Clinico-mycological study of superficial mycoses and correlation with anti-fungal susceptibility among the Candida isolates in a teaching institution of Western India

ABSTRACT

Introduction:

Most common superficial fungal infection is caused by dermatophytes, which infects skin, hair, and nail in all age groups. Candida spp. is also a known causative agent for superficial mycoses. The prevalence also depends on different demographical variations.

Aim and Objectives:

The present study was conducted to isolate and identify the fungal species by phenotypic and culture methods from patients suffering from superficial fungal infections and establish in vitro anti-fungal susceptibility testing on candida isolates.

Materials and Methods:

The skin scrapings, hair, nail clippings were collected from 330 patients, and KOH mount and fungal culture were performed. The culture-positive samples were subjected to lactophenol cotton blue (LPCB), slide culture and CHROM agar for species-level identification. Anti-fungal susceptibility testing was done on the candidaisolates.

Results:

Out of 330 specimens collected, 161 (49%) were skin scrapings, 42 (13%) were hair samples, and 127 (30%) were nail clippings. Most commonly isolated was Candida spp. in nail and Trichophyton mentagrophyte in skin. Anti-fungal susceptibility was performed on the candida isolates, and caspofungin was found to be the most susceptible anti-fungal agent.

Conclusion:

All dermatophytes, non-dermatophyte molds (NDMs), and yeasts can cause infections of the skin, hair, and nails. The present study has highlighted the growing importance of NDMs as a cause of dermatomycoses. Among the Candida species, the most susceptible drug was caspofungin. The use of fluconazole in the prophylaxis of fungal infections in patients may be the cause of the rising fluconazole resistance rate among them and, hence, should be used cautiously.

Introduction

Superficial fungal infections affect the outer layers of the skin, the nails, and hair. The main groups of fungi causing superficial fungal infections are: dermatophytes (tinea or ringworm), yeasts, e.g., Candida (moniliasis) including non-albicans Candida species; Malassezia (pityriasis versicolor); Piedra; and molds.

Generalized cutaneous candidiasis is an unusual form of cutaneous candidiasis that manifests as a diffuse eruption over the trunk, thorax, and extremities. The patient presents with a history of widespread pruritus in genitofemoral folds, anal region, axillae, hands, and feet showing greater severity.[1] C. albicans coexists in equilibrium with other bacteria of the microbiota in the majority of immunocompetent people. 80–90% of infections are caused by Candida albicans.[1,2] However, several studies reported that there has been a progressive shift from a predominance of C. albicans to non-albicans Candida species (NAC) such as C. tropicalis, C. glabrata, and C. krusei.[2]

Dermatophytosis is named as per the involved area of the body, e.g., tinea corporis, tinea capitis, tinea pedis, and tinea unguium. Tinea capitis is the most common (about 25%) dermatophyte infection worldwide and is mainly caused by Trichophyton rubrum.[3] Dermatomycoses and dermatophytosis are two categories of cutaneous mycoses. Agents from the genera Epidermophyton spp., Microsporum spp., Trichophyton spp. are responsible for causing dermatophytosis. However, dermatomycoses are cutaneous infections caused by other fungi, most commonly by Candida spp.

The prevalence and etiological agents of dermatophytic infection also differ according to geographical location, socioeconomic status, climatic conditions, and the lifestyle of the population. According to World Health Organization (WHO), the prevalence rate of superficial mycotic infection worldwide is 20-25%.[4,5] However, it differs in tropical, sub-tropical, and winter zone of climatic change. The same diversity can be found in different regions and states of India.

Hence, keeping this as a front-runner, the present study was conducted in Thane district of Maharashtra state, located to the north of Mumbai at an altitude of 8-11 meters above sea level. The relative humidity varies between 60% and 70% for most of the year due to heavy rainfall. Therefore, the setting was favorable for the emergence of superficial mycoses. The present study was conducted to isolate and identify the fungal strains by phenotypic and culture methods from patients suffering from superficial fungal infections, to study these fungal isolates in relation to the site of infection, and in vitro anti-fungal susceptibility testing on candida isolates.

Materials and Methods

The study was a prospective type of study including 330 patients with clinically suspected superficial fungal infections, who attended the outpatient department of dermatology, Rajiv Gandhi Medical College and CSMH, during the period July 2021 to June 2022. The samples were collected from the patients who were referred by the dermatologist for microbiological evaluation of superficial fungal infection and given their consent. Ethical clearance was obtained from the Institutional Ethical Committee prior to the start of the study.

