to 6). Over all, the Trichophyton genera dominated in the fungal isolates (92.35%, 174/188) followed by Microsporum 11cases (5.8%) and Epidermophyton 3cases (1.5%). Trichophyton rubrum (71) was the most common species isolated followed by Trichophyton violaceum in 65 cases.

DISCUSSION

There has been a changing pattern of the dermatophytes isolates in India in the past 5–7 years, with an alarming rise in overall number of case and increasing proportion of atypical clinical types and unusual manifestations, due to a complex interplay of host, agent, environmental or pharmacologic factors.¹³

In the present study of 210 cases, dermatophytoses was more common in male; similar findings have been reported by other workers.^2-4 The higher incidence in male could be due to greater physical activity and increased sweating.

While maximum number of cases were encountered in the age group of <10 yrs (35.2%) followed by 31-40 years (15.2%). Although majority of studies done observed higher incidence in the 3^rd decade, a study done at Calicut observed higher incidence in the 2^nd decade.¹ These differences may be due to regional variation in the prevalence of various dermatophyte species and affinity of certain species for particular anatomical sites. The higher frequency in males may be because they frequently visit saloons for their hair dressing compared with females, where they use contaminated razors and hence spreading infection from one child to another. The lower frequency in females could be due to the custom of regular application of vegetable oil over the scalp, which has fungistatic properties.¹⁴

Mixed clinical types were seen among the enrolled patients. Tinea corporis (85) was the highest (40.5%), followed by 74 cases of tinea capitis (35.2%). This is in accordance with the findings of Bindu et al and other workers.¹ In a study done at Kolkata tinea capitis was the commonest dermatophytic infection in children.⁵ In a study by Sardari et al it has been reported that tinea cruris was the commonest clinical type.⁶ In another study in North East India, Tinea pedis (29.2%) was the most common dermatophytoses followed by tinea cruris (26.2%).⁷

The laboratory diagnosis of dermatophytoses relies first on the KOH mount on direct microscopic observation of the pathogen in samples from the affected area and identification of the fungal agents can be done based on the colony morphology by the macroscopic characteristics: upper and bottom side of colonies as well as pigmentation and slide culture growth forms by lactophenol cotton blue (LPCB) tease mounts for microscopic characteristics: formations of macro and microconidia, respectively.

For the laboratory to provide the optimum performance, the quantity and the quality of the material examined are critical. Because for most samples, both culture and microscopic examination will be performed, it is essential that as much clinical material as possible is submitted to the laboratory to allow both diagnostic methods to be carried.¹⁵

In the present study out of 210 clinical cases it was possible to demonstrate fungi on direct microscopy with potassium hydroxide in 81%. Fungal element from skin samples was detected by three methods (standard-97%, SSB-98%, Cellophane tape method-96.2%), (Figure 1-3). However, cellophane method and SSB method were noted to be much better in both collection and transport of samples, over the standard method, especially in lesion with minimal scales.

A few studies in the past have demonstrated the usefulness of alternative methods of sample collection for KOH preparation and fungal culture, but data on sensitivity and specificity of these methods is lacking.

Overall positivity by culture was 89%. In comparison, a study done at Thane showed culture positivity of 71%, but a study done at Aurangabad showed very low rate of culture positivity (22.8%).^4,11

Correlating clinical and mycological data it shows that in T. rubrum (Figure 4) was isolated in most cases followed by T. violaceum. T. rubrum was the common isolate in cases of tinea cruris. Out of 14 isolates from tinea unguium, 8 were T. rubrum. And out 74 cases of tinea capitis most common isolate was T. violaceum (55.4%) followed by T. rubrum (17.6%). It was therefore concluded that T. rubrum was the main organism isolated from the infections of glabrous skin and groins followed by tinea capitis, which was again supported by other studies from India and other countries.^8,9,12 Overall, the Trichophyton species dominated with 90% of the isolates followed by Epidermophyton and Microsporum. The reports from other parts India showed T. rubrum as the commonest species, 87.12% in Bombay, 80% in Punjab, 73.27% in Baroda, 66.2% in Calicut.^1,3,11 This variance is possibly due to the different geographical regions in these studies which have different prevailing dermatophyte flora.

