Fungal foes in the nail unit: Non-dermatophytic molds in onychomycosis - A cross-sectional study

INTRODUCTION

Onychomycosis is a fungal infection of the nail unit caused by dermatophytes, yeasts, and other non-dermatophytic molds (NDMs) and is associated with considerable morbidity. A hot and humid climate, occlusive footwear, trauma, exposure to soil, spas, swimming pools, and immunosuppressive states are responsible for the incidence of onychomycosis.^[1] Recent trends indicate a shift in the spectrum of causative organisms, with an increasing number of cases attributed to NDMs rather than dermatophytes.^[2] Nail involvement by NDMs is relatively rare, as these organisms are often considered contaminants or opportunistic pathogens. Prevalence rates of molds range from 1.45% to 17.6%.^[1] As environmental saprophytes or plant pathogens, molds typically colonize the nail only after keratin damage, since they lack keratolytic activity and rely on unkeratinized intercellular components.^[1]

Many dermatological conditions, such as psoriasis, lichen planus, and onychogryphosis, may be clinically confused with onychomycosis.^[3] The clinical features of NDM-induced onychomycosis are indistinguishable from those caused by dermatophytes. Therefore, it is not possible to identify the causative agent by a mere examination of the diseased nail; rather, a thorough clinicomycological evaluation is necessary before initiating the treatment. Variability in the prevalence and distribution of causative agents may be attributed to geographical and environmental factors, differences in diagnostic criteria, and the inconsistent use of mycological techniques optimized for NDM detection.

MATERIALS AND METHODS

This study was conducted over a 1-year period, from January 2023 to December 2023, among patients with clinically suspected onychomycosis attending the outpatient department of dermatology, venereology, and leprosy at a tertiary care hospital. Patients who had taken either topical or oral antifungal medication within the past 3 months as well as those diagnosed with other dermatological diseases having nail changes, were excluded. After obtaining informed consent from each patient, demographic data and relevant medical history, including risk factors (such as immunosuppressive states and peripheral vascular disease), were recorded. A thorough physical and cutaneous examination, including assessment for concurrent fungal infections (e.g., tinea pedis and tinea manuum), was conducted and documented using a standardized pro forma.

A nail examination was performed to identify the clinical type, associated periungual inflammation, site, and total number of nails involved. In all cases, onychoscopy was performed using a handheld Model- Illuco IDS-1100 at 10x magnification, in both polarized and non-polarized modes, and digital photographs were taken. Under aseptic precautions, nails were cleaned with 70–80% alcohol to remove contaminants, and then nail clippings and scrapings were collected from the involved nail bed. Two nail samples were taken from each patient at the same time. In case of multiple nail involvement, clinically the most affected nail was selected for sampling. Nail clippings and scrapings were subjected to 40% potassium hydroxide (KOH). Overnight incubation with KOH is required for the digestion of hard keratin. For cultures, each specimen was inoculated on two Sabouraud’s dextrose agar (SDA) media (without actidione) and one SDA media with actidione, to inhibit the growth of non-dermatophytes and kept in biochemical oxygen demand incubator at room temperature for 3–4 weeks. The cultures were regularly examined every week. When culture showed NDM, the second sample was used for culture to confirm the same and rule out contamination. Whereas in the case of dermatophytes, a single sample was considered. Species identification was accomplished through examination of colony characteristics, pigment production, slide culture, and lacto phenol blue (LPCB) stain.

The criteria used to conclude NDMs as causing onychomycosis were as follows:

1. Clinical presentation of onychomycosis

2. Positive KOH mount

3. Growth of the same NDM in at least 2 nail samples from the same individual

4. Failure to isolate dermatophytes in culture.

Non-dermatophytes were considered as pathogens only when the growth was seen on both SDA agar media without actidione, and dermatophytes were considered when the growth was seen on all three media - Two SDA (Sabouraud’s agar media without actidione) and one SDA with actidione.^[4]

RESULTS

Among 156 patients enrolled in the study, 76 were male and 80 were female. Among 156 patients, 92 were culture positive, 38 were males, and 54 were females. Among 92 culture-positive patients, the maximum number of patients belonged to the age group of 45-64 years, followed by the older adults. Manual laborers, agricultural workers, and homemakers were the most commonly affected groups, which correlated with the predominant age group observed in the study. Toenails were affected in 106 patients (68%), fingernails in 41 patients (26%), and both toenails and fingernails in 9 patients (6%). Single nail involvement was observed in 23 cases (14.74%).

