Aspergillus fumigatus-Related Species in Clinical Practice

Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA). Other Aspergillus species belonging to the section Fumigati (A. fumigatus complex) may occasionally be the cause of IA. These strains are often misidentified, as they cannot be distinguished from A. fumigatus by conventional morphological analysis and sequencing methods. This lack of recognition may have important consequences as these A. fumigatus-related species often display some level of intrinsic resistance to azoles and other antifungal drugs. A. lentulus, A. udagawae, A. viridinutans, and A. thermomutatus (Neosartorya pseudofischeri) have been associated with refractory cases of IA. Microbiologists should be able to suspect the presence of these cryptic species behind a putative A. fumigatus isolate on the basis of some simple characteristics, such as defect in sporulation and/or unusual antifungal susceptibility profile. However, definitive species identification requires specific sequencing analyses of the beta-tubulin or calmodulin genes, which are not available in most laboratories. Multiplex PCR assays or matrix-assisted laser desorption ionization – time-of-flight mass spectrometry (MALDI-TOF MS) gave promising results for rapid and accurate distinction between A. fumigatus and other Aspergillus spp. of the section Fumigati in clinical practice. Improved diagnostic procedures and antifungal susceptibility testing may be helpful for the early detection and management of these particular IA cases.


INTRODUCTION
Aspergillus fumigatus is the most important pathogenic filamentous fungus in humans causing a spectrum of diseases including allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis and invasive aspergillosis. Among the growing population of patients with depressed immune defenses, A. fumigatus represents one of the major infectious cause of death. Other important pathogenic Aspergillus spp. include A. flavus, A. niger, A. terreus, A. versicolor, A. calidoustus, and A. nidulans (Steinbach et al., 2012). These designations actually represent sections (or complexes) of closely related species (also referred as cryptic species) that cannot be clearly distinguished morphologically. While A. fumigatus sensu stricto represents by far the leading human pathogen, some other species belonging to the A. fumigatus sensu latu complex (i.e., section Fumigati) have been recognized as occasional causes of invasive aspergillosis in 3 to 6% of cases (Balajee et al., 2009b;Alastruey-Izquierdo et al., 2013Escribano et al., 2013; Table 1). Their actual prevalence may be underestimated because of their lack of recognition by conventional diagnostic approaches. Moreover, the diagnosis of invasive 1 Teleomorph: Neosartorya pseudofischeri. aspergillosis often relies on suggestive radiological findings and/or positive fungal biomarkers (galactomannan, beta-1,3-Dglucan;De Pauw et al., 2008), in the absence of microbiological documentation and the actual contribution of Aspergillus spp. other than A. fumigatus in this setting is unknown.
Apart from A. fumigatus, the species of the section Fumigati that are most frequently recovered in clinical specimens and associated with invasive fungal diseases are A. lentulus, A. udagawae, A. viridinutans, A. thermomutatus (Neosartorya pseudofischeri), A. novofumigatus, and A. hiratsukae (Balajee et al., 2006(Balajee et al., , 2009bAlcazar-Fuoli et al., 2008;Alastruey-Izquierdo et al., 2013;Escribano et al., 2013). Their limited pathogenic role compared to A. fumigatus may be explained by a lower thermotolerance and different profiles of secondary metabolites with decreased production of mycotoxins, such as gliotoxin (Frisvad and Larsen, 2015;Tamiya et al., 2015). However, while resistance to triazoles is rare among A. fumigatus sensu stricto, these sibling or cryptic species commonly exhibit decreased susceptibility to azoles and other antifungal agents (Alcazar-Fuoli et al., 2008;Escribano et al., 2013;Alastruey-Izquierdo et al., 2014). A recent multicentre prospective survey reported a rate of azole resistance of 3.2% among A. fumigatus isolates (van der Linden et al., 2015). Among these resistant strains, 78% were A. fumigatus sensu stricto harboring mutations of the Cyp51A gene, while the remaining 22% were actually sibling species (A. lentulus, A. thermomutatus, and A. udagawae).
Microbiologists and clinicians thus must be aware of the existence of these sibling species that may be the cause of refractory cases of invasive aspergillosis. The aim of this review is to provide an overview of the characteristics of the most clinically relevant Aspergillus spp. of section Fumigati (other than A. fumigatus).

