Treatment of Infections Due to Aspergillus terreus Species Complex

The Aspergillus terreus species complex is found in a wide variety of habitats, and the spectrum of diseases caused covers allergic bronchopulmonary aspergillosis, Aspergillus bronchitis and/or tracheobronchitis, and invasive and disseminated aspergillosis. Invasive infections are a significant cause of morbidity and mortality mainly in patients with hematological malignancy. The section Terrei covers a total of 16 accepted species of which most are amphotericin B resistant. Triazoles are the preferred agents for treatment and prevention of invasive aspergillosis. Poor prognosis in patients with invasive A. terreus infections seems to be independent of anti-Aspergillus azole-based treatment.


Introduction
Aspergillosis refers to infection by any of the Aspergillus species; the most common pathogens are representatives of the Aspergillus fumigatus species complex, Aspergillus flavus species complex, Aspergillus terreus species complex, and Aspergillus niger species complex. The A. terreus species complex is found in a wide variety of habitats, soil, compost, or dust, and the spectrum of diseases caused covers allergic bronchopulmonary aspergillosis, Aspergillus bronchitis and/or tracheobronchitis, and invasive and disseminated aspergillosis [1,2]. The section Terrei covers A. terreus sensu stricto (s.s.), A. alabamensis, A. allahabadii, A. ambiguus, A. aureoterreus, A. bicephalus, A. carneus, A. citrinoterreus, A. floccosus, A. iranicus, A. hortai, A. microcysticus, A. neoafricanus, A. neoindicus, A. niveus, and A. pseudoterreus, giving a total of 16 accepted species [2]. Infections due to the A. terreus species complex occur worldwide. However, there are certain geographic locations with frequent occurrences, such as Innsbruck, Austria and Houston, USA [3]. Within the aspergilli, the A. terreus species complex assumes a specific role, as most representatives are amphotericin B resistant [4]. A. terreus is an emerging opportunistic fungus whose clinical incidence has increased in recent years, with invasive diseases constituting nearly 4% of all invasive aspergilloses [5]. Of special concern is the high mortality in disseminated diseases caused by this species [6]. Here, we review in vitro susceptibility, experimental and clinical outcome data, and overview recommendations on how to treat infections due to the A. terreus species complex.

The Pathogen and Fungal Epidemiology
Aspergillus section Terrei is widespread throughout the world and found in arable soil, desserts, and compost heaps [2]. A. terreus usually shows cinnamon-brown colonies when growing on Saubouraud Agar at 37 • C for 48 h (see Figure 1a). The production of aleurioconidia in A. terreus, A. carneus, A. flavipes, and A. niveus seems to be unique among the Aspergillus species; these are morphologically distinct lateral conidia (aleurioconidia) which are attached directly to hyphae (see Figure 1b). Whether these features support dissemination or enhanced virulence in vivo is presently unknown. Deak et al. [7] showed that these accessory conidia induce inflammation in a pulmonary mouse model of invasive aspergillosis; amphotericin B resistance was shown not to be associated with the production of aleurioconidia. A. terreus is commonly used in industry to produce organic acids and lovastatin. In a prospective international multicenter surveillance study, including 21 countries and 38 centers, an overall prevalence of 5.2% (370/7116) of A. terreus infections among mold infections was detected [8]. Cases were reported from Europe (n = 261), Middle East (n = 70), India (n = 19), South America (n = 10), and North America (n = 10). Spain and Austria were the countries with the highest density of A. terreus isolates collected in patients [8].  The reason for this specific epidemiological situation is unclear. A. terreus strains isolated from environmental and clinical sources were genotyped using a novel panel of short tandem repeats [9]. Three major endemic genotypes collected from the Inn region and its side valleys were found to cause the majority of invasive A. terreus diseases in Tyrol, Austria. The genotypes were of the same mating type and persisted for at least 11 years.

