Clinical Spectrum of Exophiala Infections and a Novel Exophiala Species, Exophiala hongkongensis

Abstract

We characterized 12 Exophiala strains isolated from patients over a 15-year period to the species level using phenotypic tests and internal transcribed spacer (ITS) and Rpb1 sequencing and described the clinical spectrum of the 12 patients. Eight patients had nail or skin infections, two had invasive infections, and two had colonization of the gastrointestinal tract. ITS and Rpb1 sequencing showed that 11 of the 12 strains were known Exophiala species (E. oligosperma [n = 3], E. jeanselmei [n = 2], E. lecanii-corni [n = 2], E. bergeri [n = 1], E. cancerae [n = 1], E. dermatitidis [n = 1], and E. xenobiotica [n = 1]), which included the first reported cases of onychomycosis caused by E. bergeri and E. oligosperma. The 12th strain (HKU32^T), isolated from the nail clipping of the right big toe of a 68-year-old female patient with onychomycosis, possessed unique morphological characteristics distinct from other Exophiala species. It grew very slowly and had a velvety colony texture after 28 days, short conidiophores of the same olivaceous color as the supporting hyphae, numerous spores, and no chlamydospore-like cells. ITS, Rpb1, β-tubulin, and β-actin gene sequencing unambiguously showed that HKU32^T was clustered with but formed branches distinct from other Exophiala species in phylogenetic trees. We propose the new species Exophiala hongkongensis to describe this novel fungus.

INTRODUCTION

Exophiala is a genus of saprophytic fungi that have been isolated from environments rich in hydrocarbons (1–3) or from hot, humid, and oligotrophic environments, such as dishwashers (4), steam bath facilities (2), and bathrooms (5). This genus belongs to the family Herpotrichiellaceae and currently consists of more than 30 species. Traditionally, these fungi are considered dematiaceous molds (6). Due to their phenotypic characteristics at the beginning of colony formation, these fungi are often also referred to as “black yeasts” (7), a misnomer which sometimes may mislead the choice of antifungal agents. When the cultures mature, brown hyphae are formed which bear conidiogenous cells referred to as annellides, a typical characteristic of this genus of fungi (8).

Although Exophiala species are environmental fungi, they should not be disregarded as contaminants when they are isolated from clinical specimens. These fungi are causative agents of skin and subcutaneous tissue infections and of systemic infections, such as prosthetic valve endocarditis, dialysis-associated peritonitis, and disseminated infections, especially in immunocompromised patients (9–17). In this study, we report the clinical spectrum of Exophiala infections in our hospital, characterized by phenotypic examination and sequencing of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA gene cluster (referred to hereinafter as ITS) and RNA polymerase II's largest subunit gene (Rpb1). During the process, we also discovered a potentially novel Exophiala species from the toe nail clipping of a patient with onychomycosis. The strain, named HKU32^T, exhibited phenotypic characteristics that do not fit into the pattern of any known Exophiala species. Amplification and sequencing of four independent DNA regions showed that it is distinct from all other Exophiala species. On the basis of these studies, we propose a new species, Exophiala hongkongensis sp. nov., to describe this fungus.

MATERIALS AND METHODS

Patients and strains.

All Exophiala strains isolated from patients in this study were retrieved from the collection in the clinical microbiology laboratory at the Queen Mary Hospital in Hong Kong during a 15-year period (1998 to 2012). All clinical data were collected as described in our previous publication (18). Clinical specimens were collected and handled according to standard protocols. The reference strains, including E. jeanselmei (CBS 507.90^T), E. nishimurae (CBS 101538^T), E. oligosperma (CBS 265.49^T), E. spinifera (CBS 899.68^T), and E. xenobiotica (CBS 118157^T), were purchased from The Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre.

Phenotypic characterization.

