Nondermatophytic Filamentous Fungi Infection in South America—Reality or Misdiagnosis?

Dermatol Clin 26 (2008) 271–283

Nondermatophytic Filamentous Fungi Infection in South AmericadReality or Misdiagnosis? Regina Casz Schechtman, MD, PhDa,b,* a University of Gama Filho, Rua Ribeiro de Almeida 44, Apt. 102, 22240-060, Rio de Janeiro, Brazil Instituto de Dermatologia Prof. Rubem Azulay, Santa Casa da Misericordia do Rio de Janeiro Hospital, Rua Santa Luzia 206, 20020-022, Rio de Janeiro, Brazil

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The term ‘‘Dermatomycosis,’’ better defined as superficial infections caused by ‘‘nondermatophyte fungi,’’ characterizes a variety of infections of the skin, nails, ears, eyes, and hair. These fungi can be organized into three groups: The first group represents the genus Scytalidium sp, which comprises two species: Hendersonula toruloidea or Scytalidium dimidiatum and Scytalidium hyalinum. The second group represents other hyphomycetes. Some of these agents can cause onychomycosis, otomycosis, or keratitis. They are Acremonium sp, Fusarium sp, Scopulariopsis brevicaulis, Aspergillus sp, and Penicillium sp. The third group is characterized by nonfilamentous fungi; these are yeasts that produce superficial cutaneous infections, which are mainly represented by Candida sp and Trichosporon sp. They may also be encountered as frequent contaminants in the laboratory, for they can exist in the environment as saprophytes on a variety of substrates. In these cases, the identification of an isolate does not necessarily confirm a pathogenic role, and the observation of the fungus in tissue often is of greater value than the verification of the species [1]. The microscopic features shown in pathologic material from South American frequent superficial mycosis like tinea nigra, black piedra, and white piedra are so typical that a precise diagnosis can usually be made before cultures are obtained. In spite of this, the identification of the pathogen in these infections is crucial, because

* University of Gama Filho, Rua Ribeiro de Almeida 44, Apt. 102, 22240-060, Rio de Janeiro, Brazil. E-mail address: [email protected]

the observed clinical features look a lot like those of ringworm [2]. Hendersonula and Scytalidium may not be distinguishable from dermatophytes on direct microscopy. Typically, however, their hyphae vary in width and are more irregular than those of dermatophytes, and Hendersonula hyphae may be seen to be pigmented [3,4]. These organisms are sensitive to cycloheximide and may be missed if that agent is used in routine media. Even if isolated, they might easily be dismissed as a contaminant by the unwary. Moreover, either of these infections may coexist with ringworm, and double infections with Hendersonula and Scytalidium have been reported [4]. Hendersonula toruloidea, a weak secondary pathogen of higher plants in mainly tropical areas, is a gray to black mold and is recognized as the cause of ringwormlike infections of the hands, feet, and nails. These two molds invoke the same clinical picture [5–7]. Scytalidium hyalinum, a similar but nonpigmented organism, can also mimic tinea pedis and manum and invade the nail plate [3,4]. It has been reported that nondermatophyte onychomycosis accounts for 1.5% to 6% of all cases of onychomycosis, but there is debate on the significance of the nondermatophyte molds and yeasts when they are isolated along with dermatophytes [8]. These ‘‘mixed infections’’ are increasing in frequency, and the true causative organism is still unknown. In almost all cases they present only as secondary invaders living saprophytically in nail plates already damaged by trauma, ischemia, or other disease; indeed, nails rendered abnormal by a primary dermatophyte infection may also be secondarily invaded by such fungi.

