Aspergillus Versicolor As Cause of Onychomycosis : Report of 12 Cases and Susceptibility Testing to Antifungal Drugs

Journal of the European Academy of Dermatology and Venereology ELSEVIER 11 (1998) 25-31

Aspergillus versicolor as cause of onychomycosis : report of 12 cases and susceptibility testing to antifungal drugs

J.M. Torres-Rodriguez", N. Madrenys-Brunet, M. Siddat, 0. L6pez-Jodra, T. Jimenez

RG Experimental urid Clinicul Myculogy, IMIM, Universitut Autonoma de Barcelona, Avdu. Dr. Aiguader 80. Barcelona, Spain

Abstract

Background Onychornycoses caused by opportunistic moulds are not well understood, and many are due to Scopulariopsis Orevicaulis and other species. Aspergillus versicolor is not documented as an etiological agent in most studies. We have found an increasing prevalence of this species which is involved in 5.8% of all fungal infections of toe nails. Objective To study the clinical and mycological characteristics of the onychomycosis caused by A. versicolor and the in vitro susceptibility of this mould to antifungal agents. Results Onychomycosis due. to A. versicolor is mainly seen in people over 60 and presents with chronic involvement of the big toe nails. Predisposing factors are not always present and the infection does not respond to conventional topical antifungals. In vitro, A. versicolor has been shown to be resistant to griseofulvin and fluconazole as well as to amphotericin B, whereas MICs for itraconazole and ketoconazole are variable but within a range of 0.50-4.0 pg/ml; on the contrary, MICs for rerbinafine are very low (<0.125 pg/ml). Discussion Aspergillus versicolor could be considered as an emergent pathogen causing toenail onychomycosis. Local treatment seems not to be effective. Of the various systemic antifungal agents studied terbinafine appears to be the most effective in treating onychomycosis. 0 1998 Elsevier Science B.V. All rights reserved

Keywords: Aspergillus versicolor; Onychomycosis; MIC; Terbinafine; Azoles; Griseofulvin; Amphotericin B

1. Introduction second fungus in terms of frequency. It is worthwhile noting the high incidence of A. versicolor in nail Nail infections caused by opportunistic fungi repre- lesions, as this is not a common laboratory contami- sent, in our environment, around 7.6% of all cases of nant species nor has its presence been described in onychomycosis, the most common causative agent aerobiological or environmental studies published; being Sropulariopsis brevicaulis, found in 4.8% of at most it has a very lop and sporadic incidence in 354 samples studied between 1986 and 1993 [I]. Dur- human fungal infection all year round [2,3]. ing the same study nine cases of toenail infections Together with other species of the genus Aspergil- were recorded caused by Aspergillus versicolor, the lus, such as A.jlavus and A. fumigatus, A. versicolor is frequently found in stored cereals, hay, cotton, cheese, meat and other foods in a state of decomposition, as * Corresponding author. Fax: +34 3 2213237; well as in various types of soil. A. versicolor has been e-mail: jmtorres iniim.es classified within the section versicolor in the subgenus

