Scedosporium and Lomentospora Infections: Contemporary Microbiological Tools for the Diagnosis of Invasive Disease

Abstract

Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods.

Keywords: Lomentospora prolificans; MALDI-TOF MS; PCR-based diagnosis; Scedosporium; antifungal susceptibility; culture; histopathology; whole genome sequencing.

Figure 1

Phylogenetic tree of Scedosporium species based on 60 tubulin sequences (TUB, exon 5 and 6), representing the known genetic variation within the genus, using Maximum Likelihood with the K2+G model and 500 bootstrap replications. Bootstrap values >50 are given. The tree is rooted with the outgroup species Petriellopsis africana and Lomentospora prolificans. GenBank accession numbers are given in brackets behind the strain numbers. Bold font indicates sequences of type strains.

Figure 2

(A): Flat, spreading olive-grey to black colored and suede-like surface (obverse view) of Lomentospora prolificans on Sabouraud dextrose agar (SDA) after four days growth at 28 °C. (B): Greyish-white cottony to woolly surface with concentric growth pattern and white margins (obverse view) of Scedosporium aurantiacum on SDA after 3 days growth at 28 °C. (C): Brown orange center with pale yellow pigment (reverse view) of Scedosporium aurantiacum on potato dextrose agar after 3 days incubation at 28 °C.

Figure 3

A and B: Microscopic morphology of Scedosporium aurantiacum (A) and Lomentospora prolificans (B) 40x magnification. (A) Hyphae are septate with simple long conidiophores (arrow) bearing conidia singly or in small groups. Conidia (4–9 × 6–10 μm) are single-celled, pale brown ovoid in shape and are rounded above with truncated bases. (B) Hyphae are hyaline and septate with conidia born in small groups on basally swollen, flask-shaped conidiophores (arrow). Conidia (2–5 × 3–7 μm) are hyaline to pale brown, one-celled and oval-shaped with smooth walls.

Figure 4

Workflow for diagnosis of invasive Scedosporium and Lomentospora prolificans infections. CLSI = Clinical and Laboratory Standards Institute; EUCAST = European Committee on Antimicrobial Susceptibility Testing; rDNA = ribosomal DNA; ID = identification; ITS = internal transcribed spacer; MALDI-TOF MS = matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PCR = polymerase chain reaction; PDA = Potato dextrose agar; TUB = Tubulin.

Figure 5

Distribution of interspecies (blue line) and intraspecies (red line) pairwise Kimura 2-parameter genetic distances in the genus Scedosporium based on the primary fungal DNA barcode the ITS1/2 region, based on 60 ITS1/2 sequences of Scedosporium angustum, Scedosporium aurantiacum, Scedosporium apiospermum, Scedosporium boydii, Scedosporium cereisporum, Scedosporium dehoogii, Scedosporium desertorum, Scedosporium ellipsoideum, Scedosporium fusoideum and Scedosporium minutisporum. No DNA barcoding gap was identified using the ITS1/2 region. All taxa were subjected to pairwise sequence divergence calculations using the Kimura 2-parametric distance model (K2P) in MEGA ver. 5.2.2.