Objective: To study the frequency and diversity of fungi involved in onychomycosis.
Method: The cross-sectional study was conducted from February 2018 to February 2019 at the Department of Microbiology, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi, and the University of Karachi. Specimens were taken from patients suffering with onychomycosis attending the Dermatology Department of Jinnah Postgraduate Medical Centre and were processed for the isolation of fungi. Specimens were processed for the diagnosis by potassium hydroxide mount for microscopic evaluation and mycological culture to determine the type of fungus involved in onychomycosis. Fungi were isolated using Sabouraud Dextrose Agar with antibiotics cycloheximide and chloramphenicol, and without antibiotics. Plates were observed for the growth periodically until 4th week. Appearance of any colony was studied further for identification on the basis of cultural characteristics and microscopy. Precise identification of Candida species was done.
Results: Of the 230 samples, 85(36.96%) were from males and 145(63.04%) from females. Potassium hydroxide mount showed fungal element in 134(58.26%) specimens, while in 111(48.26%) patients the fungal culture was positive. Nine (3.9%) cases were culture-positive but potassium hydroxide-negative, while among the 134(58.26%) potassium hydroxide-positive cases, 32(23.9%) were culture-negative.
Keywords: Candida, Dermatophytes, Fungal infections of nail, KOH mount, Non-dermatophyte molds, Onychomycosis, Tinea unguium. (JPMA 71: 51; 2021)
Onychomycosis is a fungal disease of nail plate caused by either dermatophytes or non-dermatophytes. When the onychomycosis is due to invasion by dermatophytes, the condition is termed "tinea unguium".1 Among all the nail diseases, proportion of onychomycosis is 50%, and while just 5% of the population suffers from this disease,2 in old age the occurrence is as high as up to 48%.3 It affects the quality of life, as the nail is disfigured and the patient feels discomfort which ultimately influences the patient’s physical and psychological health.1
Determining factors for prevalence of the disease include age, social status, nature of job and environmental conditions.4 With increasing age, the peripheral circulation becomes poor, and chance of diabetes increases. In addition, growth rate of the nail slows down and consequently the exposure of nail to pathogenic fungi is increased. Low immunity at old age is also a factor and there is increased chance of onychomycosis in the elderly if sufficient hygiene is not maintained.5
Among the dermatophytes, the common organisms involved in onychomycosis are Trichophyton (T.) rubrum, T. mentagrophytes and Epidermophyton (E.) floccosum. Non-dermatophytic molds (NDMs) include acremonium, alternaria, aspergillus, botryodiplodia, fusarium, onycochola canadensis, scytalidium dimidiatum, scytalidium hyalinum, geotrichum candidum, cladosporium carrionii, and scopulariopsis brevicaulis. Among yeasts, Candida (C.) albicans is the main pathogen responsible for onychomycosis, while less frequent are C. krusei, C. parapsilosis, C. glabarata, C. guilliermondii, C. tropicalis and blastoschizomyces capitatus.6
In the local setting, the most common pathogen isolated in Lahore, Pakistan, was Candida spp. (46%), followed by dermatophytes (43%) which include T. rubrum, T. violaceum, T. mentagrophytes, T. tonsurans and E. floccosum. NDMs were isolated in 11% of cases, including Fusarium spp., Aspergillus spp., scopulariopsis brevicaulis, Acremonium spp., scytalidium dimidiatum and Alternaria spp. Yeast and dermatophytic infections were not significantly different. Both yeasts and dermatophytes were significantly more prevalent than NDMs.7
In India, NDMs predominated (55%) and included Aspergillus (A.) niger, A. flavus, Mucor spp., alternaria alternata, Rhizopus spp., Penicillium spp., Fusarium spp., Bipolaris spp., A. fumigatus and Curvularia spp. Amongst the dermatophytes, T. verrucossum and T. rubrum were commonly isolated followed by T. schoenleinii T. violaceum, Microsporum spp and T. mentagrophytes. Amongst the yeasts, C. albicans was the most commonly isolated species.8
Dermatophytes get involved in superficial keratinised structures (nails, hair and skin) and do not penetrate deeper tissues. Hyphae of the organism penetrate the stratum corneum, secrete keratinolytic enzyme and thereby enter into the living cells.6
Apparently onychomycosis seems like a cosmetic problem, but in old patients it may get complicated with cellulitis, while in diabetics there may be foot ulcers.9 The aim of treatment is to improve the shape of the nail and to avoid complications. Many drugs cannot penetrate the layer of keratin, and therefore it is very difficult to achieve the target. The choice of treatment depends on the severity of disease, type of organism involved, adverse effects of the drugs and their interactions.10 Commonly, topical or oral antifungal medications are used. Different topical medicines, such as ciclopirox 8% nail lacquer, tavaborole, amorolfine or efinaconazole, are prescribed for onychomycosis.11 Oral medications include terbinafine, itraconazole and fluconazole. These antifungals penetrate the nail bed promptly and persist there for months after discontinuation of therapy.12 Comparatively, terbinafine has fewer side effects than itraconazole. Ketoconazole is not recommended orally due to its side effects.13
The current study was planned to observe the diversity of fungi involved in onychomycosis.