Nidulantes [4]; also, some potentially toxic products, patients selected were positive in direct microscopy such as 6-methoxysterigmatocystine and aversine, an and A. versicolor was the only microorganism isolated antraquinone derivative, as well as the yellow-orange from the nail. A case card was completed for each pigment known as versicolorine [5], have been iso- patient where all relevant data, specifically age, sex, lated from its metabolites although their significance origin, description of lesions and nails affected, time as pathogenic agents in infectious processes is practi- of evolution, associated conditions and treatments cally unknown. instituted, was recorded. In an earlier publication we described one case of The isolated strains were identified using the meth- bilateral toenail onychomycosis due to Aspergillus ods described by Raper and Fenell [5] and Hoog and versicolor in an elderly patient with a peripheral cir- Guarro [9], their identification being confirmed in the culatory disorder 161. Since then, we have had the Mycology Laboratory, Microbiology Department, opportunity to examine new cases of nail infections; Medicine School, Universitat Rovira i Virgili, Reus. a relationship between A. versicolor and onychomyco- All isolates were maintained by periodic replanting in sis has been reported in the related species A. sydowi or Czapek agar medium in our laboratory’s collection, A. versicolor itself [7] although neither the clinical nor until they were used for in vitro studies of sensitivity the epidemiological characteristics were given. to antifungals, at which time one strain per patient was The purpose of this paper is to present a series of 12 replanted, after ensuring that it was a pure A. versico- new cases of onychomycosis due to A. versicolor, lor culture. Aspergillus versicolor CECT (Spanish evaluating their clinical and epidemiological charac- Collection of Standard Strains) 2814 was used as the teristics. Furthermore, we present the results obtained reference control strain. from the in vitro sensitivity study of this fungus Following the method recommended by the Anti- against systemic antifungals which could be useful fungal Standardization Subcommittee of the NCCLS in the treatment of this condition. [lo], the minimal inhibitory concentrations for each strain were determined in microplates using a RPMI + 20% glucose medium. The inoculum was 2. Material and methods prepared using the method of Espinel-Ingroff et al. [l 11 and the antifungals studied were amphotericin B, Between 1988 and 1995, 205 patients with lesions itraconazole, fluconazole, ketoconazole, griseofulvin in the toenails which were clinically compatible with and terbinafine; the product was supplied as a powder onychomycosis were positive for fungi. The species by the manufacturing laboratories (Janssen, Ketoco- isolated in 12 patients (5.8%) were identified as nazole and Itraconazole; Pfizer, Fluconazole; Squibb, Aspergillus versicdor in two or more successive sam- Amphotericin B; Sandoz, Terbinafine). Griseofulvin ples; Scopulariopsis brevicaulis was isolated in 7.5% was obtained from Laboratorios Sigma (G 4753). cases, Acremonium spp. and Fusarium solani in two The MIC was determined 72 h after incubation at (I%), while dermatophytes accounted for the remain- 30°C by visual reading on an inverted mirror, making ing 67.7% and yeasts 18%. a spectrophotometric reading also at 405 nm. The All samples were planted in Petri plates with MICs end-points were defined as the lowest drug con- Sabouraud dextrose agar with chloramphenicol centration which resulted in 80% reduction of fungal (Difco, Detroit, MI, USA) and added in the same growth compared with the drug free control. medium cycloheximide. In each plate, the nail flakes All determinations were done in duplicate. were distributed in 12-20 inoculum spots. Part of the material was used for fresh microscopic examination with 40% potassium hydroxide. 3. Results In order to confirm the etiological role of a mold as the causative agent of an ungual lesion, we followed 3.1. Clinical characteristics the criteria proposed by English et al. [8]; thus, in the 12 cases where A. versicolor was isolated, culture was Six of the twelve patients were male and six female. repeated two or more times to confirm the result. All Except for two cases, they were all from the Barcelona J.M. Torres-Rodriguez er (11. / J. Eur. Acad. Dermarol. Venereal. 1 I (1998) 25-31 27

Table I

Cases of onychomicosis due to Aspergillus wrsicolor diagnosed between 1988 and 1995 Patient Sex Age Nail Microscopic Evolution Associated (years) affected examination (years) conditions

SH Male 83 v1 Hypha + conidia 20 Heart disease JP Male 60 I.VI Hypha + conidia 3 Trauma LZ Female 33 VI HYPha 3 - JPA Male 54 I.VI HYPha 7 - EG Female 60 I.VI Hypha + conidia 8 Diabetes GP Female 60 I,II,III HYPha 8 Diabetes. arthritis ISM Male 57 1.VI HYPha I - CER Female 69 I.VI HYPha 20 Venous insuffic. EMZ Male 70 1,VI HYPha 5 - FBP Female 58 I.VI. HYPha ? ,? CMS Female 59 I.VI,VIII HYPha 4 - DVR Female 63 I.VI HYPha ? -

metropolitan area; the other two had permanent Time of evolution of nail lesions could be deter- addresses in Valencia and Madrid, respectively. The mined in 10 patients and it was longer than 3 years in demographic and clinically significant data for all 12 most of them; one case had a history of more than 20 patients studied is summarised in Table 1. years. The nail lesions were similar in all cases: pachyo- Most patients had undergone treatment with topical nychia with subungual hyperkeratosis and distal ony- antifungals (mostly azole compounds) in one or more cholysis, which was dark brown in colour, in most occasions, without clinical improvement. cases covering 75% or more of the ungual surface Associated conditions were found in four patients, (Fig. 1). The only nails involved in all patients were with a predominance of diabetes and venous circula- the big toenail in three cases and bilateral in all nine tory insufficiency in the lower limbs. One patient had remaining cases. Most nails were classified as disto- a history of significant local trauma prior to develop- lateral onychornycosis but in four patients total dys- ment of infection. trophic onychomycosis was found. No clinical Samples from the internal plateau of the nail were differences from other etiological agents including easily obtained by scraping with a sterile scoop or dermatophytes and Scopulariopsis brevicaulis could scalpel; direct examination was positive in all cases. be observed. In nine cases there were hyphal fragments, and hypha together with conidial structures in samples from three patients. In two cases, nail biopsy was per- formed and examination showed the presence of PAS-positive branched hyphae (Fig. 2).