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Among this group of molds is S brevicaulis, one species of which does appear capable of attacking undamaged nails or ones showing only trivial abnormalities. The great controversy is based on whether these organisms are true causative pathogenic isolates or simply contaminates from a nonsterile environment [9]. Some investigators [10] attribute misinterpretation of the collected sample as a contributing factor in the debate. They present with several examples: (1) 20% of all samples with dermatophytes present have ‘‘dead filaments’’ included, which can lead to false-negative results when there is still an active dermatophyte colony; (2) when dead filaments of dermatophytes are seen under microscopy in the presence of spores, this could be misinterpreted as a nondermatophyte infection; (3) if dead filaments are interposed with living elements of nondermatophytes, the incorrect diagnosis of a pure nondermatophytic infection could be made; and (4) a true nondermatophyte infection can be missed when nondermatophyte filaments are interpreted as dead dermatophyte filaments, and the nondermatophytes are mislabeled as contamination, suggesting that growth of a nondermatophyte on a culture after positive findings on microscopy is not enough to diagnose a nondermatophyte infection. A recent study found that nondermatophytic mold could invade healthy nails and that local factors were not important for the occurrence of this type of onychomycosis [11]. Current medical literature implies that nondermatophytes are simply opportunists that invade an already dermatophyte-degraded nail [8]. They have reported that even if the mixed infection is the cause of onychomycosis, the treatment for the patient still will not change. In another study, they have reported that the treatment for dermatophytes successfully cleared the nails of infection in all cases [9]. Tosti and colleagues [11] reported that all of the patients in their study with Aspergillus-induced onychomycosis were cured after systemic or topical treatment. Furthermore, the eradication of the mold produced a complete cure of nail abnormalities in all of the patients who responded to treatment. These conflicting reports fuel the ongoing debate on the interactions of dermatophytes and nondermatophytes in the diagnosis of onychomycosis. Scherer and colleagues [12] have studied a South Florida geriatric population with onychomycosis. This study found an increase of mixed fungal infections and saprophytes. The investigators believe that age, circulation, health, environment,

and geography all may play an equal role in the pathogenesis of onychomycosis. The study’s results are contradictory to most reports in the medical literature that state that onychomycosis is mainly caused by dermatophyte infection. However, most current medical studies do not restrict age as a parameter. The results of this investigation show that there may be a shift from isolated dermatophyte infection to mixed saprophyte infections in a geriatric population with onychomycosis. Actually, the main focus of attention of some researchers has been the current changes in the etiology of onychomycoses. The accurate determination of the etiology of the injured nail that clinically resemble those of onychomycosis constitutes an important clue for the establishment of an adequate and efficient therapeutic approach, because different organisms respond differently to the antifungal agents [13].

Etiology Scytalidium dimidiatum, formerly known as H toruloidea, is a plant pathogen of fruit trees found in the tropics and subtropics. Scytalidium hyalinum, which already has been isolated in humans, causes a chronic disease of the soles, toe spaces, palms, and nails that is clinically indistinguishable from Trichophyton rubrum infections [14]. The infection presents with scaling of the soles and palms and cracking between the toes. These fungi seem capable of producing toe cleft changes and involvement of the palms and soles, very similar but not identical to those produced by dermatophytes (Figs. 1–5). Nail involvement of both toes and fingers typically is present; some brown discoloration starting at the lateral edges of the nail and spreading centrally has been observed in some cases caused by Hendersonula (Fig. 6) [15]. There seems to be the tendency for swelling of the nail fold to be present too. Whether this is an independent nail fold infection or is directly caused by Hendersonula or Scytalidium infection itself is not clear. The clinical features of S dimidiatum and S hyalinum infections are identical. Lesions may often be asymptomatic. It is essential to recognize these infections because they show a poor response to most antifungal drugs [3]. Tinea capitis has not been seen, although the fungus will degrade hair weakly in vitro [16]. All patients with this condition seen in the United

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Fig. 1. Scytalidium sp infection of the hand. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Fig. 3. Scytalidium sp infection of the hand. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Kingdom so far have come from Africa, Asia, or the Caribbean, and it has been assumed that they have acquired the infection in their countries of origin [4,6]. Recently, similar cases have been reported from North America [16] and India [17] and facial infection from Algeria [4,6]. Several nondermatophyte molds are known to be able to invade the superficial nail plate. The most common are Fusarium sp and Acremonium sp (Figs. 7–10). Mold white superficial onychomycosis (WSO) usually affects a single toenail, mainly in adults. Patients sometimes report barefoot gardening, suggesting a possible contact with fungi via the soil. Clinically, mold WSO may be indistinguishable from dermatophyte

WSO, or it may show a diffuse involvement of the nail both in width and depth. This is especially seen in Fusarium and Aspergillus WSO [18]. In these cases, the nail is diffusely opaque and friable, with a pigmentation varying from homogeneous white to patchy yellow brown. The color of the pigmentation may result from the production of pigmented conidia within the nail. Nail plate discoloration involves the entire nail surface and often extends beneath the proximal nail fold; in this case, the pigmentation is visible through the cuticle. Mechanical removal of the invaded nail plate reveals deep penetration of hyphae that may sometimes even reach the ventral portion of the nail. Progression is usually rapid,

Fig. 2. Scytalidium sp infection of the hand. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Fig. 4. Fingernail infection caused by S dimidiatum. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

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Fig. 5. Toenail infection due to S dimidiatum. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

and patients often complain that entire nail has become pigmented in a few months, as seen in the other types of onychomycosis caused by nondermatophyte molds [11]. Periungueal inflammation is commonly associated, usually without pus discharge. Pigmentation of the proximal nail and deep invasion of the nail plate often make it difficult to distinguish between WSO and proximal subungual onychomycosis (PSO) caused by molds [11]. Histologically, mold WSO is characterized by the presence of short-branched filaments in splits in the nail plate from the nail surface to the deep nail plate.

