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INTRODUCTION
Mucormycosis is a life-threatening fungal infection that occurs in patients who are immunocompromised. It refers to infections caused by members of the order Mucorales. While most human infections are caused by Rhizopus, Mucor, and Rhizomucor, other clinically relevant organisms within this, include Actinomucor, Apophysomyces, Cunninghamella, Lichtheimia (previously known as named Absidia), Saksenaea, and Syncephalastrum [1]. To date, twelve species of the genus Lichtheimia have been reported: L. corymbifera, L. ramosa, L. ornata, L. hyalospora, L. sphaerocystis, L. brasiliensis, L. koreana, L. alba, L. blakesleeana, L. globospora, L. hongkongensis, and L. piauiensis [2–4]. L. corymbifera and L. ramosa, the most important pathogenic species, are also the most frequently isolated from a variety of substrates, including farming products such as decaying vegetation and rotting fruits [5]. Infection experiments in chicken embryos have shown a lower virulence potential for L. hyalospora and L. sphaerocystis [6]. The clinical importance of the genus Lichtheimia varies by geographic region. In Europe and Africa, it is the second most frequently reported causative agent of mucormycosis after the genus Rhizopus, whereas in America, the number of cases is relatively low. Most infections caused by Lichtheimia manifest as cutaneous or pulmonary infections, although rhinoorbital-cerebral and disseminated infections also occur [7].
Phenotypic differentiation and identification of Mucorales is challenging due to their similar morphologies shared by various species. Here, we report two cases of fungal infection confirmed through molecular techniques: pulmonary mucormycosis and otomycosis caused by L. ramosa and L. hyalospora, respectively. Notably, this is the first reported case of otomycosis caused by L. hyalospora.
CASE REPORT
1. Case 1
A 74-year-old female was admitted with a two-day history of hemoptysis, dyspnea, and cough in April 2023. Her medical history included type 2 diabetes mellitus, hypertension, chronic kidney disease, and gout. On presentation, blood tests revealed a white blood cell count of 5.94×109/L with 65.3% neutrophils. Serum C-reactive protein (87.9 mg/L; reference interval: 0.0–5.0 mg/L) and D-dimer (3.29 nmol/L; reference interval: 0.00–3.00 nmol/L) levels were elevated. Tests for tuberculosis infection, including acid-fast bacilli (AFB) stain, culture, and polymerase chain reaction (PCR), were negative. Chest computed tomography (CT) revealed an air crescent sign in the superobasal segment of the right lower lobe and a fungal ball. Tests related to fungal infections showed positive results for the Aspergillus galactomannan antigen test (0.56; positive ≥0.50 index) and (1,3)-β-D-Glucan (159.2; positive ≥80.0 pg/mL), but a negative result for Aspergillus antibody IgG (5.0; positive ≥40.0 mg/L). Sputum culture on Sabouraud dextrose agar (SDA) yielded rapidly growing whitish cottony mold after 48 hr of incubation at 30°C (Fig. 1A, B). Lactophenol cotton blue (LPCB) stain revealed broad and aseptate fungal elements with ribbon-like hyphae. Additionally, microscopy showed branched sporangiophores with characteristic circinate side branches, ellipsoid sporangiospores, and pleomorphic giant cells with finger-like projections (Fig. 2A–D). Voriconazole was initiated for suspected invasive pulmonary aspergillosis. However, molecular identification using internal transcribed spacer (ITS) and D1/D2 region of the large subunit ribosomal RNA gene sequencing confirmed the causative agent as L. ramosa (Table 1). After two months of oral voriconazole medication, the patient’s respiratory symptoms were resolved, and treatment was discontinued. At her last outpatient visit in November 2024, she remained asymptomatic.
2. Case 2
A 58-year-old male presented to the otolaryngology clinic in March, 2023 with left-sided otorrhea lasting two days at the site of a surgical wound. His medical history included chronic obstructive pulmonary disease (COPD), asthma, and chronic otitis media. He had undergone foreign body removal (ear mold) and ossiculoplasty of the left ear in January 2022. Blood tests showed a white blood cell count of 7.44×109/L with 53.2% neutrophils and a normal serum C-reactive protein level (1.3 mg/L, reference range: 0.0– 5.0 mg/L). Examination of the left ear revealed yellowish-sticky discharge and necrotic tissue. Right ear findings were normal. Ear discharge samples were cultured on SDA media at 30°C. After 48 hr, a white woolly growth was observed, which turned grey with further incubation and rapidly filled the entire culture plate. LPCB staining showed ribbon-like hyphae with irregular diameters and branching at approximately 90°. Long hyaline sporangiophores with prominent funnel-shaped apophyses, ellipsoid sporangiospores, and giant cells with thick walls and finger-like projections were observed (Fig. 2E–H). ITS and D1/D2 sequencing confirmed the isolate as L. hyalospora (Table 1). The patient was treated with dressing and topical terbinafine hydrochloride antifungal cream for six months, with complete symptom resolution.