3.2. In vitro sensitivity to antifungals

MICs were highly variable depending on the antifungal under test (Table 2). Griseofulvin and fluconazole showed the highest MICs, thus it was assumed that all strains were resistant to these antifungals. In the case of amphotericin B, seven strains had MICs higher than 64 pg/ml and the rest always showed Fig. 1. Onychomycosis caused by Aspergillus versicolor affecting values >4 pg/ml. both big toenails. MICs for ketoconazole and itraconazole were 28 J.M. Torres-Rodriguez et al. / J. Eur. Acad Dermatol. Venereol. I I ( I998) 25-3 I

role is debatable. The same is true of some mycelial fungi such as Fusarium, Acremonium and Aspergillus. There is merit in using English’s proposals [8] when it comes to considering whether these fungi are pathogenic or not. In the 12 patients analyzed in this study, which represented 5.8% of the total onychomycosis of the feet diagnosed, A. versicolor was isolated in cultures of several samples taken on different days, thus ful- filling the criteria that define this as a mycosis. The first case detected in our environment was a male patient with onychogryphosis and peripheral circulatory insufficiency from whom A. versicolor was iso- Fig. 2. Nail biopsy of onychomycosis due to Aspergillus versicolor. lated on three occasions, and it was also isolated from Several fragments of irregular septate hyphae can be seen. PAS the toenails after removal, with histological verifica- I OOOX. tion of invasion by filaments from this species [6]; since then our laboratory has had more experience similar, although some strains were more susceptible in identifying this species in cases of onychomycosis, to itraconazole; on the contrary, susceptibility to and in determining their clinical characteristics. Nail terbinafine was very high since the highest MIC lesions caused by A. versicolor were indistinguishable was I Fg/ml and seven strains had values below from those produced by other moulds and dermato- 0.125 pg/ml. phytes, especially when total dystrophia was observed. In the series described in this report, a similar inci- 4. Discussion dence was found for both sexes with an average age of 60.5 years (33-83 years). The only nails affected were Most fungal nail infections affecting the feet corre- toenails, primarily the big toe, as often occurs in spond to unguium tinea, with Trichophyton rubrum almost all cases of onychomycosis by opportunistic and T. mentagrophyres as the main etiological agents molds [ 121 if we exclude ScyralidiumlHendersonula [ 121, but is not uncommon either to find in the affected spp that also affects fingernails [13]. Most patients nails yeasts of the Candida genus, specifically C. studied (nine cases) presented with bilateral involve- parapsilosis, although in some cases their etiological ment.

Table 2

MIC distribution of I2 Aspergillus versicolor strains isolated from onychomycosis against six antifungal drugs Strain Amphot B Griseof Terbinaf Itracon Flucon Keto

~ 4456 >64 >64 0.25 1 64 2 4484 >64 %4 64 0.25 2 64 2 95.210 >64 >64 0.50 I 64 2 CETC 28 14 >64 %4 0.50 1 >a I J.M. Torres-Rodriguez er a/. / J. Eur. Acad. Dermatol. Venereol. I1 (1998) 25-31 29

