Clinical Spectrum, Molecular Characterization, Antifungal Susceptibility Testing of Exophiala spp. From India and Description of a Novel Exophiala Species , E. arunalokei sp. nov

Abstract

Introduction: Exophiala spp. are important opportunist pathogens causing subcutaneous or even fatal disseminated infections in otherwise both immunosuppressed and healthy individuals but there are no systematic studies on the isolates of Exophiala species from India.

Methods: Twenty-four isolates of Exophiala species were retrieved from the National Culture Collection of Pathogenic Fungi (NCCPF) and identified phenotypically and by molecular methods (ITS region sequencing) followed by antifungal susceptibility testing (AFST) as per CLSI-M38A3 guidelines. A review of the literature of cases from India was performed up to 1^st January 2021 using the Medline and Cochrane database.

Results: E. dermatitidis (n = 8), E. jeanselmei (n = 6), E. spinifera (n = 6), E. mesophila (n = 1), E. oligosperma (n = 1), E. xenobiotica (n = 1) were identified and the sequencing of ITS, β-tubulin and β-actin revealed a novel species, E. arunalokei sp. nov. (n = 1). The ITS sequence phylogram of E. jeanselmei revealed that the majority (83%) formed a separate cluster close to type A while majority (75%) of E. dermatitidis were type B. The MIC50 (mg/L) of amphotericin, itraconazole, voriconazole, micafungin, caspofungin, anidulafungin, and posaconazole, was 1, 0.25, 0.125, 0.12, 0.125, 0.062, and 0.062, respectively. Sixteen more cases were identified on the literature review and a significant association of E. dermatitidis with history of surgical procedures (p = 0.013), invasive disease (p = 0.032) and of E. mesophila with tuberculosis (p = 0.026) was seen.

Conclusion: This, to the best of our knowledge is the first study from India elucidating the molecular and clinical characteristics of Exophiala species and the first Indian report of human infection due to E. xenobiotica and E. arunalokei.

Keywords: Exophiala; India; antifungal susceptibility; molecular; novel species.

Figure 1

Representative clinical images from subcutaneous infection due to Exophiala species. (A). Case no. NCCPF 106025 multiple subcutaneous nodular lesions over dorsum of left-hand (B). Case no. NCCPF 106030 nasal polyps (arrow) in patient with allergic fungal rhino sinusitis (C). Case no. NCCPF 106028 single subcutaneous cystic swelling skin over chest (left side).

Figure 2

Microscopic findings on lactophenol cotton blue mount from slide culture and gross morphology on obverse surface of Sabouraud’s dextrose agar after 3–7 days at 25°C (inset). (A) E. dermatitidis, (B) E. mesophile, (C) E. jeanselmei, (D) E. spinifera, (E) E. xenobiotica, (F) E. mesophila.

Figure 3

Phenotypic characteristics of E arunalokei sp. nov. Gross morphology on obverse surface culture growth after 14 days at 28°C on (A) Potato dextrose agar, (B) Oatmeal agar, and (C) Sabouraud’s dextrose agar. (D) Microscopic findings from slide culture (day 10) on lactophenol cotton blue mount showing branched annellophore, (E) terminal prominence (arrow), (F) LCB from slide culture on day 21. Scanning electron microscopy (SEM) of (G) E oligosperma, (H) E. jeanselmei, and (I) E. arunalokei sp. nov. day 10, ×2,500; (J) ×5,000 (day 10); (K) day 21, ×2,500, (L) day 21, ×2,500 with size measurements.

Figure 4

Phylogenetic tree showing the relationship of E. arunalokei to closely related species using (A) β-actin gene and (B) β-tubulin gene sequencing data by using the Neighbor-Joining method clustered together in the bootstrap test (1,000 replicates); conducted in MEGA 7.

Figure 5

Evolutionary relationships of 24 isolates of Exophialia species with Neighbor-joining algorithm with 1,000 bootstrap replicates. Phaeococcomyces mexicanus CBS 127164 is taken as out-group. ITS, internal transcribed spacer.