Treatment Recommendations
The effective management of invasive aspergillosis includes strategies to optimize prevention and early antifungal treatment, immunomodulation, and in some cases, surgery. Overall, treatment strategies should be based on the institutional epidemiology of infections and assessment of individual risk factors.
Triazoles are the preferred agents for treatment and prevention of invasive aspergillosis in most patients [11]. Patients receiving triazole-based therapy for treatment of invasive diseases or prolonged prophylaxis should undergo therapeutic drug monitoring (TDM). This approach supports enhancing therapeutic efficacy of voriconazole and posaconazole. Echinocandins have been shown to be effective only in salvage therapy. Amphotericin B is not recommended; however, in selected cases [12,13], improved treatment outcome was observed under high dose amphotericin B treatment. Combinations of polyenes or azoles with echinocandins suggest additive or synergistic effects in some preclinical studies [14,15]. However, variable assay conditions and conflicting results have led to uncertainty as to how to interpret these findings. Early initiation of adequate antifungal therapy is warranted in addition to a diagnostic evaluation [11]. Treatment should be continued for a minimum of 6-12 weeks [11], depending on degree and duration of immunosuppression and the site of infection. Patients with chronic aspergillosis and either pulmonary or general symptoms or progressive loss of lung function should be treated for a minimum of 6 months. Oral itraconazole and voriconazole are the preferred drugs [11]; posaconazole is another option for individuals with adverse events or clinical failure. Hachem et al. investigated 513 patients suffering from hematological malignancies and compared baseline characteristics, antifungal treatment, and outcome between patients infected with A. terreus and non-terreus Aspergillus species [10]. A. terreus diseases were associated with a lower response rate to treatment, and a higher rate of mortality when compared to non-terreus infections. In addition, fungal breakthrough infections occurred more often in the A. terreus arm; independent factors associated with final outcome included treatment with azoles (p < 0.0001) and Aspergillus species involved (p = 0.043).

Amphotericin B
Amphotericin B, a polyene antifungal drug, was developed in the 1950s and is used intravenously to treat invasive aspergillosis, cryptococcal meningitis, candidiasis, and mucormycosis. In vitro resistance to amphotericin B is rare; few fungal species such as A. terreus, Aspergillus tanneri, Fusarium species, and Scedosporium prolificans show polyene resistance [16,17]. The major clinical interest in A. terreus infections is related to its amphotericin B resistance (see Figure 1c); retrospective studies revealed high rates (80-90%) of treatment failure with amphotericin B [18]. The Clinical Laboratory Standard Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have categorized A. terreus as resistant to amphotericin B [17,19]. Furthermore, 70% of A. terreus isolates investigated showed minimum fungicidal concentrations (MFCs) to be twofold higher than the obtained minimum inhibitory concentrations (MICs). With these findings, the authors underline the poor efficacy against A. terreus infections, specifically in neutropenic patients [15]. Animal studies confirmed resistance of A. terreus infections even to high doses of amphotericin B or its liposomal formulation in murine and rabbit models. However, a single experimental study in neutropenic mice suggested A. terreus to be susceptible to high doses of liposomal amphotericin B, achieving high concentrations in the lungs (4-8 mg/L) [13]. A. terreus is intrinsically resistant to amphotericin B, and the underlying mechanisms may be associated with the level of catalase production by this species when compared to A. fumigatus [20][21][22]. Studying amphotericin B resistant (ATR) and amphotericin B susceptible (ATS) clinical A. terreus isolates showed ATR to possess doubled basal superoxide dismutase (SOD) activity when compared to ATS; in addition, ATR presented an enhanced oxidative stress response (OSR), with significantly higher sod2 mRNA levels and increased catalase transcripts during amphotericin B treatment. The inhibition of SOD and catalase (CAT) proteins rendered ATR to ATS in vitro [20][21][22].
Hypoxia seems to have a major influence on the activity of amphotericin B against A. terreus [23]. A significant decrease in the MICs against amphotericin B was detected, mainly due to the loss of mycelium sterillium zones applying the Etest ® . In addition, the fungicidal activity of amphotericin B against A. terreus was significantly decreased under hypoxia, suggesting a shift to fungistatic activity under low-oxygen conditions. Whether these findings have any clinical relevance needs to be investigated in more detail. The alternative in vivo model Galleria mellonella was applied to study the pathogenicity of ATS and ATR, and to evaluate amphotericin B efficacy in vivo [24]. Larvae were susceptible to A. terreus infection in an inoculum-size and temperature dependent manner, and in vitro susceptibility to amphotericin B correlated with in vivo outcome, defining an amphotericin B susceptible strain cluster of A. terreus [24]. Usually, the MICs of amphotericin B are generally ≥2 mg/L (see Figure 1c), but low MICs (0.06-0.125 mg/L) have also been reported for the A. terreus species complex. To our knowledge, the real origin of ATS is unknown; ATS may exist in the environment or may reflect an accidental product of sectoring of ATR on drug-free medium, or may result from both [25]. In a clinical setting, ATS occurs rarely, and we have shown that spontaneous culture degeneration of ATR leads to the emergence of multiple sectors (see Figure 1d), which in turn displayed low MICs against amphotericin B, hence featuring ATS. Susceptibility and virulence studies of sector subcultures (ATSec), ATR, and ATS strains showed ATSec harbouring amphotericin B MICs ranging from 0.12 to 0.5 µg/mL, and G. mellonella survival studies revealed an enhanced virulence of ATSec [25]. With regard to antifungal susceptibility testing, this is a very important finding; any colony selected for inoculum preparation should be free of sectoring. Otherwise, MIC data may be unstable in repeating tests, further complicating the interpretation of MIC and in vivo outcome. We noticed that sectoring frequently takes place on various culture media in ATR.