TAXONOMY AND DIVERSITY OF Aspergillus spp. IN SECTION Fumigati
The genus Aspergillus is divided in subgenera and sections (Peterson, 2008). A. fumigatus belongs to the section Fumigati, in which 63 species have now been described, although some of them are doubtful because of possible synonymy with other species (Samson et al., 2007;Frisvad and Larsen, 2015). Species delimitation within section Fumigati relies on a polyphasic approach including phenotypic characteristics, rate of growth at variable temperatures, extrolite patterns, and genotyping characterization by random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) and multilocus sequence typing (Hong et al., 2005;Samson et al., 2007). Based on this approach, Yaguchi et al. (2007) have proposed a subdivision of the section Fumigati into five clades: (I) A. fumigatus, (II) species including A. lentulus and A. fumisynnematus, (III) species including A. fumigatiaffinis and A. novofumigatus, (IV) A. viridinutans, A. udagawae, and other atypical strains, and (V) species including A. hiratsukae, A. brevipes, A. duricaulis, and A. unilateralis.
Teleomorphic homothallic species producing ascomata (i.e., sexual stage of reproduction) were initially classified under the genus name Neosartorya. However, many species thought to be strictly anamorphic further demonstrated the ability to reproduce sexually with an opposite mating type (e.g., A. fumigatus, and A. lentulus;O'Gorman et al., 2009;Swilaiman et al., 2013). From 2012, taxonomists recommend to apply a single-name nomenclature keeping the name Aspergillus for all species of this genus . This system will be used in this review with reference to the old teleomorphic name in brackets when applicable.

IDENTIFICATION OF Aspergillus spp. OF SECTION Fumigati
Morphological examination of macroscopic and microscopic features remains the standard method for identification of filamentous fungi. While phenotypic characteristics allow distinguishing Aspergillus spp. at the section level, accurate species identification is usually not possible. Compared to A. fumigatus sensu stricto, other Aspergillus spp. of the section Fumigati may exhibit distinct morphological features, such as loss of pigmentation, poor sporulation, presence of ascomata, or variable growth at different temperature (Balajee et al., 2005a;Hong et al., 2005). These distinctive features, summarized in Table 2, are non-specific and variable, according to the growth media and conditions. Molecular methods are increasingly used as an adjunctive diagnostic tool in medical mycology. However, sequencing methods usually targeting fungal ribosomal DNA [internal transcribed spacer region (ITS), 18S rDNA, 26S/28S rDNA] are reliable for distinction at the section level, but not at the species level for Aspergillus spp. (Balajee et al., 2007). Phylogenetic studies integrate data of partial DNA sequences of various genes, such as beta-tubulin (benA), calmodulin, actin and hydrophobin (rodA), to distinguish the different species within the section Fumigati (Hong et al., 2005;Samson et al., 2007;Yaguchi et al., 2007). This approach is fastidious and not convenient for rapid identification in clinical practice. Currently, experts recommend comparative sequence analyses of the ribosomal ITS region (ITS1 and ITS2 flanking regions of the 5.8S rDNA) for intersection identification of Aspergillus spp. and of the beta-tubulin or calmodulin genes for intrasection identification at the species level (Balajee et al., 2007(Balajee et al., , 2009aSamson et al., 2014). Multiplex PCR assays targeting microsatellite markers or specific gene sequences (ITS2 region, benA, rodA) have been developed for rapid detection and discrimination of Aspergillus species within the section Fumigati in clinical samples (Serrano et al., 2011;Araujo et al., 2012;Fernandez-Molina et al., 2014). Other techniques have also been described, such as PCR-restriction fragment length polymorphism (PCR-RFLP) of the benA gene (Staab et al., 2009) and microsphere-based Luminex assay (Etienne et al., 2009).
Matrix-assisted laser desorption ionization -time-of-flight mass spectrometry (MALDI-TOF MS) also gave promising results for the distinction between A. fumigatus and A. lentulus in clinical isolates (Verwer et al., 2014). This approach demonstrated accuracy for identification of Aspergillus spp. at the species level (Sanguinetti and Posteraro, 2014). Mass spectra of A. lentulus and other cryptic Aspergillus spp. have been included in different commercial or in-house libraries. However, protein extraction procedures for filamentous fungi are not standardized and further analyses of mass spectra of these fungal species, performed under the same experimental conditions, are warranted in order to obtain a complete reference database.