All strains were inoculated onto Sabouraud dextrose agar (SDA) (Sigma-Aldrich, St. Louis, MO) for fungal culture. Slides for microscopic examination were prepared using the agar block smear method we described recently (19). The enzyme activity test was performed using the API-ZYM system (bioMérieux SA, Marcy l'Etoile, France). All tests were performed in triplicate. The effects of different temperatures on growth on potato dextrose agar (PDA) (Becton, Dickinson and Company, Franklin Lakes, NJ) and comparison of growth rates on PDA, brain heart infusion (BHI) agar (Becton, Dickinson and Company, Franklin Lakes, NJ), cornmeal agar (CMA) (Sigma-Aldrich, St. Louis, MO), and oatmeal agar (OMA) (Becton, Dickinson and Company, Franklin Lakes, NJ) at 24°C were studied using published protocols, with slight modifications (20–22). Briefly, conidia were harvested in distilled water from 7-day cultures on SDA. Concentrations of conidia were determined using a hemocytometer. A circular cavity was made at the center of each agar plate using a Pasteur pipette. Five thousand spores were inoculated into the central cavity of each agar plate. PDA plates were incubated at temperatures ranging from 24 to 37°C, and other agar plates were incubated at 24°C. The radii of colonies were measured in four directions after 14 days of incubation. All assays were performed in triplicate, and the mean radii were calculated. Scanning electron microscopy of strain HKU32^T was performed according to the protocol described in our previous publication (23).

DNA extraction and ITS, Rpb1, β-tubulin, and β-actin gene sequencing.

Fungal DNA extraction, PCR amplification, and DNA sequencing of the ITS and partial Rpb1 gene for all 12 Exophiala strains isolated from our patients and the reference strains and of the partial β-tubulin and β-actin genes for HKU32^T and the reference strains were performed according to the protocols described in our previous publication (24). Briefly, fungal cells on SDA were harvested with a sterilized cotton swab and suspended in 1 ml of autoclaved distilled water. DNA was then extracted from the fungal cells using the DNeasy plant minikit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted DNA was eluted in 40 μl of distilled water, and 1 μl of the extracted DNA was used for PCR. For PCR, each 20-μl PCR mixture contained diethyl pyrocarbonate (DEPC)-treated water (Invitrogen, Carlsbad, CA), fungal DNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 3.125 mM MgCl₂) (Applied Biosystems, Foster City, CA), 1 mM each primer (Invitrogen, Carlsbad, CA) (Table 1), 200 μM each deoxyribonucleoside triphosphate (dNTP), and 0.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). The PCR mixtures were first heated at 95°C for 10 min, then heated in 45 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and finally incubated at 72°C for 10 min in an automated thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA). Preparation of PCR master mix, addition of DNA samples into the reaction tubes, and post-PCR steps, including gel electrophoresis, purification of PCR products, and DNA sequencing were performed in separate rooms to avoid possible contamination. Autoclaved distilled water was used as the negative control in each run of PCR. Ten microliters of each amplified product was electrophoresed in 1.5% (wt/vol) agarose gel (SeaKem LE agarose), and the PCR products were purified using the QIAquick gel extraction kit (QIAgen, Hilden, Germany). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA), using the PCR primers. The sequences of the PCR products were processed using BioEdit 7.1.3.0 (25) and compared with sequences of closely related species in GenBank by multiple sequence alignment using MUSCLE 3.8 (26).

Table 1.

Primer sequences used for PCR amplification in this study

Target region	Primer, sequence
	Forward	Reverse
ITS	ITS1, 5′-TCCGTAGGTGAACCTGCGG-3′	ITS4, 5′-TCCTCCGCTTATTGATATGC-3′
Rpb1 gene	LPW20506, 5′-TYMTGRSNARRGTCAAGAAGAT-3′	LPW20507, 5′-GGNAGBGMVGABARRATCATCCA-3′
β-tubulin gene	LPW17603, 5′-SWVRTCTCDGGMGAACAYGGTCT-3′	LPW17604, 5′-HRTKKCTTACCAGCACCGCT-3′
β-actin gene	LPW17499, 5′-CGTGTCGAYATGGCTGGTCG-3′	LPW17500, 5′-GGHGCRATRATCTTGACCTTCAT-3′

Phylogenetic characterization.

Poorly aligned or divergent regions of the aligned DNA sequences were removed using Gblocks 0.91b (27, 28) with relaxed parameters. Testing for the substitution model and phylogenetic tree construction, by the maximum likelihood method, were performed using MEGA 5.0.5 (29). Phylogenetic analyses included 468 nucleotide positions of the ITS, 371 nucleotide positions of the partial Rpb1 gene, and 641 nucleotide positions of the concatenated partial β-tubulin gene and the partial β-actin gene sequence.

Nucleotide sequence accession numbers.