Azoles
Voriconazole is the drug of choice for first-line treatment of invasive aspergillosis including A. terreus [26]. Limited data are available for itraconazole, posaconazole, and isavuconazole. Four (44%) out of nine patients were treated with posaconazole (salvage therapy) and responded favorably, whilst the success rate of lipid amphotericin B formulations was only 19%. There is limited clinical experience on the efficacy of the echinocandins in the treatment of A. terreus infections. A murine model of A. terreus disseminated infection, with isolate MICs ranging from 0.12 mg/L to 4 mg/L in evaluated voriconazole treatment [27]. Improved survival and a reduced fungal load in brain and kidney were given in strains displaying MICs ≤1 mg/L. Few experimental data showed that high itraconazole doses may be effective in the treatment of animals infected with A. terreus. Posaconazole has shown efficacy in treating invasive infections by A. terreus in rabbits and mice.
Recently, a considerable amount of azole resistance in Aspergillus section Terri was observed; however, whether the latter finding correlates with in vivo results is presently unknown [28]. The emergence of A. terreus sensu lato (s.l.) isolates with reduced azole-susceptibility was reported in patients suffering from cystic fibrosis [29]. In a worldwide study performed, approximately 5% of all tested A. terreus s.s. isolates were resistant to posaconazole in vitro [28]. In Austria, Germany, and the UK, posaconazole resistance was higher than 10% in all A. terreus s.s. isolates. The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey, to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare, and was observed only in one A. citrinoterreus and one A. alabamensis isolate. Resistance against itraconazole and voriconazole was not detected [28]. Azole resistance in A. terreus s.s. and A. fumigatus is associated with mutations and alterations of the lanosterol-14-α steroldemethylase gene (Cyp51A), a key protein in the ergosterol biosynthesis pathway [30]. However, aside from mutations in the primary target gene, other less known mechanisms (e.g., efflux pumps, overexpression of cyp51) were also found to be involved in azole resistance.

Conclusions
The effective management of invasive aspergillosis includes strategies to optimize prevention and early antifungal treatment, immunomodulation, and in some cases, surgery. Overall, treatment strategies should be based on the institutional epidemiology of infections and assessment of individual risk factors. Triazoles (Vorriconazole and isavuconazole) are the preferred agents for the treatment and prevention of invasive aspergillosis due to A. terreus in most patients [11]. The underlying resistance mechanisms of A. terreus against amphotericin B are only partly understood. The mode of action of amphotericin B seems to be complex and multifaceted (see Figure 2); amphotericin B interacts in various ways with ergosterol, and hence results in fungal cell membrane disruption leading to intracellular leakage and cell death [22]. In addition, amphotericin B may directly act as a pro-oxidant or anti-oxidant, and contribute to an accumulation of free radicals which have multiple deleterious effects. We demonstrated that the co-application of anti-and pro-oxidants significantly affects amphotericin B efficacy in an antithetic manner [21]. Antioxidants and ROS-scavenging agents increased amphotericin B tolerance in susceptible A. terreus strains, while pro-oxidants rendered them resistant to susceptible strains; this in vitro data was confirmed in vivo by applying Galleria mellonella animal models. Whether such management is of clinical interest needs to be studied in detail.  (3,4,5) causing cell permeability and cell death. Amphotericin B may act as an oxidant as well as antioxidant [22]. Several AmB molecules penetrate the cell, where oxidation takes place, and radicals emerge.