Introduction

The onychomycosis (from the Greek onychos—nail and mycosis—fungal infection) is produced by three types of microorganisms: dermatophytes, yeasts, and non-dermatophyte molds (NDM). According to the Society for Human and Animal Mycology, the term onychomycosis is exclusive for the infections caused by dermatophytes, while the ones caused by yeasts are known as onyxis; if it is Candida, they are called nail candidiasis, and those caused by an opportunistic mold are known as nail mycosis [1•, 2••].

Onychomycosis is one of the main nail infections at a global level; it represents up to 50% of all onychopathies, and of this, 33% is related to diabetic patients and 30% to HIV-positive patients [3••, 4]. In recent years, an increase in the incidence of this disease has been reported due to various factors, such as the trimmings of nails, use of artificial nails, advanced age, peripheral vascular disease, diabetes, autoimmune diseases, and swimming regularly [5••].

Currently, the frequency of onychomycosis by NDM has increased appearing in 1 to 45.8% of the population, depending on the geographical region, and exceeding 20% in population over 60 years [2••, 6, 7, 8•, 9, 10, 11•, 12, 13••, 14•]. Among the main NDM, there is Scopulariopsis brevicaulis, Aspergillus spp., Fusarium spp., Neoscytalidium spp., Acremonium spp., Paecilomyces spp., Penicillium spp., Rhizopus spp., Alternaria alternata, Tritirachium oryzae, Ulocladium spp., Trichoderma spp., and Nattrassia mangifereae. In recent years, there has been an increase in the diagnosis of onychomycosis by NDM in dermatology services, where Aspergillus species have been considered as emerging and represent between 34 and 60% of diagnoses [1•, 3••, 12, 15•, 16•, 17•].

The species of genus Aspergillus are saprobes and are found in the environment (soil, air, water, and vegetation). This group of fungi is normally considered as a contaminant as it does not produce keratinases and it depend on other conditions to cause nail onychomycosis, such as a previous nail trauma, anatomical alterations, bacterial infections, circulatory alterations, and immunosuppression; however, it has also been found causing damage in immunocompetent patients [1•, 3••, 12, 15•, 16•, 17•].

The first case of onychomycosis due to Aspergillus was reported by Émile-Weil and Gaudin in 1919. Subsequently, Sartory (1920) and Ota (1923) reported other cases. However, Thom and Church (1926) strongly questioned the relationship between onychomycosis and Aspergillus because it was considered a contaminant fungus; in addition, the clinical manifestations were similar to those caused by yeasts, so there was a lack of reports in the literature. Despite this controversy, cases such as those of Sartory et al. (1930) and Smith (1934) continued to be reported, and the reports remained controversial as the genus Aspergillus was still considered as a simple pollutant. From 1935 to 1941, no case was reported [18]. In this paper, we present a review on the behavior (epidemiology, clinical characteristics, diagnostic methods, treatment, and identified species) of the onychomycosis produced by Aspergillus spp. as primary agents.

Epidemiology

There are remarkable geographical differences in the epidemiology of onychomycosis, being the heat and humidity of the tropical and subtropical regions responsible for promoting the broad dissemination of the same [16•, 19•]. It is known to the present date that dermatophytes are the main cause of onychomycosis; however, it has been reported in some countries that the NDM have increased. Malaysia is one example, where in 1999, its main etiologic agents were dermatophytes, but in 2012, the NDM (45.4%) ranked first as responsible agents for onychomycosis being Aspergillus spp. the most common one (59.8%) [20•].

The onychomycosis caused by NDM is present throughout the world; in European countries, it has been reported between 5 and 17.2%, in North America between 4.3 and 33%, in South America 1 and 9.5%, in Central America 0.76%, East Asia 12 and 45.8%, Africa 2.78 and 9.0%, and in Mexico 1.49% (Fig. 1) [17•, 20•, 21•, 22•, 23••].

Fig. 1
figure 1

Distribution of onychomycosis caused by non-dermatophytic molds and Aspergillus spp.

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Among the NDM, genus Aspergillus presents a variable frequency according to the geographical region, being found in a 5% in North America, 0.6% in Brazil, 22 to 35.33% in India, 13.15% in Italy, 0.26% in Guatemala, 9.5–50% in Iran, 30.3% in Turkey, 2.0% in Pakistan, 63% in England, and 59.8% in Malaysia (Fig. 1) [1•, 16•, 17•, 20•, 24•, 25••, 26•, 27•, 28•, 29•, 30••, 31•].