Complete details of the patient and clinical history were recorded including age, gender of the patient, occupation, site and type of the lesion, any contact with pet animal or household contacts, and any immunosuppressive illness or therapy and treatment taken earlier. The patients who took topical or systemic antifungal therapy recently, i.e., within a duration of 7 days, were excluded from the study.[6]

The specimen was collected after cleaning the area with normal saline and allowed to dry. The specimen from central scarring area was collected by scrapping or scotch tape method for KOH. The culture specimens were collected by skin scraping methods using a sterile blunt blade. The potassium-hydroxide (KOH) wet preparation (10% KOH for skin and hair; 20% KOH for nails) was performed to check the presence of fungal elements and scanned under a microscope (10x and 40x). Irrespective of the demonstration of fungal elements on KOH, the specimens were inoculated into two sets of test tubes of Sabouraud’s dextrose agar (SDA) (total of 4 tubes), one set without antibiotic and the second set with SDA with 0.05% chloramphenicol and 0.5% cycloheximide. This was incubated at 37°C and 25°C for up to 4 weeks. If no growth was found after 4 weeks, it was reported as negative for fungi. The growth-positive fungal isolates were identified based on colony morphology, pigmentation, growth rate, microscopy (lactophenol cotton blue mount), slide culture, and urease hydrolysis test.[6]

For better sporulation and conidiasis, the culture tubes containing the mold group of fungal growth were inoculated into potato dextrose agar. The molds are also inoculated in dermatophyte test medium. The lactophenol cotton blue stain was used for phenotypic identification. Dermatophytes were identified based on the characteristics of mycelium, macroconidia, and microconidia. Slide culture was also used to identify the fungi. Urease test was performed to distinguish between the several Trichophyton species. The yeast colonies were inoculated for slide culture and CHROM agar for speciation of Candida spp.[6] [Figure 1]

Figure 1.

Showing Candida species growth on CHROM agar

Antifungal drug susceptibility testing for non-dermatophyte isolates of candida species was done by disk diffusion method using the following drugs: fluconazole (25 mcg), voriconazole (1 mcg), and caspofungin (5 mcg).[7]

The data were entered and statistically analyzed using Microsoft Excel (version 2211). A percentage distribution was calculated for discrete data. Suitable statistical tests (chi-square test) were applied to determine statistical significance at P < 0.05.

Results

The study population of 330 patients with clinically suspected superficial fungal infections was subjected to mycological examination. Out of 330 patients, 143 (43%) were females and 187 (57%) were males, out of which the maximum cases of infection were observed between 31-40 years of age (22%). Out of 330 patients, 174 (53%) cases were unemployed (i.e., housewives, babies, students, and senior citizens), 68 (21%) from elementary occupations (i.e., watchmen, vendors, cleaning staff, farmers, washermen, etc.), 32 (10%) from technicians and associate professionals, and 29 (9%) from shop and market sales workers [Table 1]. Most of the patients infected in the study were in the 31–40 years age group [Table 2].

Table 1.

Occupation of the study group

Occupation	No. of study group
Employed	156 (47%)
Unemployed	174 (53%)
Total (n)	330

Table 2.

Age- and gender-wise distribution of clinically diagnosed fungal infections (n=330)

Isolates	Age distribution (years)				Gender
	<20 year	21 to 40	41 to 60	61 to 80	M	%	F	%
Tinea corporis	6	39	34	10	49	55%	40	45%
Tinea cruris	0	4	1	3	5	63%	3	38%
Tinea pedis	0	1	1	1	2	67%	1	33%
Tinea mannum	0	2	1	0	1	33%	2	67%
Tinea capitis	29	7	2	1	23	59%	16	41%
Tinea unguim	0	0	0	1	1	100%	0	0%
Tinea icognito	1	1	1	0	3	100%	0	0%
Seborrhoeic dermatitis	1	0	0	1	1	50%	1	50%
Pityriasis	4	2	0	0	5	83%	1	17%
Onychomycosis	10	41	51	14	70	60%	46	40%
Mixed	7	30	19	4	27	45%	33	55%
Grand Total (n=330)	58	127	110	35	187		143

Out of the total 330 specimens collected, 174 (53%) cases were from Thane rural areas, followed by Thane urban areas with 101 (31%) and 3 (1%) from outside Maharashtra. Among the collected samples (n = 330), 161 (49%) were skin scrapings, 42 (13%) were hair samples, and 127 (30%) were nail clippings. All were screened for fungal elements on the KOH mount. Irrespective of KOH positive or negative results, these samples were inoculated for culture. A maximum number of specimens were skin scrapings. Out of 330 specimens, the fungal elements were observed in 131 (40%) samples, and culture positivity was 96 (29%), as shown in Table 3.