Materials and Methods
The cross-sectional study was conducted at the Department of Microbiology, Basic Medical Sciences Institute (BMSI), Jinnah Postgraduate Medical Centre (JPMC), Karachi, and the University of Karachi from February 2018 to February 2019. Specimens were taken from patients suffering with a clinical diagnosis of onychomycosis attending the Dermatology Department of JMPC and were processed for the isolation of fungi.
After approval from the Pakistan Medical Association (PMA) ethics review board, the sample size was calculated using OpenEpi calculator14 at confidence level 95% and bound of error 5% and taking relevant references from literature.15
All patients with a clinical diagnosis of onychomycosis, irrespective of age and gender, were included, while all lesions other than onychomycosis were excluded.
The patients were also categorised into socioeconomic status (SES) as poor, average or fair, according to the Demographic and Health Survey 2012-13.16
Specimens were processed for diagnosis by potassium hydroxide (KOH) mount for microscopic evaluation and mycological culture to determine the type of fungus involved in onychomycosis. Fungi were isolated by spreading the specimen on Sabouraud Dextrose Agar (SDA) plated with cycloheximide and chloramphenicol antibiotics, and without antibiotics. The plates were observed for the growth periodically for 4 weeks. Appearance of any colony was studied further for identification on the basis of cultural characteristics and microscopy. For observing the microscopic features of the isolates, lactophenol cotton blue stain (LPCB) was used. Precise identification of candida species was done using API 20C AUX system (Bio Merieux, USA).
Of the 230 samples, 85(36.96%) came from males and 145(63.04%) from females (Table 1).
The age of the subjects ranged from 11 to 75 years; and he highest number of patients 77(33.5%) were aged 31-40 years (Table 2).
KOH mount showed fungal element in 134(58.26%) specimens, while in 111(48.26%) patients the fungal culture was positive (Table 3).
Nine (3.9%) cases were culture-positive but KOH-negative, while among the 134(58.26%) KOH-positive cases, 32(23.9%) were culture-negative (Table 4).
Of the 111 fungi isolated, 44(39.64%) were dermatophytes, 26(23.42%) NDMs and 41(36.94%) were yeast belonging to Candida species. Among dermatophytes, T. rubrum 26(59%), T. mentagrophytes 7(16%), T. tonsurans 4(9%) T. soudanense 1(2.27%), T. schoenleinii 1(2.27%), T. verrucosum 1(2.27%) and epidermophyton 4(9%) were isolated. Among the NDMs, there were Fusarium spp. 7(27%), aspergillus 6(23%), Cephalosporium spp. 4(15.38%), Penicillium spp. 4(15.38%), Alternaria spp. 2(7.69%), Cladosporium spp. 2(7.69%) and Chrysosporium spp. 1(3.84%). Among the Candida species, there were 27(65.85%) C. albicans and 14(34.14%) non-albicans, including C. tropicalis 10(71.42%), C. parapsilosis 3(21.42%) and C. glabarata 1(7.14%).
