Neoscytalidium dimidiatum as onychomycosis causative agent in an Iranian patient: a case report and literature review.

A 38-year-old healthy male presented to our medical mycology center with whitish opaque discoloration of the right toenail. He reported a history of some sand scratches subsequent to walking barefoot on the beach two years ago and wearing hard safety shoes for a period of two years. On clinical examination, onycholysis, onychodystrophy, and apparent thickening of the ungual bed in the left big toe were found. The microscopic examination of nail clippings using 15% potassium hydroxide (KOH/) revealed the presence of septate pigmented hyphae. The fungus was identified as Neoscytalidium dimidiatum based on the cultural characteristics, the arrangement of arthroconidia on lactophenol cotton blue (LPCB) staining, blocky-brown pigmented hyphae on serum physiology mounts, and sequencing. Susceptibility of the isolated fungi to amphotericin B, itraconazole, voriconazole, and terbinafine was tested using the standard broth microdilution M38-A2 method developed by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of the four antifungal drugs used in this study were: amphotericin B: 1 mg/L, itraconazole: 2 mg/L, voriconazole: 0.25 mg/L, and terbinafine: 1 mg/L. The patient underwent terbinafine and clobetasol topical treatments for 6 months.

Introduction

Onychomycosis is a term that refers to all nail disorders caused by fungal etiologic agents with a prevalence of 18–40% of all onychopathies [1]. The main types of onychomycosis have been described as distal and lateral subungual onychomycosis, superficial white onychomycosis, proximal subungual onychomycosis, endonyx onychomycosis, and total dystrophic onychomycosis [2,3]. Etiologic agents of onychomycosis mostly consist of three different groups of fungi; dermatophyte, molds, and yeasts. The prevalence of etiologic fungi varies and depends on different factors like sex, age, occupation, climate, health situation, cultural habits, country, geographic (urban and rural) area, underlying disease, and diagnostic evaluation methods [3]. Saprophytic fungi divided into hyaline and dematiaceous fungi. Dematiaceous or black refers to melanin-producing yeasts, yeast-likes, and filamentous fungi. Mostly they live in soil and plants. Several of those can cause disease in humans and animals with a spectrum of superficial mycosis to severe encephalitis [4]. Neoscytalidium sp. (formerly: Scytalidium) are ascomycetous fungi belong to a large and heterogeneous group of fungi that cause Phaeohyphomycosis. Some though can infect humans via direct contact, sometimes traumatic, with colonized plants or vegetable residues [5]. Neoscytalidium dimidiatum and its hyaline mutant, N. dimidiatum var. hyalinum can cause clinical lesions in nail and skin similar to those produced by dermatophytes [6]. Here, we report the case of a 38-year-old man with onychomycosis caused by N. dimidiatum in Iran.

Case report

A 38-year-old electrical engineer male presented to our medical mycology center with whitish opaque discoloration of the right toenail for a duration of two years. His medical history showed some sand scratches subsequent to walking barefoot on the beach two years ago and wearing hard safety shoes for a period of two years. Also, his medical reports showed that his allergic rhinitis due to concha surgery (nose flesh surgery) had been under control. Furthermore, the laboratory reports showed negative results for viral hepatic tests (HBC -Ab and HIV-Ab, HBs-Ab > 400 mIU/mL) and normal results for routine laboratory tests. On clinical examination, onycholysis and onychodystrophy, with apparent thickening of ungual bed in the left big toe were found (Fig 1: A). He underwent terbinafine and clobetasol topical treatments after initial diagnosis using potassium hydroxide (KOH) smear (Fig 2) and culture. No evidence of progress in treatment was seen. With the rest of the nail clipping, the second culture was conducted about two months later of the first visit. The microscopic examination of nail scrapings using 15% KOH revealed the presence of septate hyaline hyphae. The material was cultured on Sabouraud's dextrose agar (SDA) with and without cycloheximide and cornmeal agar. The SDA without cycloheximide grew woolly brown colonies after seven days of incubation at room temperature. There was no growth on SDA with cycloheximide. Greyish black colonies grew on SDA (Fig. 3: A&B). Normal saline mounts showed numerous cylindrical or globose, one-to-two-celled barrel-shaped arthroconidia with dark walls and many wide and septate brown and hyaline hyphae (Fig 4). The fungus was identified as Neoscytalidium sp. based on the cultural characteristics, the arrangement of arthroconidia on lactophenol cotton blue (LPCB) staining, and dark hyphae on serum physiology mounts. Accurate identification was accomplished via sequencing analysis. Briefly, genomic DNA was isolated from fresh colonies using a method described previously [7]. Then, PCR was carried out bilaterally to amplify the ITS regions of the rDNA gene using the pan fungal primers: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [8]. The amplicon was sent for sequencing, which showed 100% similarity with the sequence of Neoscytalidium dimidiatum clone URF_Pt01 (accession number MT010216) deposited in NCBI BLAST (https://www.blast.ncbi.nlm.nih.gov/Blast.cgi) database. The relevant sequence extracted from current study (Neoscytalidium dimidiatum) has been deposited in GenBank under accession number MZ377100. The in-vitro antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) document M38-A2 method [10]. The minimum inhibitory concentrations (MICs), the result of the four antifungal drugs used in the study were: amphotericin B: 1 mg/L, itraconazole: 2 mg/L, voriconazole: 0.25 mg/L, and terbinafine: 1 mg/L. The treatment has changed to itraconazole 100 mg/day and amorolfine nail lacquer for 10 weeks subsequently. Drug tolerance was well and toenail infected areas were reduced (Fig. 1B). Written informed consent was obtained from the patient. All data were de-identified. This Health Insurance Portability and Accountability Act–compliant study received approval from the institutional review board (IR.TUMS.SPH.REC.1398.197).