CLINICALLY RELEVANT Aspergillus spp. OF SECTION Fumigati (OTHER THAN A. fumigatus)
Various cases of fungal infections involving Aspergillus spp. of section Fumigati other than A. fumigatus have been reported in the medical literature (Table 3). Clinical presentation is often similar to that of other invasive aspergillosis and positive Phenotypic characteristics are expressed in comparison to A. fumigatus reference wild-type strain (Af293). Max T, maximal temperature at which growth may be observed; AMB, amphotericin B; TAZ, triazoles (voriconazole, posaconazole, and itraconazole), ECH: echinocandins (caspofungin, micafungin, and anidulafungin); NA, no data available 1 Teleomorph: Neosartorya fischeri/pseudofischeri. 2 Aspergillus felis exhibits better growth at 37 • C and is able to growth at 45 • C, while A. viridinutans cannot. fungal biomarkers (galactomannan, beta-1,3-D-glucan) may also be observed. A. lentulus is the most frequent species recovered in clinical specimens and mixed infections with concomitant A. fumigatus are also reported. Invasive fungal infections due to A. lentulus or other A. fumigatus-related species are associated with a particularly high mortality rate (about 60%). In addition, these cryptic species have also been associated with allergic fungal diseases and chronic colonization in patients with cystic fibrosis (Shivaprakash et al., 2009;Symoens et al., 2010). Some microbiological and clinical features of the most clinically important species will be discussed in this chapter.

Aspergillus lentulus
Aspergillus lentulus was first identified in 2004. While retrospectively screening for itraconazole resistance in a collection of 128 A. fumigatus isolates obtained from hematopoietic stem cell transplant recipients between 1991 and 2000 in Seattle, Balajee et al. (2004) described an A. fumigatus variant exhibiting poor sporulation and decreased susceptibility to azoles, amphotericin B and echinocandins. These atypical isolates were found to be the cause of invasive fungal infections in 4 (5%) of these patients. On the basis of phylogenetic analyses, these isolates were assigned to a new species within the section Fumigati under the name of A. lentulus (Balajee et al., 2005a). Phenotypic characteristics of A. lentulus compared to A. fumigatus are poor sporulation, smaller conidial heads and inability to grow at temperature above 48 • C. Mass spectrometry methods (MALDI-TOF, SELDI-TOF) showed promising results for rapid discrimination between A. lentulus and A. fumigatus (Pinel et al., 2011;Verwer et al., 2014).
A. lentulus was also pathogenic in larvae of Galleria mellonella who did not respond to voriconazole therapy (Alcazar-Fuoli et al., 2015). Decreased susceptibility to all three antifungal classes (azoles, polyenes and echinocandins) are reported in most clinical isolates (Alcazar-Fuoli et al., 2008;Alastruey-Izquierdo et al., 2013Escribano et al., 2013). The level of resistance of A. lentulus to triazoles is variable, with higher MICs observed for voriconazole and itraconazole compared to posaconazole. The new triazole isavuconazole displays good in vitro activity against A. lentulus (Datta et al., 2013). Intrinsic azole resistance in A. lentulus is dependant of the Cyp51A gene (AlCyp51A), which shares 92% homology with its A. fumigatus ortholog (AfCyp51A). Substitution of AfCyp51A by AlCyp51A induced the same phenotype of azole resistance in A. fumigatus . Molecular dynamics modeling suggested that some differences in the BC-loop between AfCyp51A and AlCyp51A may affect the closed form of the protein upon voriconazole binding . Among echinocandins, decreased susceptibility of A. lentulus is mainly observed for caspofungin (Alcazar-Fuoli et al., 2008;Alastruey-Izquierdo et al., 2014). However, there was no polymorphisms in the known hot spot regions of the A. lentulus fks gene (encoding for the β-1,3-glucan synthase target of echinocandins), which shares 98% homology with A. fumigatus fks (Staab et al., 2010).