Overall, 81(35.21%) samples came from the dermatology clinic within 6 months of the disease; 70(30.43%) patients consulted dermatologist in 6-12 months of the symptoms 43(18.69%) waited 1-3 years; 17(7.39%) took 3-5 years; and 19(8.26%) took >5 years to consult a dermatologist.
In terms of SES, 181(78.69%) belonged to the poor class, 45(19.56%) to average class, and 4(1.74%) belonged to the fair class. Of the 111 culture-positive specimens, 95(85.58%) came from patients of poor class and 16(14.41%) from individuals of the average class. No samples belonged to a patient of the fair SES.
Overall, 25(10.86%) patients were diabetics. The occurrence of culture-positive specimen among the diabetics was 23(92%). Among the diabetics, 11(47.82%) showed growth of Candida spp., 7(30.43%) dermatophytes and 5(21.73%) NDMs.
Onychomycosis occurs in all ages but children have infection rates lower than the adults.17 Female preponderance was observed in our study. In contrast, other studies1,18 showed a male preponderance. One study19 reported female preponderance of 80% which was higher than the current study.
The reason for the female preponderance in our study can be linked to multiple factors, such as majority of the patients belonging to low SES and unable to afford the treatment. Most of the male patients attended the dermatology clinic after a long duration of the disease when they had some secondary infection.
In the current study the age ranged 11-75 years with a mean of 36.08 years and a median of 35 years. One study1 observed that among 200 patients suffering from the disease, none was <20 years and almost 24% were aged >70years. Age-related incidence of onychomycosis has also been reported5 that could be related to various physiological problems faced by the elderly.
The current study had most of the patients belonging to the poor community. In old age, patients are usually dependent on children/siblings. Treatment in such cases is sought if there is any other complaint along with onychomycosis. Therefore, the data of old patients attending dermatology clinics may not be a true reflection of the number of cases in the community.
In the current study, five patients aged up to 12 years were found to be very poor and were not attending any school, roaming around most of the time bare-footed. A study17 has also reported prevalence of molds, particularly T. rubrum, in such cases.
Dermatophytes were less in number than NDMs in the current study, while yeasts were more frequently isolated amongst non-dermatophytes than molds. One study20 reported dermatophytes in 50%, yeast in 25.55% and mold in 24.45% of its sample.
Since onychomycosis requires long-term antifungal therapy, therefore, it is imperative to have precise and rapid diagnostic methods to avoid side effects of the drugs. While comparing the results of KOH mount with culture positive results, it inferred that more samples could be diagnosed by KOH method (58.26%) than obtaining fungal cultures (48.26%), which was in line with earlier findings.20 Indeed several studies2,15,18 also reported variation in culture positivity with a consistent sensitivity of KOH mount.21 In our study, laboratory evidence of fungal infection was obtained in 143 samples by at least one of these methods. Using this as the denominator, KOH mount had a sensitivity of 93% while mycological culture had 78% sensitivity. A slight variation to this data has been reported in literature.22,23
In the current study, 25(10.86%) patients were diabetics, and 23(92%) of them were culture-positive which accounted for 10% of the total cases. This reflects that although diabetes is not a causative factor for the disease, there is association between the two. Candida species were most frequent organisms (47.83%) isolated from these 23 culture-positive cases, amongst which C. albicans was the most predominant. A study24 found a higher coincidence (22.9%) of diabetes in cases of onychomycosis, and reported a higher occurrence of C. albicans which is in accordance with the current study.
There was a high prevalence of dermatophytes, followed by Candida species whose involvement was much higher in diabetics. Antifungal drugs should be started, but it is necessary to diagnose the infection with some laboratory evidence due to a significant proportion of non-albican Candida species. Their treatment regime is different from the traditional treatment of C. albicans.
Conflict of interest: None.
Source of Funding: None.
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