Table 3.

Comparison of KOH mount with fungal culture (n=330)

KOH Findings (n=330)	Culture positive (n=96) (29%)	Culture negative (n=234) (71%)
KOH positive (n=131) (40%)	78 (24%)	53 (16%)
KOH negative (n=199) (60%)	18 (5%)	181 (55%)

Among the 96 culture-positive isolates, 26 (8%) were dermatophytes and 38 (11%) were non-dermatophytes. Table 3 shows that among the 330 samples collected, 199 (60%) were negative for fungal elements in microscopy and 234 (71%) were not grown in culture.

Among the culture positives, 26 were dermatophytes and 38 were non-dermatophytes, which includes 4 isolates of Candida albicans and 30 isolates of non-albicans Candida. Other than Candida species, other 32 isolates of non-dermatophytic fungi were grown in this study and were considered contaminants [Tables 4 and 5].

Table 4.

Incidence of various species isolated in samples (n=330)

Species	Number of fungal isolates (n)	%
(A) Dermatophytes (n=26, 29%)
1. T. mentagrophytes	12	13%
2. T. rubrum	3	3%
3. T. violaceum	1	1%
4. T. interdigitalis	10	11%
(B) Non-dermatophytes (n=4, 4%)
5. Malessezia spp.	4	4%
(B. i.) Yeast and yeast like (n=4, 4%)
6. C. albicans	4	4%
(B. ii.) Non albicans candida (n=30, 34%)
7. C. glabrata	12	13%
8. C. gulliermondi	2	2%
9. C. krusei	3	3%
10. C. tropicalis	11	11%
11. Geotrichum spp.	1	1%
12. Trichosporon spp.	1	1%
(C) Contaminants (n=32, 33%)
Total growth	96	100%
Total no growth	234

Table 5.

Distribution of culture-positive isolates as per the site of collection (n=330)

Name of the species	Skin	Hair	Nail	Total number	Total percentage
Trichophyton mentagrophytes	9	2	1	12	3.6%
Trichophyton rubrum	-	1	2	3	0.9%
Trichophyton violaceum	-	-	1	1	0.3%
Trichophyton interdigitalis	9	-	1	10	3%
Trichosporon spp.	-	-	1	1	0.3%
Malessezia spp.	3	-	1	4	1.2%
Geotrichum spp.	-	-	1	1	0.3%
Candida spp.	4	-	28	32	9.6%
Contaminants	11	2	19	32	9.6%
No growth	125	36	73	234	70.9%

In the total of 26 dermatophytes, 12 were Trichophyton mentagrophytes, followed by Trichophyton interdigitalis (10), Trichophyton rubrum (3), and Trichophyton violaceum (1).

Antifungal drug susceptibility testing for candida isolates was performed by the disk diffusion method. Out of these, 33 isolates were of yeast like species. Antifungal susceptibility testing for Candida species showed resistance to fluconazole in C. krusei, C. glabrata, and C. albicans, whereas all isolates of Candida species were sensitive to caspofungin [Figures 2 and 3].

Figure 2.

Antifungal susceptibility pattern of candida species

Figure 3.

Antifungal resistance pattern of Candida species

Discussion

In the present study, out of 330 samples processed 57% were from males and 43% were from females. Men may be more susceptible due to prolonged outdoor exercise and increased sweating, which creates an environment favorable to the growth of certain fungal infections. Male hormones may also have role in increased sweating.[8,9] Their profession, constant contact with packed crowds of people, and poor personal hygiene could be the reason for the same. However, the lack of reporting of female patients to hospitals due to widespread social stigma could also be resulted in lower prevalence reported among females.

Although all age groups can be affected, most patients in the study were in the 31 to 40 years age group. Chaudhary et al.[10] and Poluri et al.[11] observed that dermatophyte infections were more common in the age group of 21-40 years in the North and South India, respectively. These findings of prevalence of fungal infection in most common age group are similar in different regions of India.

In this study, 18 (5%) cases were negative for fungal elements in KOH mount but culture positive. Khadka S et al.[12] study also showed that 48 (24%) were KOH negative with culture positive. For clinical diagnosis, direct microscopy of KOH mounts is necessary, but microbiological confirmation of fungal speciation requires fungal culture. Lack of standard methods for fungus identification and the use of antifungal agents prior to specimen collection were likely to be responsible for a major portion of the cases where fungus growth failed.