Aspergillus (Neosartorya) udagawae
Aspergillus udagawae was first isolated in 1995 from the soil of a sugar cane plantation in Brazil (Horie et al., 1995). Morphological features that may help distinguishing A. udagawae from A. fumigatus are the decreased spore production and the secretion of a pink-purple pigment on agar plates. A. udagawae also exhibits a different range of thermotolerance, being able to grow at 10 • C, but not at ≥42 • C, with an optimal growth at 30-35 • C (Sugui et al., 2010). Decreased rates of germination and increased susceptibility to neutrophils and hydrogen peroxide may explain the lower virulence of A. udagawae compared to A. fumigatus in murine models (Sugui et al., 2010;Gyotoku et al., 2012).
Molecular analyses suggest that A. udagawae may account for a small proportion of the Aspergillus spp. of section Fumigati isolated from clinical specimens (Balajee et al., 2006;Vinh et al., 2009b;Escribano et al., 2013). Few clinical cases have been reported in the literature. A. udagawae may cause a chronic form of pulmonary aspergillosis with a propensity for contiguous spread to adjacent structures, which may be refractory to antifungal therapy (Vinh et al., 2009b). Cases of endobronchial infection and post-traumatic keratitis have also been reported in immunocompetent patients (Posteraro et al., 2011;Gyotoku et al., 2012). A. udagawae isolates usually exhibit higher MIC (1 to 3 dilutions) to amphotericin B and voriconazole compared to A. fumigatus sensu stricto (Balajee et al., 2006;Vinh et al., 2009b). The new triazole isavuconazole demonstrated increased activity (MIC ranging 0.031-0.25 µg/ml; Datta et al., 2013).
Aspergillus (Neosartorya) fischeri/Aspergillus thermomutatus (Neosartorya pseudofischeri) Aspergillus fischeri (teleomorph: N. fischeri) is a ubiquitous fungus producing thermotolerant ascospores, which is responsible of food spoilage of fruit juices and other heat-processed fruit products. A. thermomutatus (teleomorph: N. pseudofischeri) can only be differentiate from A. fischeri by electron microscopic analysis of the ascospores (Peterson, 1992). These homothallic fungi produce whitish fast-growing colonies with few sporulation at 37 • C and can tolerate higher temperatures (48 • C). Ascomata of the sexual form are usually abundant, but may be scarce or appear at later stages, especially at 37 • C.

CONCLUSION
Recent advances in phylogenetic analyses and molecular methods have revealed the important diversity of Aspergillus species within the section Fumigati. Some clinical isolates may actually be misidentified as A. fumigatus by conventional diagnostic methods, which may result in inappropriate antifungal therapy because of the decreased susceptibility of these cryptic species to many antifungal agents. Azole resistance among A. fumigatus isolates is an emerging problem, which has been highlighted by multiple recent reports throughout the world, but its prevalence is still low. Actually, in case of refractory invasive aspergillosis, clinicians should suspect that a sibling species of A. fumigatus may be the causal agent. Slow or poor sporulation is usually the first hint that should alert the microbiologist. Sequencing of the ITS region, followed by targeted sequencing of the beta-tubulin or calmodulin genes seems to date the most appropriate method for reliable species identification. However, these procedures are not available in most institutions. MALDI-TOF MS may be a very convenient alternative for the rapid detection of A. lentulus and other A. fumigatus-related species, but further investigations for standardized sample treatment procedures of filamentous fungi and spectra characterization are required. Antifungal susceptibility testing may be useful because of the unpredictable susceptibility profile of these species. However, results should be interpreted with caution because of the lack of clinical breakpoints and the absence of data correlating MICs with clinical outcomes. Recommendations for the antifungal management of such cases cannot be clearly defined on the basis of limited published data consisting in case reports of refractory invasive aspergillosis with often fatal outcomes despite the use of multiple antifungal agents. Decreased susceptibility to azoles is common and higher MICs to other antifungal drugs (amphotericin B, caspofungin) may also be observed, especially for A. lentulus. Among triazoles, posaconazole displays better in vitro antifungal activity compared to voriconazole and itraconazole, but is not yet approved as first-line therapy of invasive aspergillosis. In addition, the new extended-spectrum isavuconazole, which has recently been approved for the treatment of invasive aspergillosis (Miceli and Kauffman, 2015), may become an interesting alternative as both A. lentulus and A. udagawae displayed low MICs comparable to that of A. fumigatus (Datta et al., 2013). Combination therapies may be considered in refractory cases, although data are lacking to demonstrate a benefit in this setting.

AUTHOR CONTRIBUTIONS
FL: Design and redaction of the manuscript.