The association of Yarrowia lipolytica with onychomycosis.

A 20-year-old white woman from the state of Idaho in the United States presented with chronic proximal subungual onychomycosis and tinea pedis ( Fig. 1 A). The patient reported a history of frequent bacterial infections of the feet, as well as endometriosis, adenomyosis, and Ehlers-Danlos Syndrome. In the initial stages of the condition, the patient reported some thickening and yellowing of the nails, but with primarily normal growth. After prolonged infection, onycholysis developed, leading to nail ridging and detachment of the nail plate from the nail bed, with recurrent shedding. At the time of examination, the infection was limited to the right foot, but the patient had previously described the exact etiology on the left foot and other toes of the right foot. Beyond the toenail infection, the patient's feet showed signs of tinea pedis with cracking and peeling skin. No signs of fungal infection were found in the fingernails. Since the initial infection, the patient was prescribed various antifungal treatments without resolution ( Table 1 ). Additionally, an oral fluconazole course was discontinued due to elevated liver enzymes and the development of frequent nausea. No attempts were made by physicians to identify or isolate the causative pathogen. The patient approached the researcher at the University of Idaho seeking to identify the fungal pathogen and develop a more effective treatment plan. Fig. 1 Onychomycosis and the in situ observation of fungal mycelia in nail material. (A) The patient's right foot exhibited abnormal nail growth in the hallux, third, and fourth toes. (B) Nail samples were stained with calcofluor white and examined at 200x magnification with illumination with ultraviolet light. Scale bar = 20 μm. Fig. 1 Table 1 Case history. Table 1 Date Significance Notes Year −5 Onset of infection – Year −4.5 First diagnosis and treatment 10 % efinaconazole; topical application twice a day for two weeks. Year −4 End of efinaconazole treatment No change in condition. Year −3.5 Terbinafine treatment 250 mg terbinafine; oral once daily for 6 weeks. End of terbinafine treatment No change in condition. Year −3 Combined azole treatment 300 mg fluconazole; oral once weekly with daily 1.6 % topical itraconazole. Year −2 End of combined azole treatment Treatment was discontinued due to elevated liver enzymes and nausea. Day 0 Patient approaches researchers at the University of Idaho. - Day 10 Sample collected from patient - Day 32 First isolation of Yarrowia lipolytica - Year 1 Sample collected from patient - Onychomycosis and the in situ observation of fungal mycelia in nail material. (A) The patient's right foot exhibited abnormal nail growth in the hallux, third, and fourth toes. (B) Nail samples were stained with calcofluor white and examined at 200x magnification with illumination with ultraviolet light. Scale bar = 20 μm. Case history.

Julia R. Major: Writing – original draft, Validation, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Claire A. Warren: Validation, Investigation. Paul A. Rowley: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

This publication was made possible by an Institutional Development Award (IDeA) from the 10.13039/100000057 National Institute of General Medical Sciences of the 10.13039/100000002 National Institutes of Health under Grant #P20GM103408 . We would also like to thank the Dyess family for their support of this research project.

The clinical and laboratory Y. lipolytica were sent to the Fungus Testing Laboratory at the University of Texas San Antonio, for antifungal susceptibility testing using a panel of azole antifungals following CLSI M27 methodology ( Table 2 ). Due to the novelty of Y. lipolytica being associated with onychomycosis, no breakpoints could be established. Compared to the known breakpoints of common fungal pathogens in the Candida genus, the isolate of Y. lipolytica would be considered resistant to posaconazole, itraconazole, and fluconazole. Consistent with our findings, fluconazole can be ineffective against specific isolates of Y. lipolytica , whereas resistance to posaconazole and itraconazole has been reported, but is less common [ [6] , [7] , [8] ]. The observed resistance profile of Y. lipolytica was consistent with the patient's clinical history, with the apparent ineffectiveness of prior treatments with fluconazole and itraconazole. Voriconazole, an antifungal typically used to treat serious fungal infections such as invasive aspergillosis and candidiasis, was deemed the best future treatment option for the patient. Other drug susceptibility surveys have also shown Y. lipolytica is particularly susceptible to voriconazole [ 8 ]. However, due to the patient's previous adverse reaction to oral azoles and hesitance from physicians to prescribe the medication, this treatment option was not pursued. Table 2 Minimum inhibitory concentration (MIC) values for clinical and laboratory strains of Y. lipolytica . Table 2 Drug Clinical Yarrowia Fluconazole 2 μg mL −1 Itraconazole 0.5 μg mL −1 Posaconazole 0.5 μg mL −1 Voriconazole 0.03 μg mL −1 Minimum inhibitory concentration (MIC) values for clinical and laboratory strains of Y. lipolytica .