CONCLUSION

The present study has given us a clear insight regarding the mycological pattern of dermatophytoses in this region, tinea corporis as the commonest clinical pattern followed by tinea capitis. Trichophyton rubrum was commonest fungal isolate.

The fact that laboratory diagnosis of dermatophytoses has been generally a neglected area in clinical practice. The present study shows KOH mount is a useful rapid diagnostic method. Cellophane tape and SSB are equally effective alternate methods to standard scraping method of specimen collection for KOH mount with added advantages of lesions with minimal scaling can be easily collected and transported, scales which can lost and lead to air born infection is prevented.

Culture is not essential for diagnosis but it is useful in identifying the species. The collaborative effort involving dermatologists, microbiologists, and public health professionals is required to address this emerging public health problem.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the institutional ethics committee

REFERENCES

1. 1. Bindu V, Pavithran K. Clinico-Mycological study of dermatophytosis in Calicut. Indian J Dermat Venereol Leprol. 2002;68:259-61.
2. 2. Sentamilselvi G, Kamalam A, Ajithadas K, Janaki C, Thambiah AS. Scenario of chronic dermatophytosis. An Indian study Mycopathologica. 1997;140:129-35.
3. 3. Mehta JP, Deodhar KP. A study of dermatophytes in Bombay. Indian J Pathol Microbiol. 1977;20:23.
4. 4. Belukar DD, Barmai RN, Karthikeyan S, Vadhavkar RS. A mycological study of dermatophytosis in Thane. Bombay Hospital J. 2004;46:2.
5. 5. Barbhuiya JN, Das SK, Ghosh A, Dey SK, Lahiri A. Clinico mycological study of superficial fungal infection in children in an urban clinic in Kolkota. Indian J Dermatol. 2002;47(4):221-3.
6. 6. Sardari L, Sambhashiva RR, Dandapani R. Clinico mycological study of dermatophytes in a coastal area. IJDVL. 1983;49:2:71-5.
7. 7. Grover SCW, Roy PCL. Clinic mycological profile of superficial mycosis in a hospital in North East India, MJAFI. 2003;59:114-6.
8. 8. Sodera OJ, Elewski EB. Fungal diseases. In: Bolognia LJ, Jorizzo LJ, Rapini RP editors. Dermatology. 2nd end. Okhla: Elsevier; 2009: 1135-1164.
9. 9. Kanwar AJ, Mamta, Chander J. Superficial fungal infections in: Valia GR editor. IADVL text book and Atlas of Dermatology 3rd ed. Mumbai:Bhalani Publishing House; 2007: 215-258.
10. 10. Singh S, Beena PM. Profile of dermatophyte infections in Baroda. Indian J Dermatol Venereal Lepro. 2003;69:281-3.
11. 11. Rani V. Study of dermatophytoses in Punjabi population. Ind J Pathol Microbiol. 1983;26(4):243-7.
12. 12. Alsogair SM, Moawad MK, Al-Humaidan YM. Fungal infection as cause of skin disease in the eastern province of Saudi Arabia prevailing fungi and pattern of infection. Mycosis. 1991;34:337.
13. 13. Dogra S, Narang T. Emerging atypical and unusual presentations of dermatophytosis in India. Clin Dermatol Rev. 2017;1:12-8.
14. 14. Karmakar S, Kalla G, Joshi KR, Karmakar S. Dermatophytoses in a desert district of Western Rajasthan. Indian J Dermatol Venereol Leprol. 1995;61:280-3.
15. 15. Rudramurthy SM, Shaw D. Overview and update on the laboratory diagnosis of dermatophytosis. Clin Dermatol Rev. 2017;1:3-11.