The ITS and partial Rpb1 gene sequences of the Exophiala strains and the partial β-tubulin and β-actin gene sequences of HKU32^T and E. nishimurae CBS 101538^T have been deposited in GenBank under the accession numbers listed in Table 2.

Table 2.

GenBank accession numbers of the ITS and partial Rpb1, β-tubulin, and β-actin gene sequences of the 12 Exophiala strains from patients and the control strains

Strain (case)	GenBank accession no.
	ITS	Partial
		Rpb1 gene	β-Tubulin gene	β-Actin gene
PW2461 (1)	JX473275	JX498926
PW2482 (2)	JX473282	JX498933
PW2462 (3)	JX473276	JX498927
PW2464 (4)	JX473278	JX498929
PW2534 (5)	JX473283	JX498934
PW2468 (6)	JX473281	JX498932
PW2465 (7)	JX473279	JX498930
PW2535 (8)	JX473284	JX498935
PW2466 (9)	JX473280	JX498931
HKU32T (10)	JN625231	JX498924	JN625236	JN625241
PW2642 (11)	JX473285	JX498936
PW2643 (12)	JX473286	JX498937
CBS 101538T	JX473274	JX498925	JX482552	JX482553

RESULTS

Phenotypic characterization and ITS and Rpb1 sequencing of Exophiala strains and identification of a novel Exophiala species.

On SDA, the colonies of all 12 strains were initially brown to black, moist, and yeastlike. All mature colonies became velvety, with the front color gray-to-black-olivaceous and the reverse black. Microscopic examination of the young cultures of all the 12 strains showed subspherical, budding, yeastlike cells. As the cultures became mature, septate hyphae which bore tubular and rocket-shaped annellides that tapered to form narrow elongated tips were observed. Ellipsoidal conidia, in clusters at the apices of the annellides or at the sides of the conidiophores, were produced from the annellides.

PCR of the ITS and partial Rpb1 gene of the 12 strains and the reference strains showed bands at about 600 bp and 500 bp, respectively. Sequencing of the ITS showed that 11 of the 12 strains were known Exophiala species (Fig. 1). Sequencing of the partial Rpb1 gene revealed results concurring with those of ITS sequencing (data not shown). As for HKU32^T (case 10), although it is most closely related to E. nishimurae CBS 101538^T, there was a 46 (8.0%)-base difference between the ITS of HKU32^T and that of E. nishimurae CBS 101538^T and a 37 (8.1%)-base difference between the partial Rpb1 gene of HKU32^T and that of E. nishimurae CBS 101538^T.

Fig 1.

Phylogenetic trees showing the relationships of the 12 strains of Exophiala species in this study to known Exophiala species. The tree was inferred from ITS sequence data by the maximum likelihood method with the substitution model GTR+G+I (general time-reversible model, with gamma-distributed rate variation [G] and an estimated proportion of invariable sites [I]). The scale bar indicates the estimated number of substitutions per 50 bases. Numbers at nodes indicate levels of bootstrap support calculated from 1,000 trees. All names and accession numbers are given as cited in the GenBank database.

Clinical spectrum of Exophiala infections.

The clinical characteristics of the 12 patients with Exophiala species isolated are shown in Table 3. The male-to-female ratio was 1:1. The median age was 66 years (range, 3 to 88). Eight patients had infections of the nails or skin (four had onychomycosis, two had skin nodules, and two had chronic skin infections), two patients had invasive infections (one had continuous ambulatory peritoneal dialysis [CAPD] peritonitis and one had pneumonia), and two had colonization of the gastrointestinal tract. All patients recovered from the Exophiala infections.

Table 3.