Fig. 6. Direct examination with potassium hydroxide preparation of a nail infected with Scytalidium sp shows characteristic irregular and distorted hyphae. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Fig. 7. Fusarium sp culture shows white-colored colonies. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

A variety of filamentous nondermatophyte organisms may cause nail infections, particularly after damage to the tissue by trauma or disease as secondary invaders. Many of these fungi are present in the environment, and care must be taken in the interpretation of their recovery from infected material. It may be possible to correlate the morphology of the fungus in the specimen with that of the cultural isolate and, in any case, it is essential to confirm the presence of these fungi in nails by repeated cultures whenever possible. Cultures of nails suspected of a nondermatophyte infection should be made on a medium without cycloheximide.

Fig. 8. Fusarium sp culture shows pink-colored colonies. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

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Fig. 9. Fusarium sp culture shows pink-colored colonies. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Onychomycosis represents one of the most common nail diseases. The studies of prevalence concerning the general population consulting a medical practitioner or specialist show a rate of 2% to 13% according to the different investigators [19]. Recent surveys done in Europe on a large scale in adults indicated a high prevalence; 20% to 30%, depending on whether the consultant is a general practitioner or a dermatologist [19]. In any case, all studies concur in the opinion that onychomycosis has been in constant

Fig. 10. Microscopy of Fusarium sp shows a curvedshaped and multicellular macroconidia typical of the genus. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

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progression over the last 20 years. Rarely observed in children, frequent in adults, onychomycosis affects particularly the elderly. In Western Europe and in North America, onychomycosis involves principally the feet, especially in men. By contrast, in Southern Europe and in the Middle and Far East, the prevalence is highest in women’s fingernails, often associated with paronychia [19]. Toenails, especially hallux nails, are affected more than fingernails, and there are no associated skin lesions. Among the factors promoting fungal nail invasion, some are local (trophic troubles, circulatory impairment, and overlapping of digits), others are caused by general factors such as immunosuppression, diabetes mellitus, and psoriasis. There are also behavioral factors such as occupation, lifestyle, and sports, which favor the meeting with the pathogenic fungus [19]. Investigations of abnormal toenails particularly in the elderly, have found that an appreciable minority are colonized by nondermatophyte fungi usually molds, often Aspergillus sp. Fingernails are much less often invaded. These molds are generally accepted as existing saprophytically. They frequently live in the outer or dorsal areas of the nail plate, and although they may conceivably add to primary damage caused by ischemia, trauma, or a dermatophytosis, they are, in general, of little practical importance to the patient. For the dermatologist, their significance lies in the fact that they must be distinguished from dermatophytes. Moreover, a dystrophic nail caused by ischemia secondarily colonized by Aspergillus sp, is likely to regrow abnormally even if the fungus is eliminated by avulsion. Unlike Scopulariopsis infections, secondary mold invasion often affects several nails and indeed all may be invaded. According to English and Atkinson [20], the distinction between T rubrum and Trichophyton interdigitale is important. The first one is a fairly potent pathogen capable of invading skin and attacking nails deeply in the more ventral areas and the nondermatophyte molds that appear to prefer the dorsal plate well away from living cells and defense mechanisms. T interdigitale is regarded as an intermediate between these two classes, capable of primary parasitism but sometimes behaving almost as a saprophyte invading the already abnormal nails of elderly subjects. It is worthy of note that in white superficial onychomycosis, where the dorsal surface of the nail plate is eroded by fungi, the organisms implicated are not only the saprophytic molds like Fusarium