where no specific morphological features were seen in the hyphae. / Therapeutic failure was documented in all patients since all had received local treatment mainly with topical azoles, for years or months prior to cultures with no noticeable improvement. The poor sensitivity of the Aspergillus fungi to the antifungals used in the treatment of onychomycosis encouraged studies of their in vitro susceptibility using a standar- dised method based on NCCLS recommendations [lo]. Although amphotericin B is not used in the treatment of this type of mycosis, it was taken as the reference antifungal; also the possibility of A. versi- Fig. 3. Pure culture of Aspergillus versicolor on agar-Sabouraud color causing systemic infections that could be treated with cloramphenicol. with this polyene cannot be ruled out. It is not surprising that all strains, including the one Only in one of the five patients, was a history of used as reference, are resistant to griseofulvin and significant toenail trauma (case no. 2) documented fluconazole since these antifungals are not active which appeared to have been the predisposing factor; against Aspergillus fumigatus [ 15,161, and although in the other four cases underlying conditions were there is no specific data available on A. versicolor, it present that have been described previously as favour- can be assumed that the absence of activity will also ing the development of nail infections. These condi- be the rule for this species. tions were diabetes and circulatory disorders, which When itraconazole was evaluated, only two strains are in general the same as those described for other showed MICs higher than 2 pg/ml; this data coincides onychomycoses [ 141. The absence of local or general with that from Espinel Ingroff et al. [ 1 I], for 10 strains factors that favour onychomycosis in most patients of A. fumigatus in which MIC values ranged from studied, would support a primary pathogenic potential 0.063-2 Fg/ml, even though these values are higher for this species in the healthy nail; however, consider- than those found for A. flavus in the top range of the ing the prolonged evolution of this disease, more than MIC which was 0.13 pg/ml. The range for MIC90 3 years in nearly all patients, and also the multiple described by Van Cutsem et al. [17], however, was antifungal treatments applied, one cannot rule out much higher for species such as A. niger ( 10- 100 pg/ the possibility that the primitive nail lesion was ml). caused by a dermatophytic fungus, as has been pro- The range found for ketoconazole in A. versicolor posed by several authors [ 121; in such cases secondary was lower than that described for A. fumigatus (4- 16 colonization by Aspergillus would be possible. pg/ml) [ 1 I, 181. Mycological diagnosis of infection by Aspergillus With regard to amphotericin B, there is no infor- was suspected in two cases where direct microscopic mation on A. versicolor. The most commonly studied examination showed large quantities of conidia, species within the Aspergillus genuq have been A. together with fragments of hyphae. Since the charac- fumigatus and A. Jlavus, with MICs usually in the teristics of these hypha did not differ from those seen range from 0.1 to 1.0 pg/ml, and therefore it is sur- in the unguium tinae, the definite diagnosis can only prising that all A. versicolor strains have presented be confirmed by mycological culture. with MICs higher than 4 pg/ml, and three of Repeated isolation in pure culture of a considerable them equal to or higher than 64 pg/ml. According to number of colonies (Fig. 3) warrants the diagnosis these results all strains studied are resistant to this onychomycosis due to A. versicolor. Nail invasion pol yene. was histopathologically confirmed in the two cases The most remarkable finding in this study was the where it was possible to conduct a biopsy, and extreme susceptibility of all strains to terbinafine, with 30 J.M. Torres-Rodriguez et al. / J. Eur. Acad. Dermatol. Venereol. I1 (1998)25-31