In the present study, 53 (16%) were KOH positive, but culture negative. According to Khadka S et al.[12] study 26 (13%) were KOH positive with culture negative. These above findings could be explained that direct microscopy is an easy and reliable test, an false-negative results in the current study may have been occurred because insufficient or inappropriate specimen was collected for analysis. Cotton wool fibers, synthetic material, starch, lipid droplets, vegetable waste, and mosaic fungus (network of material including cholesterol crystals) could be a cause of false-positive results in KOH wet mount tests.[10]

In the present study, there was no past history of infection by the same isolates in 50% of cases, and 31% of the patients had contact history with an infected person in their house. Another study by Ganesan et al.[13] showed that 48% of cases did not suffer from the same infection previously, and 22% of cases have a family history of the illness.

In the present study, onychomycosis was the commonest lesion (35%) followed by Tinea corporis 27% and Tinea capitis 12%. This observation correlated with Kaur et al.[14] study showing the high frequency of onychomycosis compared to other forms of ringworm. Miklić P et al.[15] in Croatia reported that the most common clinical forms of dermatomycosis were onychomycosis followed by tinea corporis. The high frequency of onychomycosis among the cases can be explained by long walks, typically wearing closed rubber shoes while daily travelling, working in fields or offices for long hours. The combination of occlusion, perspiration, and occupational damage favors the growth of fungus.

Out of the total fungal culture-positive cases, 40.6% were dermatophytes and 59.3% non-dermatophyte molds. Similar to a study done by Kumaran et al.[16] where 49% culture-positive cases were dermatophytes. Hazarika et al.[17] observed 43.54% of dermatophytes and 14.51% non-dermatophyte molds of the fungal culture-positive cases. These findings were different from the present study. The different geographical locations could be one of the reasons for such findings in different studies.

In the present study, T. mentagrophytes (13%) was the most common dermatophyte isolate followed by T. interdigitalis (11%). In the study by Kaur et al.[18], Trichophyton rubrum (4.6%) was the most common isolate. Other studies conducted by Noronha et al.[5] and Abu-Elteen[19] isolated T. mentagrophytes as the most frequent dermatophyte isolate which is similar findings as the present study.

In the present study, the total percentage for the isolates of yeast and yeast-like was 38%. In others study by Gupta et al.[20] also showed that yeasts were the most common isolates. Out of 330 isolates, Non-albicans Candida species predominated with 34% of isolates, whereas 4% of isolates were Candida albicans. Similar finding was reported by another study where non-albicans Candida species predominated over the Candida albicans.[17] In the present study, Candida glabrata (13%) was the major species isolated among the non-albicans Candida species. C. tropicalis was commonly isolated species in other studies.[21,22] These differences may be due to geographical variation in the prevalence of various species and affinity of certain species for particular anatomical sites.

In the current study, the susceptibility pattern of C. albicans and non-albicans Candida species was observed. All the isolates of Candida albicans showed 100% susceptibility to Caspofungin. Amongst the isolates of non-albicans Candida species, 100% isolates were susceptible to caspofungin.

The isolates of C. albicans showed 75% susceptibility to Voriconazole and 50% susceptibility to Fluconazole. The study by Shaik N et al.[23] reported 76.6% susceptibility to Voriconazole and 63.3% susceptibility to Fluconazole. Another study by Edula AR[22] isolated 95.83% Candida albicans which were susceptible to Fluconazole. Among non-albicans Candida spp., maximum resistance was shown by C. krusei. In the present study, Voriconazole seemed to be superior to Fluconazole with a better susceptibility in the Fluconazole-resistant strains. The isolates of Candida species (n = 34) showed 88.2% susceptibility to Voriconazole and 58.8% susceptibility to Fluconazole. 75% C. albicans isolates were susceptible to Voriconazole, and 50% were susceptible to Fluconazole. Among the non-albicans Candida spp., 90% were susceptible to Voriconazole and 60% were susceptible to Fluconazole. However, the P value was insignificant. Edula AR study also demonstrated better voriconazole susceptibility pattern among Fluconazole-resistant Candida species.[22]

Conclusion

The finding of our study is that all dermatophytes, non-dermatophyte molds (NDMs), and yeasts can cause infections of the skin, hair, and nails. The present study has highlighted the growing importance of NDMs as a cause of dermatomycoses, taking the place of the previously dominant dermatophytes.

Fluconazole use in the prophylaxis of fungal infections in patients may be the cause of the rising fluconazole resistance rate among Candida species. Therefore, early detection and identification of the causal agent is the best strategy for the effective management of a fungal infection, allowing for the earliest possible initiation of suitable treatment in patients.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.