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sp., Aspergillus sp., and Acremonium sp., but also the dermatophyte fungus T interdigitale is present. Species of the genus Fusarium are phytopathogens with a wide distribution. The main characteristic of this genus is the production of multiseptated conidia in spindle form with pointed extremities. These conidia are produced in basipetal succession and accumulate in a gelatinous mass in the phialides. There are also unicellular microconidia, some of which present a greater variety of forms and can be found grouped in a mass or knot. The taxonomy of this genus is complex because of the great number of species existent and to the complicated conidiogenesis that differentiates one species from another. Identification is difficult, especially when macroconidia are not produced, because they can be confounded easily with other species such as Acremonium. The species of nondermatophytic fungi that most frequently causes onychomycosis are Fusarium solani and Fusarium oxysporum that also cause other diseases, such as dermatomycosis and systemic infections. In culture they are very sensitive to cycloheximide [21]. Fusarium solani and F oxysporum led to proximal involvement of the nail, associated with periungual pain and inflammation. The affected nail presents a white-yellowish color and, frequently, an opaque surface. The proximal fold of the nail and the cuticle develop a yellowish-white color, indicating the proximal origin of the infection. The distal nail may take on a yellowish coloration when there is a progression of the mycosis. The course of onychomycosis caused by Fusarium is from 1 month to 15 years (mean, 3 years) [21]. A total of 79 patients with WSO were seen at the Department of Dermatology of Bologna University from 1994 to 2002 [22]. Responsible agents included T interdigitale in 73%, T rubrum in 5%, Fusarium sp in 11%, Aspergillus sp in 6%, and Acremonium strictum in 3%. Nondermatophyte molds were responsible for 17 cases of WSO: Fusarium sp (F solani and F oxysporum) in nine cases, Aspergillus sp (Aspergillus candidus and Aspergillus terreus) in five, and Acremonium strictum in three. These patients were healthy adults with no associated cutaneous mycoses, and none was used to walking barefoot in the grass. Acremonium sp may also, but rarely, be responsible for a classic WSO. A strictum WSOs usually are cured by mechanical removal of the affected nail plate followed by applications of topical antifungal agents [23].

Scytalidium dimidiatum may cause a superficial black onychomycosis characterized by small opaque black patches on the dorsal nail plate that are easily scraped away. Hyaline fungi and dematiaceous fungi have been described, representing a mixed infection. There also have been reports of these fungi associated with yeasts or dermatophytic fungi. In the latter case, they are considered to be merely contaminants of tinea unguium [21]. The genus Scopulariopsis presents a wide geographic distribution. The soil is its main habitat, although it has even been found in caves, along with Histoplasma. They are filamentous hyaline fungi; the colonies are of yellowish color and never green. Conidiogenesis is in the form of a brush, reminiscent of Penicillium; the main difference is the colors of their colonies, at first white and over time becoming a brown or cinnamon color. The phialides are clearly formed in a bottle shape; the conidia present with annelids that possess a thick and wrinkled wall, being arranged in chains, with the youngest in the bases [21]. Among the filamentous nondermatophytic fungi, the species S brevicaulis is the most frequent causal agent of onychomycoses of the feet in temperate climates, most often involving the nail of the hallux [1–3]. The proximal location is more frequent and is characterized by white, yellow, or orange coloration that starts in the lunula and extends to the distal area of the nail. The disease duration before examination ranges between 1 month and 12 years (mean, 2 years) [21]. Epidemiology The nondermatophyte filamentous fungi are seen frequently in endemic areas where the rate of infection may equal or even exceed that of dermatophytes. They are found in Africa, the West Indies, the Indian subcontinent, and the Far East as well as parts of the United States. In temperate countries, they are seen mainly in immigrants from tropical regions [3]. In countries such as Australia, the United Kingdom, and the United States, the incidence of onychomicosis had been estimated previously to be about 3% in the general population, increasing to 5% in elderly individuals. In some subgroups such as miners, servicemen, and individuals participating in sports, this incidence may increase to 20% because of the use of communal showers and changing rooms. It is important to stress, however, that only 50% of dystrophic nails have a fungal cause [23–25], and it is therefore essential to establish a correct