Fig. 1

A: Toe nail day 0; B: After 10 weeks treatment with oral itraconazole & Amorolfine nail lacquer.

Fig. 2

KOH mount.

Fig. 3

A:N. dimidiatum SDA culture, forward view; B: N. dimidiatum SDA culture, reverse view.

Fig. 4

Normal saline mount.

Discussion

Onychomycosis challenges such as aesthetic problems, difficulty in running or rotating, and more make discomfort situations for patients. Causative agents of onychomycosis include molds, dermatophytes, and yeasts [35]. Studies demonstrate that the prevalence and distribution of causative agents of onychomycosis can vary according to geographic location, climate, and cultural customs [11,35]. Non-dermatophyte molds (NDMs) are one of the etiologic agents of onychomycosis which divided into hyaline and dematiaceous fungi. The most common hyaline NDMs are different species of Aspergillus, Scopulariopsis, Alternaria, Acremonium, and Fusarium [[12], [13], [14]]. A. flavus and A. niger complex are generally isolated from abnormal nails [15], but there are also reports of onychomycosis due to rare species of Aspergillus like A. candidum [16] or A.sydowii [15].

Among dematiaceous NDMs Cyphellophora and Phialophora are involved in onychomycosis and mild skin infections [4]. Also, Neoscytalidium sp. is a genus of dematiaceous fungi that has been isolated from several nail infections [11]. N. dimidiatum and its mutant N. hyalinum can both play a similar role in causing onychomycosis or superficial skin infections and are clinically indistinguishable from dermatophytosis. N. dimidiatum more commonly invade the nails, toe tissues, and soles of the feet with hyperkeratosis; however, hands are rarely affected [17]. The fungi first inhabit toenails by a laterodistal development. Paronychia may also occur as total nail dystrophy. Palm lesions are mainly unilateral although single affecting is classically bilateral reproducing the two-foot one-hand syndrome [6,[17], [18], [19]].

Neoscytalidium infections have a different prevalence in the world that it seems is more dependent on the geographic area than the susceptible population. Mostly there are two clinical forms of Neoscytalidium infections in published articles, onychomycosis, and systemic disease [20]. A systematic review conducted on NDMs etiologic agents of onychomycosis in 2012 among nine countries (Canada, Colombia, Spain, Italy, Greece, Pakistan, Thailand, and Brazil) shows that 4% of onychomycosis etiologic agent belong to N. dimidiatum which occurred only in the study conducted in Brazil and none in the other countries contribute in the article [13]. In São Paulo, Brazil from 588 cases of onychomycosis, NDMs were isolated in 7.4% of positive cultures related to toenail onychomycosis with 2.3% Nattrassia mangiferae (Neoscytalidium) [21]. Furthermore, in Alagoas, Brazil during 2009-2016 only 0.1 percent of each annual sampling refers to Neoscytalidium sp. [22]. One report from Thailand showed Fusarium and Scytalidium dimidiatum are two major etiologic agents of onychomycosis due to NDMs [23]. Deep infection in scytalidiosis is mostly accompanied by immune suppression in situations like lung and renal transplant, diabetes, or further underlying disease. There are several reports about profound infection as central nervous system abscesses, endophthalmitis, sinusitis, osteomyelitis mycetoma, subcutaneous lesions, and disseminated infection caused by Neoscytalidium sp. [22,[24], [25], [26], [27], [28], [29], [30], [31]]. N. dimidiatum is involved more than N. hyalinum in deep mycosis [32] and its related mortality is about 50% [17]. A study from Iran presented a fatal case of Nattrassia mangiferae (Neoscytalidium sp.) encephalitis after thorn eye penetration in a diabetic 60-year-old male with cirrhosis and heart failure [33]. In another study, a cerebral phaeohyphomycosis due to Nattrassia mangieferae (Neoscytalidium sp.) reported in Iran. This case was a 17-year-old male with a history of systemic lupus erythematous and renal involvement. The diagnosis was based on histopathologic and mycologic evidence after craniotomy [27]. In onychomycosis, oral drugs in combination with nail lacquer should offer better results than single treatment with antifungals [5]. A professional removal of the infected area along with antifungal therapy is recommended as a perfect therapeutic method [34]. Also, several case reports had assessed in vitro antifungal susceptibility testing of different antifungal agents against Neoscytalidium sp. isolated from clinical specimens (Table 1).