To identify the species of the dimorphic fungal isolates, single colonies were inoculated into YPD broth and incubated overnight at 30 °C with shaking at 200 rpm. Genomic DNA was extracted using the phenol-chloroform method [ 4 ], followed by PCR amplification of the internal transcribed spacer (ITS) region using specific primers (forward: 5′-TCCTCCGCTTATTGATATGC; reverse: 5′-CTTGGTCATTTAGAGGAAGTAA). Sanger sequencing of the amplified ITS region revealed identical sequences across all fungal isolates. Species identification was confirmed using BLASTn analysis, which showed a 98 % nucleotide identity of the isolated fungus to the dimorphic yeast of the Ascomycota, Y . lipolytica . Comparison of the growth of the clinical isolate of Y. lipolytica to a laboratory strain of the species ( Y. lipolytica strain 038) revealed significant differences in colony morphology ( Fig. 2 ) [ 5 ].

Toenails were removed from the patient's right foot using sterile forceps. To prepare the toenail samples for direct microscopic examination, they were incubated for 1 minute with a 10 % (w/v) potassium hydroxide solution and mixed with a 5 mM calcofluor white solution. Septate fungal hyphae (∼2 μm in diameter) with no observable branching were observed in toenail samples ( Fig. 1 B). Nail samples were also suspended in sterile phosphate-buffered saline (pH 7.0), mixed by vigorous vortexing, and the resulting solution was applied to yeast extract, peptone, and dextrose (YPD) agar with (15 μg mL −1 ) chloramphenicol. Inoculated agar plates were incubated at 25 °C for up to 14 days. Patient samples collected at day 10 and after 1 year exhibited microbial growth during laboratory culture on YPD media, with the repeated isolation (n = 17) of circular raised colonies that were colored beige with a smooth texture ( Fig. 2 A). The colony morphology was consistent with the isolation of a budding yeast when compared to a laboratory yeast culture ( Fig. 2 B). Microscopic analysis of the cells in cultures grown on agar confirmed them to be a unicellular budding yeast ( Fig. 3 A). From the same nail samples, there was only a single isolation of a filamentous fungus that could not be subcultured. After isolating clones of the yeast, followed by two weeks of incubation, all yeast growing on agar appeared textured and had spread away from the center of the colony. Microscopy revealed a mixture of budding yeasts and long septate hyphae with few branches and a diameter of ∼2–3 μm ( Fig. 3 B). This analysis confirmed the isolation of a dimorphic yeast and was the same as the fungus associated with the patient's nail samples ( Fig. 1 B). Fig. 2 Colony morphology of a clinical isolate and laboratory strain of Y. lipolytica. YPD agar plates with chloramphenicol growing ( A) Y. lipolytica associated with onychomycosis and (B) the laboratory strains Y. lipolytica 038 for two weeks at 25 °C. Fig. 2 Fig. 3 Cell morphology of a clinical isolate of Y. lipolytica. Cells visualized at 400x magnification from Y. lipolytica associated with onychomycosis grown on YPD agar at 25 °C for (A) one week and (B) four weeks. Scale bar 10 μm. Fig. 3 Colony morphology of a clinical isolate and laboratory strain of Y. lipolytica. YPD agar plates with chloramphenicol growing ( A) Y. lipolytica associated with onychomycosis and (B) the laboratory strains Y. lipolytica 038 for two weeks at 25 °C. Cell morphology of a clinical isolate of Y. lipolytica. Cells visualized at 400x magnification from Y. lipolytica associated with onychomycosis grown on YPD agar at 25 °C for (A) one week and (B) four weeks. Scale bar 10 μm.