The presence of Aspergillus as primary agent of onychomycosis was demonstrated in 1941 by Bereston and Keil, whom described a case of aspergillosis in a 30-year-old woman that referred a deformation of the first toenail of her right foot, which started with a dark spot in the proximal portion and a subsequent discoloration. Dr. Charles Tom isolated and identified A. flavus as the causative agent [32]. From this demonstration, numerous cases of onychomycosis caused by several species of Aspergillus have been reported in various parts of the world, among which the following have been identified: A. niger, A. sydowii, A. flavus, A. fumigatus, A. repens, A. sclerotiourum, A. versicolor, A. terreus, A. candidus, A. nidulans, A. clavatus, A. melleus, A. uvarum, A. nomius, A. ochraceopetaliformis, A. persii, A. tamarii, A. tubingensis, Emericella quadrilineata (Teleomorph of A. tetrazonus), A. hongkongenesis, A. unguis, A. welwitschiae (synonym A. awamori), A. austroafricanus, A. protuberus, A. alliaceus, and A. ochraceus (Table 1) [30••, 33••, 34••, 35•, 36•, 37•, 38•, 39•, 40•, 41, 42•, 43•].

Table 1 Microscopic characteristics of Aspergillus species by section
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The onychomycosis due to Aspergillus occurs in both sexes; however, there is no accurate data on which group is more vulnerable as men generally do not seek medical attention due to esthetic disinterest unlike women, which entails that in some reports females have higher incidence [21•, 29•].

As for age, onychomycosis due to Aspergillus can occur in any age group; there are reports in both pediatric and geriatric patients [43•]. However, it has been observed that the most affected group is the one between 30 and over 60 years [16•, 24•].

Clinical Manifestations

The clinical manifestations of onychomycosis caused by Aspergillus are quite varied, depending on the host and the species. In general, onycholysis, inflammation of the periungual fold, leukonychia, onychomadesis, onychodystrophy, hyperkeratosis, onychoclasis, brittle nails, changes in the coloration, and melanonychia in some cases, both in feet and hand nails (Table 2) [48], can be found. With regard to classification, it has been observed that the most frequent types of onychomycosis caused by Aspergillus spp. are distal lateral subungual onychomycosis (DLSO), white superficial onychomycosis (WSO), and proximal white subungual onychomycosis (PSO) [34••].

Table 2 Clinical manifestations of onychomycosis caused by Aspergillus spp.
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Diagnostic Methods

To diagnose onychomycosis due to Aspergillus spp., conventional techniques are used such as direct examination, considered the gold standard, with KOH 10 to 40%, alone or with dimethyl sulfoxide (DMSO). The most used mycological culture media are Sabouraud dextrose agar with and without antibiotics (cycloheximide, chloramphenicol, and gentamicin), modified Sabouraud agar, Czapek agar, and malt extract medium, being these two last specific for the isolation of Aspergillus. It can also be used potato dextrose agar which favors the sporulation of fungus. All cultures are incubated at temperatures ranging from 25 to 30 °C in a time range from 3 days to 3 weeks [14•, 15•, 38•, 51•, 52•, 63].

After it was demonstrated that genus Aspergillus is a primary pathogen in onychomycosis, certain criterion was established to discard it as a contaminant and determine the accurate diagnosis: presence of hyphae or spores in the direct microscopic examination of the clinical sample, isolation of the same fungal species from a second sample taken from nails with extreme hygiene measures after an interval of 5 to 9 days, and identification of Aspergillus in 5 of 20 inoculated nail fragments [14•, 36•].

Direct Examination

The observation of fungal structures with the combination of KOH 40% and DMSO enables a faster clearance of the sample, as it defragments keratin [15•]; this result is also attained by mixing KOH with chlorazol black [9]. Despite the high sensitivity of these techniques, it is possible to obtain in some cases false-negative results [64••]. In case of a negative result, the use of KOH/CFW (aqueous solution of calcofluor white at 0.1% mixed in equal volumes with KOH) allows early recognition of fungi in tissue under ultraviolet light [65•]. Systematic use of calcofluor white in the laboratories is not recommended given that it has not shown additional benefits when compared to KOH. This is of utmost importance when resources are limited [66••].