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laboratory diagnosis by microscopy or culture before a patient is treated with a systemic antifungal agent [26–29]. Data obtained from a study in Great Britain on 118 patients with onychomycosis revealed that 81% were infected with T rubrum [30]. Nevertheless, dystrophic nails harbor a large flora of saprophytic fungi, yeasts, and bacteria associated with a nonsterile site; therefore, the isolation of a yeast or other mold without supporting microscopy is not significant [28,29]. In the United States, dermatophytes accounted for 90% of onychomycosis of the toenails in one study [31]. In another study, Elewski [32] reported similar results, finding that 90% of fungal infections were caused by dermatophytes, 7% by Candida sp, and 3% by nondermatophytic molds. In Canada, a study of more than 3000 nails [33] found that 91% of fungal infections were caused by dermatophytes, 6% by Candida sp, and 3% by nondermatophyte molds. In the United Kingdom, Clayton [34] performed a study of 699 specimens and reported that 81% of nail infections were caused by dermatophytes, 17% by yeasts, and 2% by nondermatophyte molds. In a study from Poland, the investigators reported only 51.7% of the fungal infections over a 9-year period in 25,737 patients were caused by dermatophytes [35]. In this same study, nondermatophyte infections accounted for 48.3%, and it was determined that the only fungi to have a direct correlation to age were Aspergillus sp. In Pakistan, dermatophytes are the second most frequent pathogen in onychomycosis. Bokhari and colleagues [36] attributed the increase of nondermatophyte pathogens to the hot and humid climate of Pakistan. In addition, this study reported that saprophytes could primarily infect nails to produce disease. These findings disagree with those of other investigators who consider saprophyte fungi as nonpathogenic contaminants and therefore, unable to infect human nail [9]. The differences in causative organisms have been attributed to geographic and temporal variances. In tropical countries, such as Nigeria, Thailand, and Jamaica, 10% to 50% of fungal infections are caused by a saprophyte, the Scytalidium sp. Other reports indicate that yeasts, such as Candida albicans, are a common pathogenic agent of toenail infection in Egypt and Saudi Arabia, where bathing the feet is a ritualistic religious practice [33].

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The prevalence of mold WSO is not known. According to Zaias [37], 5% of all WSOs are caused by nondermatophytic molds. Piraccini and Tosti [22] found that 21% of all WSOs were caused by nondermatophytes. This high prevalence, however, may be only apparent because the severity of WSO caused by molds prompts patients to ask for dermatologic advice. Moreover, mold WSO seems to affect patients younger than those with T interdigitale WSO, and young patients are more bothered by unpleasant-looking nails than the elderly. Mold WSO affects healthy adult patients and is not associated with tinea pedis, which suggests that host factors play no role in its development [12]. In Rio de Janeiro city, Brazil, Araujo and coworkers [21] have examined 2271 patients with abnormal nails in which 400 were confirmed to have onychomycosis. Non dermatophyte filamentous fungi represented 4.5% of all onychomycosis. The most frequent molds found were Fusarium sp, Scytalidium sp, Trichosporon beigelii, Curvularia sp, and Charalopsis sp. Yet, they have recovered Scytalidium dimidiatum and Fusarium sp from 1.25% and 0.5% of the investigated cases of all cases of onychomycoses, and it represents 27.7% and 11.1%, respectively, if only the nondermatophyte filamentous fungi infections of the nail were considered. The preferential localization was the toenail. Distal and lateral subungual onychomycosis was the most common clinical presentation. The investigators suggest specific criteria to obtain an accurate diagnosis of onychomycosis caused by nondermatophyte-emerging fungi [21]. Another Brazilian study from Sa˜o Paulo [38] investigated the prevalence of onychomycosis in 104 patients with leprosy. A total of 91.3% of them presented with onychomycosis with predominance of yeasts (60.0%). Dermatophyte fungi represented 26.7% of the cases, of which, T rubrum represented 15.3%. Nondermatophyte filamentous fungi were recovered from 13.3%, and Scytalidium sp was the most frequent (10.5%). Their findings suggest mainly that the nails of these patients are susceptible to onychomycosis, with a predisposition to infection by opportunist agents. A study from Parana, south Brazil, has also found that yeasts are the main cause of onychomycosis in Brazil [39]. Despite the fact that those yeasts belong to the normal flora, they can cause opportunistic infections when local and systemic predisposing factors are met. In Brazil, the hot and humid climate can contribute to this

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increased predisposition [39]. Other studies outside Brazil, found that onychomycosis caused by dermatophyte fungi are more frequent [26]. In 2005, 878 patients with abnormal nails were examined at the Instituto de Dermatologia Professor Rubem Azulay (IDPA), Santa Casa da Misericordia do Rio de Janeiro. A total of 254 of them were confirmed to have onychomycosis with predominance of yeasts. The most frequent mold found was Fusarium sp followed by S dimidiatum and then S hyalinum. We found that onychomycosis caused by yeasts and nondermatophyte filamentous fungi were more frequent than the ones caused by dermatophyte fungi (Figs. 11 and 12). A study from another South American country has already registered an increased number of onychomycosis by nondermatophyte filamentous Fungi in the city of Rosario, Argentina [13]. Further studies are necessary to evaluate the new species described and their importance in the etiology of onychomycosis.