Nail discoloration was the common finding in 94 patients (60%), followed by subungual hyperkeratosis, onycholysis, nail dystrophy, thickening, and crumbling. The most common clinical type noted was distal lateral subungual onychomycosis (DLSO) [Figure 1], at 54.48%, followed by total dystrophic onychomycosis at 34.61%, mixed onychomycosis [Figure 2] at 7.05%, proximal subungual onychomycosis (PSO) at 2.56% and superficial white onychomycosis (SWO) at 1.28%.

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Figure 1:

Distal lateral subungual onychomycosis of both great toes.

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Figure 2:

Total nail dystrophic changes of finger nails.

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Onychoscopy revealed characteristic ruin appearance, longitudinal striae, spike pattern, and irregular termination with onycholysis [Figures 3a and b]. Direct microscopy with KOH was positive in 68 cases (43.58%) and culture in 92 cases (58.97%) [Tables 1-3]. Among the 92 culture-positive cases, the molds isolated were Aspergillus niger 16 (17.39%), Aspergillus flavus 12 (13%), Fusarium spp. 12 (13%), Aspergillus fumigatus 8 (8.7%), Pencillium spp., Scopulariopsis brevicaulis, Mucor spp., Syncephalastrum racemosum 4 (4.3%), and Trichophyton spp. 12 (7.69%). The causative agents identified are listed in Table 4.

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Figure 3:

Onychoscopy of nails, (a) Irregular termination with onycholysis, (b) Longitudinal striae (black arrows) (polarized, contact, 10x).

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DISCUSSION

Onychomycosis poses a significant cosmetic concern that can adversely affect the patient’s emotional health, social life, and occupational performance. It may also act as a chronic reservoir for fungal infection. Among the 92 mycologically diagnosed cases, the highest incidence of culture-positive onychomycosis was observed in the 45–64 years of age group (n = 36, 39.13%), followed by those above 64 years (n = 25, 27.17%). This distribution was statistically significant when compared to other age groups (χ² = 14.56, P = 0.006). Females constituted a higher proportion (n = 54, 58.70%) in the culture-positive group compared to males (n = 38, 41.30%), with a ratio of 1:1.42 indicating a significant female predominance (χ² = 4.17, P = 0.041). This female predominance is consistent with findings from other studies, such as Kabi et al. (2021),^[5] which reported a 52% females and 48% males, and Attal et al. (2015),^[6] which showed 56.8% females and 43.2% males.

Manual laborers, agricultural workers, and homemakers with constant exposure to soaps and water were the most commonly affected occupational groups. A significant number (44.21%) of patients had at least one associated risk factor. The most common was diabetes (15.38%), followed by barefoot walking (12.82%), trauma (8.97%), ill-fitting footwear (4.48%), and peripheral vascular disease (1.92%). In addition, one of the patients had human immunodeficiency virus (HIV). Toenail involvement accounted for 68%, which is higher than in studies such as Gupta et al.^[7] and Grover.^[8] This can be attributed to the reasons such as the usage of occlusive footwear, increased exposure to trauma, and local environmental conditions. This study depicted DLSO as the most common presentation, which is in accordance with other studies, such as Veer et al.^[9] and Lone et al.^[10]