Characteristics of patients with Exophiala species isolated in the present study

Case (reference)	Yr of isolation	Sexa/age (yr)	Underlying condition(s)a	Diagnosis	Clinical specimen	Identification by ITS and Rpb1 sequencing
1	1998	F/3	Cord blood transplant recipient for β-thalassemia major	Colonization of gastrointestinal tract	Stool	E. cancerae
2 (13)	2000	M/66	End-stage renal failure on CAPD	CAPD peritonitis	Peritoneal dialysate	E. xenobiotica
3	2002	F/79	Tuberculous cervical lymphadenitis, hypertension	Right wrist nodule for 5 years	Wrist nodule	E. jeanselmei
4	2008	M/86	DM, carcinoma of rectum, hypertension, ischemic heart disease, COPD	Right middle finger nodule for 3 years	Finger nodule	E. jeanselmei
5	2008	F/87	Bullous pemphigoid, DM, hypertension, Alzheimer's disease	Tinea pedis	Skin scrapping	E. lecanii-corni
6	2009	M/37	None	Onychomycosis	Toe nail	E. bergeri
7	2009	M/23	None	Onychomycosis	Big toe nail	E. oligosperma
8	2009	M/66	None	Pneumonia	Bronchoalveolar lavage fluid	E. oligosperma
9	2009	F/51	None	Onychomycosis	Thumb nail	E. oligosperma
10	2010	F/68	Hypertension	Onychomycosis	Big toe nail	Novel species
11	2012	M/88	DM, gout, recurrent cellulitis	Chronic skin infection	Skin scrapping	E. lecanii-corni
12	2012	F/43	AML, PBSCT	Colonization of gastrointestinal tract	Stool	E. dermatitidis

^aF, female; M, male; CAPD, continuous ambulatory peritoneal dialysis; DM, diabetes mellitus; COPD, chronic obstructive pulmonary disease; AML, acute myeloid leukemia; PBSCT, peripheral blood stem cell transplant.

Enzymatic activities of HKU32^T.

The API-ZYM test for HKU32^T showed that it was positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase, and N-acetyl-β-glucosaminidase in all replicates.

Effects of temperature and medium on growth of HKU32^T.

On PDA, HKU32^T grew at 24°C, 30°C, 33°C, and 37°C, with optimal growth at 30°C. HKU32^T also grew on CMA, OMA, and BHI agar at 24°C, with the fastest and the slowest growth observed on CMA and BHI agar, respectively.

Partial β-tubulin and β-actin gene sequencing of HKU32^T.

PCR of the partial β-tubulin and β-actin genes of HKU32^T and the reference strains showed bands at about 300 bp and 500 bp, respectively. Sequencing and phylogenetic analysis showed that HKU32^T is most closely related to E. nishimurae CBS 101538^T, concurring with the results of ITS and Rpb1 gene sequencing (Fig. 2). Pairwise alignment showed that there was a 28 (9.5%)-base difference between the partial β-tubulin gene of HKU32^T and that of E. nishimurae CBS 101538^T and a 36 (7.6%)-base difference between the partial β-actin gene of HKU32^T and that of E. nishimurae CBS 101538^T.

Fig 2.

Concatenated phylogenetic tree showing the relationship of E. hongkongensis HKU32^T to closely related species. The tree was inferred from actin and β-tubulin sequence data by the maximum likelihood method with the substitution model K2+G+I (K2 referring to the Kimura two-parameter model). The scale bar indicates the estimated number of substitutions per 50 bases. Numbers at nodes indicate levels of bootstrap support calculated from 1,000 trees. All names and accession numbers are given as cited in the GenBank database, with the first accession numbers in the parentheses referring to the β-actin gene sequences and the second accession numbers in the parentheses referring to the β-tubulin gene sequences.

TAXONOMY

Description of Exophiala hongkongensis Woo, Ngan, Tsang, Ling, Chan, Leung, Yuen, Lau, sp. nov. MycoBank accession no. MB563298. Teleomorph: unknown. Known distribution: Hong Kong. Etymology: hong.kong.en′sis. N.L. fem. adj., named after Hong Kong, where the type strain was isolated. Specimen examined: Hong Kong; from the big toe nail of a human presenting with onychomycosis in 2010 (holotype: dried culture in NBRC Herbarium H-13132; ex-type cultures: HKU32^T [= NBRC 109366^T = JCM 18697^T = CBS 131511^T]).