Diagnosis The diagnosis of onychomycosis must always be based on the following fundamental points: the clinical aspect of the abnormal nail, the geographic area of the patient’s origin, the antecedents of other infections correlated with onychomycosis, and possible previous specific treatments. Mycologic diagnosis is definitive and based on direct examination of the nail, results of culture, and identification of the etiologic agent, whether by morphologic aspects or with

Fig. 11. Mixed infection of the toenail caused by T rubrum and S dimidiatum. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Fig. 12. Mixed infection of the fingernail caused by C albicans and S dimidiatum. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

the aid of biochemical tests (Fig. 13). Collection of the sample should be made in the more peripheral areas of the lesion, where the fungus is most active, as represented by the boundary between the normal part and affected region of the nail. The instrument used for the sample collection must be sterile, as should the containers used to collect, conserve, and transport the specimen. In the direct examination, the finding of specific structures and the morphology of the hyphae indicate the possible fungal etiology: regular hyphae suggest dermatophytes; irregular and atypical hyphae, with or without conidia, arouse a suspicion of the presence of different fungi (see Fig. 6) [15,37]. In fact, contaminant fungi may be isolated easily, but successive sampling of the same nail will rarely show the same contaminant in the majority of the cases [9]. The key elements for the diagnosis of onychomycosis caused by emerging fungi are the collection of an adequate specimen

Fig. 13. Collection of nail material for diagnosis. The proximal portion of the nail infected provides the youngest and most viable elements. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

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and the correct processing and interpretation of both microscopy and culture results. Furthermore, given the common occurrence of incidental contaminants from nonsterile nail samples, it would be useful for clinicians if laboratory reports included a comment on the likely significance of a culture result and the result itself. Repeated collections may also be necessary to detect the presence of a dermatophyte in nail specimens, because most contaminant fungi will overgrow or mask the presence of a dermatophyte in culture. According to Araujo and coworkers [21], it is necessary have a positive direct mycologic examination and culture and confirmation by means of repeated examinations. The correct diagnosis of emerging fungi is based on a combination of the following criteria: the clinical aspect of the abnormal nail as related to this diagnosis, the finding of fungal structures in the direct examination of the nail with 20% sodium hydroxide, failure to isolate dermatophytes in the culture, and growth of a colony of the same fungus in two consecutive samples [15]. Direct examination Mycology should remain the investigation of choice in suspected onychomicosis. If negative, we would benefit from repeated nail samples and reduce the false-negative rate by taking skin scrapings from the soles or toe webs whenever possible [40]. Undoubtedly, the direct examination is merely suggestive because direct examination of the nail sample and of the hyperkeratotic skin can produce both false-positive and falsenegative results. This occurs when confusing the edges of the epithelial cells, fat droplets, or air bubbles with hyphae, or because these cannot be seen because of excessive dye color the thickness of the nail keratin, which impedes an adequate dispersion. Also frequently there are a sparse number of fungal elements. The distribution of the fungus in the affected nail material often is patchy, but where spores are found they are quite distinctive, being approximately spherical with one flat facet. The roughened surface of these spores is not always appreciable in potassium hydroxide mounts [15]. Direct examination occasionally may reveal spores, but usually only mycelium is seen. Broad fronds are sometimes said to be suggestive of saprophytic mold, but this feature does not reliably distinguish them from the dermatophytes, which, in the nail, can show this morphology. Careful culture is important.

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Culture Cycloheximide-free media are necessary for the isolation of many nondermatophytes, but it is equally important to identify positively any ringworm fungus also present. Yeasts are more often isolated from fingernails, but because they are rarely found on direct examination, it is not clear whether they should be considered as saprophytes or merely as surface contaminants. Apart from C albicans, Candida parapsilosis does seem to be a low-grade pathogen of nails, particularly toenails [15,37]. The isolation of a nondermatophytic fungus or of a yeast may be the result of environmental contamination, or it may be one originating from patient’s normal microbiota; it could also be the agent of a real infection (Figs. 14– 16). Positive direct examination of filamentous nondermatophyte fungus and the number of colonies corresponding to the number of inoculated points are significant; however, it will be necessary to request subsequent samples to confirm the initial diagnosis. The patient’s origin, his or her possible contact with other sick people or animals, work environment that favors the development of mycoses, and the geographic area of the country he or she comes from, are all factors that help in understanding the results of culture for rarely seen species. Primary dermatomycosis caused by filamentous nondermatophyte fungi are rare, with the exception of onychomycosis, which oscillate between 1% and 20%, depending on the investigators and on the geographic area from which the sample originated (see Figs. 11 and 12) [21].