The comparison between KOH microscopy and culture positivity showed that 48 cases were both KOH and culture positive, while 44 cases were culture positive but KOH negative. KOH microscopy [Figure 4] demonstrated a sensitivity of 52.17% and specificity of 68.75% when compared to fungal culture. The discordance was statistically significant (χ² = 6.72, P = 0.0095), reinforcing the need for culture confirmation. Characteristic colonies were noted in culture slants when followed up for 4 weeks. Among all clinically suspected cases of onychomycosis enrolled in the study, NDMs were isolated in 64 cases (41.02%), yeasts in 16 cases (10.25%), and dermatophytes in 12 cases (7.69%). This is similar to the findings of Kabi et al.,^[5] who reported non-dermatophytes in 24% of cases, yeasts in 66%, and dermatophytes in 12.5%, as well as Attal et al.,^[6] who observed NDMs in 75% of cases, Candida spp. in 8.3%, and dermatophytes in 10%. The prevalence of NDMs was significantly higher in this study compared to dermatophytes and yeasts (χ² = 52.17, P < 0.0001). This may be attributed to regional environmental conditions such as hot and humid weather, the use of confirmatory diagnostic methods to rule out contamination, and the nature of clinical samples. In addition, the accuracy of KOH examination, which requires expertise, may also influence detection rates. Thus, meticulous sample collection, confirmatory cultures, and skilled microscopic evaluation are crucial for the accurate identification of onychomycosis, especially in cases involving NDM. A. niger (17.39%) was found to be the most common NDM, and Candida albicans (17.39%), the most common yeast contributing to the disease.

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Figure 4:

Septate hyphae on potassium hydroxide mount (black arrows).

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A. niger was identified by black, granular growth and globose vesicles with black conidia in lacto phenol cotton blue (LPCB) mount [Figure 5]. C. albicans demonstrated budding yeast cells with pseudo-hyphae and was confirmed by the germ tube test. Candidal onychomycosis is often considered a marker of immunosuppression.^[11] In this study, Candida species showed association with PSO, observed in three patients - two were diabetic and one was HIV positive. C. albicans showed a significant association with the proximal subungual type of onychomycosis (P = 0.006), potentially indicating underlying immunosuppression. Notably, PSO itself is strongly correlated with HIV infection, further reinforcing its role as an indicator of underlying immunodeficiency. Species identification through LPCB mount revealed specific features of each fungus. In the case of S. racemosum, finger-like merosporangia were observed [Figure 6], whereas in Penicillium species, brush-like conidiophores [Figure 7] and in Aspergillus flavus, biseriate vesicles [Figure 8] were observed. Club-shaped microconidia were seen in Trichophyton tonsurans. SWO (2 cases) was found to be caused by Trichophyton mentagrophytes. Although periungual inflammation is characteristic of NDM-induced onychomycosis, in this study, only a few (7) of the patients presented with periungual inflammation. Managing NDM-induced onychomycosis is particularly challenging as it is recalcitrant to conventional treatments, often necessitating extended antifungal therapy and species-specific protocols. Systemic antifungals alone are effective in cases of Aspergillus, whereas Fusarium and Scopulariopsis additionally require interventional procedures such as surgical and chemical nail avulsion.^[12] Chemical nail avulsion with 40% urea is known to be effective, followed by 8% ciclopirox nail lacquer administration. Untreated NDM onychomycosis could be an important and dangerous portal of entry for deep-seated and disseminated mycosis, thus highlighting the importance of correct diagnosis and rapid treatment of non-dermatophytic onychomycosis.

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Figure 5:

Black granular growth and Globose vesicles, black conidia suggestive of Aspergillus niger.

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Figure 6:

Syncephalastrum racemosum identified by finger-like merosporangia on lacto phenol blue mount.

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Figure 7:

Brush-like conidiophores (black arrow) suggestive of Penicillium species.

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Figure 8:

Biseriate vesicles on lacto phenol blue, suggestive of Aspergillus flavus.

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CONCLUSION

Onychomycosis, though highly prevalent, is often underrecognized and dismissed as a cosmetic concern rather than a clinically significant infection. This misconception contributes to the delay in diagnosis and treatment, potentially leading to chronicity and complications. A thorough understanding of its epidemiology and etiological spectrum is crucial for timely intervention and mitigating its broader impact on individual well-being and public health. With the rising incidence of NDM-induced onychomycosis and the limited existing research, this study highlights the importance of culture-based diagnosis in guiding effective management.