Table 1

Case reports demonstrated in vitro antifungal susceptibility testing of different antifungal agents against Neoscytalidium sp.

Reference	Case No	Country	Sex/Age	Infection site	Treatment	Diagnosis
Garinet et al. [36]/2001	1	French Guyana	M/53	Leg & toenail	Surgical, oral itraconazole, and local amphotericin B	Neoscytalidium sp.
Garinet et al. [36]/2002	2	Ivory coast	F/64	Sole & nail of feet/	Local terbinafine	N. hyalinum
Jabbarvand [37]/2004	3	Iran	M/32	Eye	Natamycin eyedrop and oral ketoconazole	Neoscytalidium sp.
Geramishoar [27]/2004	4	Iran	M/17	Soft tissue (cranial abases)	Corticosteroid and cyclophosphamide	Neoscytalidium sp.
Sadeghi Tai [33]/2005	5	Iran	M/60	Eye	Surgery & systemic imipenem, metronidazole, and amphotericin B with	Neoscytalidium sp.
Garinet [36]/2007	6	Mauritania	M/52	Ankle	Surgery	N. dimidiatum
Elinav [31]/2009	7	Israel	M/56	Lung and pleura	Intravenous voriconazole and Amphotericin B from intra pleural root	N. dimidiatum
Garinet [36]/2011	8	Cameroun	M/59	Leg & foot	Oral voriconazole and topical ketoconazole	N. dimidiatum
Garinet [36]/2011	9	Congo Brazzaville	M/59	Elbow, back, and leg	Oral voriconazole	N. dimidiatum
Roy [39]/2013	10	India	NA/55	Toenails	Oral Itraconazole	Neoscytalidium sp.
Dionne [38]/2015	11	USA	M/50	Lung	Amphotericin B, posaconazole	N. dimidiatum
Shokoohi et al. [9]/2020	12	Iran	F/52	Finger nail	Oral terbinafine plus ciclopirox nail lacquer	N. novaehollandiae
Present case/2021	13	Iran	M/38	Toe nail	Oral terbinafine then changed to itraconazole and ciclopirox lacquer	N. dimidiatum

Abbreviations: M: male; F: Female; Neoscytalidium sp.: not molecular base diagnosis; NA: not available.

Diagnosis of onychomycosis is challenging and it starts by the rollout of other dermatology disorders which can affect healthy nail appearance. After reaching fungal etiology, there are some differential diagnosis criteria that differ molds from dermatophytes like priority of skin involvement than the nail, infection in more than one nail in dermatophytes, and other practical evidence. But historically, scytalidiosis was distinguished from dermatophytic onychomycosis by resistance to routine anti-dermatophyte treatments. It is notable that clinically differentiate between scytalidiosis and dermatophytosis is unintelligible [7]. The primary diagnosis of onychomycosis is based on potassium hydroxide (KOH) smear and culture. In NDMs diagnosis in comparison to dermatophytes and yeasts diagnosis, there is a need to more evaluations such as identification of the NDM in the nail by microscopy (KOH preparation), culture isolation, repeated isolation in culture, inoculum counting, failure to isolate a dermatophyte in culture, and histology. [13]. The most reliable methods are molecular approaches such as PCR-sequencing for molds and also Neoscytalidium. Although it is uncommon due to special equipment and experience. Furthermore, the results achieved with mass analysis through dendrogram construction of MALDI-TOF MS of N. dimidiatum showed valuable and cost-effective assessed with ITS rDNA gene analysis technics [28].

In conclusion, to the best of our knowledge, the present case is the first report of onychomycosis caused by N. dimidiatum in Iran. The precise identification of etiologic agents of onychomycosis and antifungal susceptibility testing before antifungal treatment is necessary.

Transparency declaration

Authors declare no conflicts of interest.

Informed permission was attained for experimentation with human subjects.