Fig. 14. Scytalidium dimidiatum culture shows gray to black colonies that completely fill the Petri dish. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

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can be jeopardized as a result of previous treatments. Therefore, the patients should be asked to suspend use of any topical medicine at least 1 week before the collection of the sample. Histomycology

Fig. 15. Microscopy of S dimidiatum culture shows chains of one- to two-celled pigmented arthroconidia. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

The difference between the positive results on direct examination and negative cultures can be explained by the irregular distribution of the spores in the infected nail and the difficulty of adequate collection of the material, especially when it is of the subungual type. Apart from that, it is relatively easy to contaminate the cultures with environmental fungi and bacteria. Moreover, the intense thickness of the ungueal keratin makes it difficult for the microscopic identification of the microorganisms and can produce false-negative cultures. Culture viability

Onychomycosis exhibits considerable diversity when the site of the infection is scrutinized using traditional fluorescence or confocal microscopy. Histomycology is a noninvasive assessment performed on nail clippings [41]. The location of and the density of fungal cells is variable. In some instances, these aspects remain clinically unsuspected. In vivo confocal microscopy can provide the same information. Computerized image analysis of histologic sections is the most powerful means for quantifying the fungal load. Immunohistochemistry provides information about the identity of the fungus or the association of different fungi present in the nail plate. Mixed infections may be unifocal or located at different levels in the nail apparatus. The viability of fungi as assessed by vital stains can be visualized under the microscope and quantified by flow cytometry. The different aspects of nail histomycology are complementary and may shed some light on sometimes unsuspected aspects of the onychomycosis. It is very useful in confirming the diagnosis of onychomycosis caused by more than one pathogen, especially dermatophytes, yeasts, and nondermatophyte filamentous fungi [41].

Treatment Topical therapy

Fig. 16. Scytalidium hyalinum culture shows white colonies with a powdery surface. (Courtesy of Regina Casz Schechtman, MD, PhD, Rio de Janeiro, Brazil.)

Nail avulsion may be performed surgically using carbon dioxide laser or nonsurgically by chemical maceration [42]. Avulsion of the nail plate may be a useful adjuvant to the topical or systemic management of onychomycosis, because it can shorten the course of antifungal therapy, prolong the duration of remission, and enhance the opportunity to prevent recurrence of the infection. In addition, nail plate avulsion is also helpful in treating onychomycosis caused by saprophytes and nondermatophyte molds. Because of patient discomfort and the risk of complications, surgery generally is not recommended as first-line therapy, except for severe or refractory infections [42]. Advantages of topical antifungal agents include minimal drug interactions and no endocrine-related adverse effects, which are sometimes

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associated with systemic treatment options. Adverse effects, if present, would be localized to the site of application with topical treatments. However, these products are ineffective in curing most infections because of inadequate drug penetration of the nail and poor patient compliance. Therefore, it is now more common for these products to be used as adjuncts to systemic therapy [40,42]. Griseofulvin is valueless in treating infections caused by S dimidiatum and S hyalinum. Both fungi are sensitive to imidazoles in vitro, but topical therapy even with occlusion has failed using clotrimazole and miconazole. Benzoic Acid compound ointment (Whitfield’s) may be the best approach [15,37]. Ciclopirox nail lacquer may also have a broad spectrum activity. It is possible that certain organisms such as S dimidiatum do not respond well or at all to antifungal agents [43]. Systemic therapy Oral antifungal agents now available offer significantly increased cure rates, shorter treatment regimens, and a lower level of adverse events compared with griseofulvin, ketoconazole, and other topical treatments. The response of onychomycosis to antifungal therapy may be dependent in part on the causative organism. Griseofulvin has a limited spectrum of effectiveness and is not effective against Candida sp and nondermatophyte molds. Each of the currently available antifungal agents, terbinafine, itraconazole, and fluconazole is effective against onychomycosis caused by dermatophytes and some nondermatophyte molds. The azoles are generally effective against onychomycosis caused by Candida species, which is more likely to affect fingers than toes. Terbinafine may show a relatively higher activity against dermatophytes compared with Candida sp. Its efficacy against nondermatophyte molds is not yet proved [44,45]. Combination therapy Treatment of deep and diffuse WSO caused by Aspergillus and Fusarium can be more difficult because the nail is invaded in its deepest portion [22]. Repeated mechanical removal of the invaded nail plate is advisable. Systemic antifungal agents may be necessary in recalcitrant cases that do not benefit from topical treatment [22]. According to Piraccini and Tosti [22], most cases of Aspergillus and Fusarium WSO can be treated with either systemic terbinafine at a dosage of 250 mg/d for 6