On SDA, HKU32^T initially grew slowly as black, slimy, yeast-like colonies with a diameter of 2 mm after incubation at 25°C for 14 days. Subsequently, the centers of the colonies became velvety, gray-olivaceous, and dome-shaped with black reverse, but the margin remained slimy and yeastlike after 28 days of incubation at 25°C (Fig. 3a and b). Microscopic examination of the yeastlike colonies at 14 days of incubation after lactophenol cotton blue staining revealed brown yeast cells that were ellipsoidal, had an average size of 6 by 3.5 μm, and possessed short annellated zones to form budding cells (Fig. 3c). Mycelia were usually swollen and formed torulose hyphae with annellated zones found at their ends (Fig. 3c). At the subsequent velvety stage, septate mycelia were not swollen and conidia were formed distinctively in clusters (Fig. 3d and e). The clusters of conidia were either intercalary or found at the tips of free conidiophores (Fig. 3d and e). Under high-power magnification, intercalary annellides were observed in the form of short annellated pegs (Fig. 3f). These intercalary annellides were present along the creeping septate hyphae. Free conidiophores consisting of 1 or 2 cells with inconspicuous short annellated tips were also observed (Fig. 3g). They possessed the same olivaceous color as the creeping septate hyphae and were often constricted at their bases (Fig. 3g). At both stages, conidia were formed by percurrent growth through annellated zones. Most of them were oval or subglobose, with an average size of 3 by 2 μm, and some with truncated scars. No synanamorphs of Phialophora or Rhinocladiella types were observed. Scanning electron microscopic examination of HKU32^T at 14 days of incubation revealed budding yeast cells and torulose hyphae with distinct annellated zones, consistent with the results observed on light microscopy (Fig. 3h and i). In addition, yeast cells with truncated scars were also observed (Fig. 3h).

Fig 3.

Culture of HKU32^T on SDA after 28 days of incubation at 25°C showed a black slimy colony with gray olivaceous center (a) and black reverse (b). At the initial yeast stage (14 days of incubation), yeast and torulose hyphae with annellated zones were observed with light microscopy (c) and scanning electron microscopy (h and i). Yeast cells with truncated scars were observed (h), and distinct annellated zones were well demonstrated (h and i). At the subsequent velvety stage (28 days of incubation), conidia in clusters were either intercalary or found at the tips of free conidiophores under both low-power (d) and high-power magnification (e). Intercalary annellides with short pegs (f) and free conidiophores with constricted bases (g) were observed.

DISCUSSION

Using the polyphasic approach with a combination of phenotypic and genotypic techniques, we describe a wide variety of Exophiala species associated with different forms of clinical infections. Although Exophiala can often be identified to the genus level by morphological characteristics, identification of Exophiala to the species level by phenotypic characterization alone is very difficult. This difficulty is exemplified by our description of the first case of CAPD peritonitis caused by Exophiala in 2000 (Table 3, case 2) (13). At that time, we could not identify the Exophiala to the species level. Due to the advancement of molecular techniques and availability of DNA sequences of different gene loci in sequence databases such as GenBank, identification of Exophiala to the species level has been made possible. In this study, by sequencing two independent gene loci (ITS and Rpb1), eight different Exophiala species, including E. oligosperma, E. jeanselmei, E. lecanii-corni, E. bergeri, E. cancerae, E. dermatitidis, E. xenobiotica, and a novel Exophiala species, were isolated from 12 patients (Table 3 and Fig. 1). These Exophiala species were found to be associated with both superficial infections and invasive infections, including the case of CAPD peritonitis caused by E. xenobiotica (Table 3, case 2) and another case of pneumonia caused by E. oligosperma (Table 3, case 8). In two patients with cord blood and peripheral blood stem cell transplant, respectively, E. cancerae and E. dermatitidis were the colonizers of their gastrointestinal tracts (Table 3, cases 1 and 12).

Phenotypic and genotypic analysis revealed that HKU32^T is a novel species in the genus Exophiala, which we propose to name E. hongkongensis. Using four independent DNA regions widely used for phylogenetic analysis, including ITS and three housekeeping genes (Rpb1, β-tubulin, and β-actin genes), it was shown unambiguously that E. hongkongensis is closely related to but distinct from other Exophiala species (Fig. 1 and 2). Among the Exophiala species, E. hongkongensis is most closely related to E. nishimurae, as shown in all phylogenetic trees with high bootstrap supports. Comparison of phenotypic characteristics between E. hongkongensis and those of other closely related Exophiala species shown by ITS and Rpb1 sequencing also revealed unique phenotypic characteristics of E. hongkongensis (Table 4). E. hongkongensis grew very slowly and had a velvety colony texture after 28 days, short conidiophores of the same olivaceous color as the supporting hyphae, numerous spores, and no chlamydospore-like cells.