Molecular Tests

Classic diagnosis has limitations; direct examination may show false results up to 30%, while the mycological culture is not positive in all cases. In addition, its interpretation is often complicated, particularly when the NDM are isolated, as they are generally considered to be pollutants. These limitations lead to an empirical treatment which is not always effective.

Given the taxonomical complexity of genus Aspergillus, the morphological similarity, and different susceptibility to antifungal agents among the species of the same section, its identification is difficult. For example, within the section Nigri, it includes A. welwitschiae, A. carbonarius, A. brasiliensis, and A. tubingensis, which are morphologically similar species to A. niger, but A. tubingensis presents different susceptibility to antifungals [67]. Therefore, the precise identification of species in the diagnosis of onychomycosis due to Aspergillus is necessary. The use of morphological criteria to achieve the identification of Aspergillus spp. is insufficient because it lacks accuracy and requires highly trained staff; hence, more accurate methods are required such as molecular ones. Among these, the polymerase chain reaction (PCR) technique stands out, being the gene fragments (calmodulin and β-tubulin), the 28-s region of the rDNA and the internal transcribed spacer (ITS) regions, the most common amplification targets to identify, based on the sequence of these genes, any Aspergillus species [39•, 40•, 41, 42•, 52•].

It is important to point out that even though molecular biology techniques undoubtedly represent a step forward in the direct diagnosis of onychomycosis, surpassing in occasions the sensitivity limitations of direct examination and cultures, they do not substitute conventional tests, but are complementary; that is, the sensitivity of the diagnosis is greater when conventional and molecular procedures are combined.

Treatment

The treatment modalities for onychomycosis due to Aspergillus spp. include nail avulsion, surgical debridement [51•], topical therapy, oral therapy, or a combination of oral and topical antifungal agents. When it comes to topical treatment, the Whitfield ointment has been used, tioconazole 28%, amorolfine 5%, and calcipotriol, with failed results in infections caused by A. fumigatus, A. persii, and A. clavatus, while the iodochlorhydroxyquin (Vioform) ointment, urea cream 40%, terbinafine, bifonazole, and urea 40% have achieved clinical cure in onychomycosis caused by A. fumigatus, A. tamarii, and A. niger [42•, 43•, 50•, 60]. Currently, there is a new triazole antifungal topical solution, the eficonazole, which has shown an excellent activity against A. fumigatus, A. niger, A. flavus, and A. terreus, as well as against other NDM, which turns it into an alternative for the onychomycosis treatment [68•].

Oral therapy with 100 to 200 mg daily of itraconazole for 2 to 3 months has shown successful results against A. niger, A. clavatus, and A. candidus, except A. sydowii [39•, 52•, 55•, 56••, 59]. The terbinafine at a dose of 250 mg daily for 6 weeks has shown negative results against A. candidus [55•].

The therapy that has shown best results is the combined one. Reported combinations are ciclopiroxolamine and terbinafine 250 mg/day, amphotericin B 1% and terbinafine (200 mg/3 months), terbinafine 250 mg and amorolfine 5%, itraconazole 400 mg for 4 months with amorolfine nail lacquer, ketoconazole 2% and terbinafine 250 mg for 3 months, and amorolfine and fluconazole, for A. ochraceopetaliformis, A. persii, A. niger, A. nomius, A. uvarum, and A. sclerotiorum, respectively [37•, 38•, 40•, 41, 42•, 61].

It is important to note that the clinical cure rates with systemic or combined therapy, and even more with topical therapy, may be limited due to the lack of compliance by the patient [40,41,42]. Another factor that may interfere in the response to onychomycosis treatment by Aspergillus is the etiological agent, since within the current taxonomic classification of this genera, there are several species that have different susceptibility profiles and are morphologically indistinguishable, even though they pertain to the same section; therefore, an adequate treatment of onychomycosis due to Aspergillus must be based on the identification of the fungus at the species level.

Conclusion

Nail pathology caused by Aspergillus spp. is emerging and accounts for 34 to 60% of onychomycosis caused by NDM. It is developed mainly in patients presenting some types of immunosuppression, such as diabetic patients, HIV-positive patients, and those who engage in certain sports activities such as swimming. During diagnosis, it is important to determine the species of Aspergillus to avoid therapeutic failure, since resistance has been observed in some of them. The accurate identification of species must be done through the combination of conventional and molecular methods. For the treatment of this mycosis, it is recommended to use combined therapy.