We report a unique case of onychomycosis associated with the presence of the dimorphic yeast Y. lipolytica . Fungi isolated from this type of nail infection are more often dermophytes and other species of saprophytic filamentous fungi [ 9 ]. Yeasts are less commonly isolated from infected nails, but the important opportunistic pathogen Candida albicans is the yeast species most frequently isolated [ 10 ]. Y. lipolytica is a biosafety level 1 GRAS (Generally Regarded As Safe) organism [ 11 ]. It is commonly found in oil-polluted environments and high-fat and protein-containing foodstuffs (particularly in cheese) due to its production of lipases and proteases and its affinity for hydrocarbon and alkane metabolism. It has been used in research and biotechnology due to its potential for biofuel production and environmental remediation [ 12 ]. Relative to other opportunistic fungal pathogens, Y. lipolytica is rarely associated with human disease [ [13] , [14] , [15] ]. The species is not generally found as a commensal on human skin but has been isolated from various human body locations and samples, such as stool, sputum, the mouth, the pulmonary tract, and the intestinal tract [ 16 , 17 ]. Y. lipolytica , like many other fungi, is an opportunistic pathogen that mainly threatens immunocompromised individuals or those with comorbidities, resulting in bloodstream, skin, lung, eye, and mouth infections [ [18] , [19] , [20] ]. The most frequently reported infection caused by Y. lipolytica appears to be systemic fungemia, often associated with the use of indwelling medical devices, such as catheters. Resolution of these infections is typically achieved by removing the medical device and/or treatment with a systemic antifungal drug, such as fluconazole or amphotericin B, with good cure rates. One example of cutaneous isolation of Y. lipolytica was associated with “toe lesions,” but no other clinical details were recorded [ 16 ]. The ability to induce hemolysis, the production of degradative enzymes, and the potential for the invasive growth of hyphae and pseudohyphae have all been suggested as pathogenicity factors of Y. lipolytica . This is in addition to the innate drug resistance of some clinical isolates. However, in general, Y. lipolytica is not considered an aggressive opportunistic pathogen and is often well-managed with the appropriate application of antifungal therapies and the removal of catheters. Candida species are an uncommon cause of true onychomycosis, as infections typically manifest as paronychia and onycholysis without direct invasion of the nail plate [ 21 ]. However, in rare and particularly chronic cases, such in individuals with chronic candidiasis and other comorbidities (AIDS, Cushing syndrome etc.), true onychomycosis with Candida invasion of the nail plate can occur. The case presented here is notable in that it represents a true onychomycosis, highlighting an atypical and infrequent clinical presentation of a yeast infection. Onychomycosis is challenging to treat due to the non-porous nature of the nail plate, which hinders the penetration of antifungal therapeutics, the need for long courses of antifungal treatment, the toxicity of antifungal drugs, and the innate drug resistance of some fungi. Physical methods to enhance drug penetration or non-drug therapies have shown promise, including debridement, nail plate etching, iontophoresis, and lasers [ 22 ]. Many factors can influence disease persistence, including increased susceptibility in patients with specific comorbidities, the risk of reinfection from contaminated surfaces (fomites), poor personal hygiene, and environmental factors such as high humidity. We speculate that the association of Y. lipolytica with this case of onychomycosis could be the causative agent of this infection due to its repeated isolation from infected nails. Notably, the culturing method used to isolate Y. lipolytica would have been adequate for identifying other common fungi that cause onychomycosis, particularly Trichophyton rubrum , a common cause of onychomycosis [ 23 ]. However, we recognize that the association of Y. lipolytica with this case could represent colonization rather than being the causative disease agent, due to its low pathogenicity. If Y. lipolytica is responsible for the disease, the observed antifungal drug resistance could have contributed to the failure of past therapeutic interventions [ [6] , [7] , [8] ]. Prior efforts at treating the patient in this case were further complicated by their adverse reaction to oral antifungal therapies, which has left them with limited future therapeutic options.

Onychomycosis, a chronic fungal infection of the nail, is estimated to affect ∼4 % of people worldwide, with poor treatment outcomes and high recurrence rates [ 1 , 2 ]. The majority of these cases affect the toenails, rather than the fingernails, with common symptoms including nail malformation, such as thickening, crumbling, and discoloration. The condition is often chronic, leading to ingrown toenails and nail loss. Risk factors include, but are not limited to, old age, diabetes, tinea pedis, and a weakened immune system [ 3 ]. Nail infections are typically caused by filamentous fungi, particularly the dermatophyte species of the Trichophyton genus , resulting in tinea unguium. Other fungi have also been found to cause onychomycosis, including yeasts (e.g., Candida spp.) and saprophytic molds (e.g., Fusarium spp. , Aspergillus spp.).