Table 4.

Comparison of phenotypic features of E. hongkongensis and closely related Exophiala species

Species	Colony texture after 28 days of incubation at 25°C on SDA	Colony diam after 14 days of incubation at 25°C on SDA	Average length of conidiophores (μm)	Darkening of conidiophores	Abundance of spores	Presence of chlamydospore-like cells
E. hongkongensis HKU32T	Velvety	2 mm	10	Same color as supporting hyphae	Numerous	Absence
E. jeanselmei CBS 507.90T	Velvety	60 mm	12	Inconspicuous	Normal	Absence
E. nishimurae CBS 101538T	Velvety	5 mm	12	Same color as supporting hyphae	Numerous	Presence
E. oligosperma CBS 265.49T	Velvety	50 mm	>100, branched	Same color as supporting hyphae	Few	Absence
E. spinifera CBS 899.68T	Slimy	40 mm	50	Conspicuous	Normal	Absence
E. xenobiotica CBS 118157T	Slimy	20 mm	15, branched	Same color as supporting hyphae	Normal	Absence

This study includes the first reported cases of onychomycosis associated with E. hongkongensis, E. bergeri, and E. oligosperma. Although Exophiala species are relatively common causes of subcutaneous and skin infections in the forms of phaeohyphomycosis, chromoblastomycosis, and mycetoma, they have only occasionally been reported to cause onychomycosis. Among the seven cases reported in the literature, four were caused by E. dermatitidis and three were caused by E. jeanselmei (Table 5), whereas for the four patients with Exophiala onychomycosis in the present study, two were caused by E. oligosperma, one was caused by E. bergeri, and one was caused by E. hongkongensis (Table 3). Exophiala species are global pathogens of onychomycosis, with cases reported from Asia, Europe, America, and Africa. Among the seven cases reported in the literature, underlying diseases leading to immunosuppressive states were present in three patients, including diabetes mellitus in two patients and renal transplantation in one (Table 5), whereas for the four patients with Exophiala onychomycosis in the present study, only the patient with E. hongkongensis onychomycosis had underlying hypertension (Table 3). In all 11 patients, the nails were infected by the fungi for one to a few years before presentation, indicating that the disease was a very indolent process with relatively mild symptoms and, hence, patients tended to observe them for a time and delay in seeking medical advice. The nails of the big toes were affected in eight patients (Tables 3 and 5). Due to underreporting and difficulty in making the microbiological diagnosis, the number of cases of Exophiala onychomycosis is probably underestimated. This is in line with the results of a recent study, which also suggested that the incidence of superficial infections caused by black yeastlike fungi could be underestimated (37). Another study also noted that nonthermophilic Exophiala species may expand in diabetic patients with poor blood circulation (38). A combination of phenotypic and genotypic techniques using the polyphasic approach in microbiology laboratories will facilitate the understanding of the epidemiology of Exophiala onychomycosis, as well as other infections associated with Exophiala species.

Table 5.

Cases of onychomycosis caused by Exophiala species reported in the literature

Reference	Geographical location	Sex/age (yr)a	Underlying disease	Nail(s) involved	Clinical presentation	Exophiala species isolated
Matsumoto et al. (30)	Japan	F/51	Diabetes mellitus	Both big toes	Black discoloration and subungual hyperkeratosis of nails for 8 years	E. dermatitidis
Krajden et al. (31)	Canada	M/NA	Diabetes mellitus	Middle finger	Black discoloration of nail for 1.5 years	E. dermatitidis
Hata et al. (32)	Japan	F/61	None	Big toe	Discoloration of nail	E. dermatitidis
Boisseau-Garsaud et al. (33)	France	M/60	Post-renal transplant	Two thumbs and two big toes	Hyperkeratosis and black coloration of nails for 4 years	E. jeanselmei
Oudaina et al. (34)	Morocco	F/39	None	Big toe	Discoloration of distal side of nail for 2 years	E. jeanselmei
Park et al. (35)	South Korea	M/42	None	Big toe	Linear longitudinal ridging with yellowish pigmentation of nail for 1 year	E. dermatitidis
Sharma et al. (36)	India	M/50	None	Big toe	Black discoloration and hyperkeratosis of nail for 5 years	E. jeanselmei

^aF, female; M, male; NA, not available.