DISCUSSION
Mucormycosis caused by Lichtheimia species is a rare opportunistic infection found in soil, farming products, and food. The prevalence of Lichtheimia infections varies geographically, from 5% to 29% [5]. In Korea, a recent study reported a low frequency of Mucorales (0.7%) among 4,011 fungal isolates, with L. corymbifera being the most common [8]. The clinical spectrum of Lichtheimia infections includes cutaneous, pulmonary, rhinal, rhinocerebral, renal, disseminated infections, and otomycosis. To date, six isolates from three Lichtheimia species (L. corymbifera, L. ramosa, and L. ornata) have been identified in clinical specimens from Korean patients [8, 9], of which L. corymbifera isolated from respiratory specimens was the most common (Table 1). Our case of otomycosis caused by L. hyalospora is the first reported instance in literature (Table 1).
Lichtheimia species exhibit broad and aseptate branching hyphae. Microscopically, rhizoids are rarely observed in Lichtheimia. The sporangiophores are branched and widen near the top, forming a conical apophysis just below the columella. The columella is typically semicircular with small, dome-like projections on top. The sporangia are relatively small and pear-shaped structures supported by a characteristic funnel-shaped apophysis. The sporangiospores are round to oval [2, 10]. Giant cells are swollen, branched, or unbranched, and often consist of droplet-filled hyphae with thick, refractive walls. Additionally, these cells present irregular swelling and branching [2]. Lichtheimia can be distinguished from the genera Mucor and Rhizomucor by its small, pear-shaped sporangia with a funnel-shaped apophysis [10]. However, morphological features such as sporangiophore and spore shape are insufficient for the differentiation of Lichtheimia spp. This fungus shows positive results in serum markers of invasive fungal infections, including galactomannan and 1, 3-beta-D-glucan tests. There are no serum assays specific to mucormycosis caused by Lichtheimia spp. [11]. Therefore, molecular diagnostic tests are necessary for accurate identification of Lichtheimia spp. In this study, sequencing of ITS and D1/D2 regions allowed precise identification of L. ramose and L. hyalospora. However, differences in targets for molecular identification were observed depending on the Lichtheimia spp. [2]. When beta-tubulin was used as a molecular target for the identification of L. corymbifera, it incorrectly identified the species as Aspergillus terreus [12]. Similarly, for L. ornata, ITS as a molecular target could not differentiate it from other Lichtheimia spp. [9]. Ultimately, accurate identification was achieved when sequencing was performed using ITS and D1/D2 together (Table 1). Thermotolerance is another important factor in differentiating Lichtheimia spp. L. corymbifera and L. ramosa grow at temperature up to 49°C, whereas the maximum growth temperature for L. ornata is 46°C. L. sphaerocystis and L. hyalospora grow at 37°C and 40°C, respectively, but do not grow at temperatures above 40°C [13]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can also be used for fungal identification. Although additional testing using MALDI-TOF MS was not performed in these cases, previous studies have reported the successful identification of L. ramosa using MALDI-TOF MS [14, 15].
Amphotericin B is the treatment of choice for Lichtheimia infections. Oral posaconazole and isavuconazole are also effective options. Treatment of mucormycosis caused by Lichtheimia spp. involves a combination of empirical antifungal therapy and surgical debridement of affected tissues [16–20]. Although no consistent guidelines exist for the treatment of L. hyalospora, the patient in Case 2 achieved successful treatment with topical terbinafine hydrochloride alone. Some researchers suggest that the slower growth rate of L. hyalospora compared to other Lichtheimia spp. may account for its comparatively lower virulence [6].
Mucormycosis is a rare but life-threatening fungal infection that typically affects patients that are immunosuppressed, such as those with neutropenia, corticosteroid treatment, or diabetes. Early and accurate identification of fungal pathogens, particularly members of the order Mucorales, is critical for selecting appropriate antifungal agents and avoiding an aggressive clinical course. However, identifying Mucorales species based solely on macroand microscopic features is challenging due to morphological similarities. In the case of Lichtheimia spp., species-level identification using morphological features is particularly difficult. Since virulence has been reported to vary among Lichtheimia spp., accurate species identification is crucial for determining treatment strategies and predicting outcomes [6]. When Mucorales, especially Lichtheimia spp. are suspected based on macro- and microscopic appearance, molecular techniques such as ITS and D1/D2 sequencing analysis are effective for accurate identification.
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