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months or systemic itraconazole at a dosage of 400 mg/d, 1 week per month for 6 months, in association with weekly applications of antifungal nail lacquers. Although combination therapy might seem to be a more costly option, pharmacoeconomic studies have clearly shown that treatment of onychomycosis with topical amorolfine or topical ciclopirox, in combination with either oral terbinafine or oral itraconazole, is both cost saving and cost effective compared with oral treatment alone [46]. Mixed infection is a clinical indicator that may contribute to poor response to therapy [45]. In view of the high rate of therapeutic failure and variable sensitivity to fungicides, many investigators have suggested the need for an in vitro study to test the resistance of these fungi against the various fungicides [15,21,37,45].

Discussion The ability of the emerging fungi (nondermatophytes and other microorganisms) to involve the nail fragment was described initially by English [20] and later by Roberts [26]. However, in the last few years the number of cases of onychomycosis caused by emerging fungi has increased, especially in Europe in both immunodeficient patients and immunocompetent hosts. As a consequence of intense migratory fluxes in the last decades, fungi of the genus Scytalidium, which are more frequent in Africa, Thailand, England, and Australia, and very rare in Brazil, are now being isolated more frequently in our country [21,38,39]. The greater detection of cases of onychomycosis caused by nondermatophyte fungi, particularly Fusarium sp, which is an agent relatively common in Italy, but also S brevicaulis and Aspergillus, indicates the necessity of inclusion of such fungi as possible etiologic agents of these diseases [8,15,21,37]. Another factor that has been contributing to the growth of nondermatophytic onychomycosis is increased immunodeficiency. Although the secondary colonization of nail dystrophy by nondermatophytic fungi is common, only a few patients notice alteration in the nails, such as modification of color or periungual inflammation, before the onset of the onychomycosis. Abnormal thickening of the nail is not observed in these cases. Onychomycosis by filamentous fungi has not been shown to be associated with systemic disease, but in some cases of immunodeficiency, notably AIDS, patients

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have died of a systemic infection of Fusarium that probably originated in an infected nail. Consequently, onychomycosis caused by Fusarium should be considered a serious disease in immunocompromised patients. Scytalidium sp has not been isolated in Italy, but this genus is found frequently in northern Europe, Canada, the United States, and South American countries such as Brazil and Argentina [13,21,38,39]. Recently, some investigators suggested that the failure to treat onychomycosis caused by nondermatophytes can signify an entry site for infection and the spread of mycoses, which are difficult to treat in immunocompromised individuals [44]. Onychomycosis in immunocompetent patients, particularly affects people that handle soil and plants because, in general, these fungi inhabit microecosystems where they are found as parasites or saprophytes in many plants [8,15]. Experience in the treatment of nondermatophytic onychomycosis is still limited because of the variety of etiologic agents potentially involved in addition to the low frequency of incidence and few cases reported in the literature. Treatment with terbinafine and itraconazole for 3 months can be considered effective in immunocompetent patients. The description of new fungal species producing onychomycosis is a reality, but its importance is unknown. To increase awareness, precise diagnostics are necessary, with an appropriate and standardized study methodology, together with publication of statistical studies providing detailed descriptions of the specific etiology and respective clinical presentations [40,42–46].

Summary For the last 10 years, a steady increment in the diagnosis of nondermatophyte filamentous fungal infections has been observed. This trend also applies to the valuation of the medical mycology. It can be attributed, in part, to the increased concern of the population with the aesthetic aspect, the easiest access to health information, and the increased demand for specialized jobs. Moreover, increase in the diagnosis of new emerging fungi is also caused by the improvement of the diagnostic techniques available as well as the qualification and constant update of the professionals in medical mycology [44–46]. Another relevant aspect is the valorization of medical mycology in the medical curriculum. Misdiagnosis can lead to

treatment failures, because not all nail diseases are caused by fungi. Therefore, the importance of clinical diagnosis is strictly associated with laboratory results [44].

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