MICs in four strains lower than the maximum dilution human and animal health. J Med Vet Mycol, 1994 of the antifungal taken as the starting point in this 32(Suppl. 1):17-32. study (0.125 bg/ml). Although there is no data on A. [S] Raper KB, Fennel1 DI. The Genus Aspergillus. Huntington, NY: Krieger, 1973. versicolor, the high sensitivity of Aspergillus species [6] Torres-Rodriguez JM, Balager-Meler J, Antonella Reixach J. to this antifungal is well-known but the MICs ranges Onychomycosis due to a fungus of the Aspergillus versicolor described by Clayton were less constant than those group. Mycoses 1988;31:579-583. seen in this study [19]. The results obtained show [7] Rippon JW. Medical Mycology: The Pathogenic Fungi and MIC values lower than those described for A. Jlavus, the Pathogenic Actinomycetes. Philadelphia, PA: Saunders, 1988:214-215. A. fumigatus and A. niger [20]. ’ [8] English MP. Comment: nails and fungi. Br J Dermatol This data suggests that itraconazole and terbinafine 1973;94:697-701. (the latter has in vitro fungicidal action) [21] could be [9] de Hoog GS, Guarro J. Atlas of clinical fungi. Baam-Reus. used in the oral treatment of fungal infections caused Centraalbureau voor Schimmel-cultures-UniversitatRovira i Virgili. Baarn: Centraalbureau, 1995:456. by A. versicolor. This assumption should be con- 101 Espinel-Ingroff A, Dawson K, Pfaller M, et a]. Comparative firmed in further therapeutic trials. The administration and collaborative evaluation of standadzation of antifungal of terbinafine in a few cases of systemic aspergillosis susceptibility testing for filamentous fungi. Antimicrob for ‘compassionate’ programs has been recorded [22]; Agents Chemother 1995;39:314-319. Schiraldi et al. [23] recently reported favourable ther- Ill Espinel-Ingroff A, Shadomy S, Gebhart R. In vitro studies with R 51.21 1 (itraconazole). Antimicrob Agents Chemother apeutic results with good tolerance of treatment in 1984;26:5-9. three immunocompetent patients with chronic asper- [12] del Palacio-Herranz A, Garcia-Bravo M. Onicomicosis. In: gillus empyema (one case), and chronic necrotizing Torres Rodriguez JM, del Palacio-Hemanz A, Guarro Artigas aspergillosis in the other two, all of them caused by A. J, Negroni-Briz R, Pereiro-Miguens M. Micologia Mkdica. fumigatus. This data allows us to propose the alterna- Barcelona: Masson, 1993:65-73. tive therapy of onychomycosis caused by Aspergillus [13] Moore MK, del Palacio A. Dermatomicosis por Scytalidium. En Torres Rodriguez JM, del Palacio-Hemanz A, Guarro versicolor with terbinafine, given the accumulation of Artigas J, Negroni-Briz R, Pereiro-Miguens M. Micologia this antifungal in the nail tissue and its fungicidal Medica. Barcelona, Masson SA, 1993:65-73. effect [ 20,241. [ 141 Haneke E. Epidemiology and pathology of onychomycoses. In: Nolting S, Korting HC. Onychomycoses. Berlin: Springer- Verlag, 1990: 1 - 1I. Acknowledgements [I51 Davies RR, Griseofulvin. In: Speller DCE. Antifungal Che- motherapy. New York: Wiley, 1980:149-182. [ 161 Maniot MS, Richardson K. The discovery and mode of action The authors wish to thank Professor Dr. Rod Hay of fluconazole. In: Fromtling RA, editor. Recent Trends in the from St. John’s Institute of Dermatology for his cri- Discovery, Development and Evaluation of Antifungal tical review of our manuscript and Dr. Josepa GenC Agents. Barcelona: Prous Sci, 1987:8 1-92. from the University Rovira i Virgili (Reus, Spain) for [I71 Grant SM, Clissold SP. Itraconazole. A review of its pharma- codynamic and pharmacokinetic properties, and therapeutic confirming the identification of the isolates. use in superficial and systemic mycoses. Drugs 1989;37:3 10- 344. [ 181 Kitahara M, Seth VK, Medoff G, Kobayashi GS. Antimicro- References bial susceptibility testing of six clinical isolates of Aspergil- lus. Antimicrob Agents Chemother 1976;9:908-9 14. [I91 Clayton IM. The in vitro activity of terbinafine agairlst [ 1 ] Madrenys-Brunet N, Torres-Rodriguez JM, Urrea-Arbelaez A. Estudio epidemiologico de las micosis ungueales en Bar- uncommon fungal pathogens. In: Fromtling RA, editor. celona. Rev Iberoamer Micol 1996;14:14-17. Recent Trends in the Discovery, Development and Evaluation [2] Wilken-Jensen K, Gravesen S. Atlas of moulds in Europe of Antifungal Agents. Barcelona: Prous Sci, 1987:433- 439. causing respiratory allergy. Copenhagen, ASK, 1984. [20] Schmitt HJ, Bernard EM, Andrade J, Edwards F, Schmitt B, [3] Nolard N, Detandt M, Beguin H. Ecology of Aspergillus Armstrong D. MIC and fungicidal activity of Terbinafine species in the human environment. In: Vaanden Bossche against clinical isolates of Aspergillus spp. Antimicrob H, Mackenzie DWR, Cauwenbergh G, editors. Aspergillus Agents Chemother 1988;32:780. and Aspergillosis. New York: Plenum Press, 1987:35- [21] Balfour JA. Faulds Terbinafine: a review of its pharmacody- 41. namic and pharmacokinetic properties and therapeutic poten- [41 Pitt JI. The current role of Aspergillus and Penicillium in tial in superficial mycoses. Drugs 1992; 43:259-284. J.M. Torres-Rodriguez et al. /J. Eur. Acad. Dermatol. Venereol. I1 (1998) 25-31 31

[22] Villars VV, Jones TC. Special features of the clinical use of compassionate treat with terbinafine. Br J Dermatol 1996; oral terbinafine in the treatment of fungal diseases. Br J Der- 134(S~ppl.46):30-32. matol 1992; 126(Suppl. 39):61-69. [24] Wamock DW. New pharmacological concepts in antimycotic [23] Schiraldi GF, Lo Cicero S, Colombo MD, Rosasato D, treatment. J Eur Acad Dermatol Venerol 1993;2(Suppl. Ferrarese M, Soresi E. Refractory pulmonary aspergillosis l):S19-S25.