Molecular Identification, Phylogenetic Analysis and Antifungal Susceptibility Patterns of Aspergillus nidulans complex and Aspergillus terreus complex isolated from clinical specimens

Abdolreza Mohamadniaa, Zahra Salehib, Zahra Namvarc, Payam Tabarsid, Mihan Pourabdollah

ABSTRACT

Objective. – Aspergillus sections Terrei and Nidulantes are the less common causes of invasive aspergillosis and pulmonary aspergillosis (PA) in immunocompromised patients when compared to A. fumigatus and A. flavus. Identifying these fungi as the infectious agent is crucial because of the resistance to amphotericin B (AMB) and increased lethality. The aim of this study was to identify the molecular status, evaluate the genetic diversity and examine the antifungal susceptibility profile of the uncommon Aspergillus species. Forty-five uncommon Aspergillus species were identified based on the microscopic and macroscopic criteria. Then, the molecular identification was performed using the sequencing beta tubulin (benA) gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), ravuconazole (RAV), voriconazole (VRC), caspofungin (CFG) isavuconazole (ISA) and posaconazole (POS) test was performed according to the CLSI M38-A2 guidelines.
Results. – A. terreus was the most species detected, followed by A. nidulans, A. latus, A. ochraceus, and A. citrinoterreus, respectively. The analysis of the benA gene showed the presence of 12 distinct genotypes among the A. terreus isolates. The other species did not show any intraspecies variation. CFG exhibited the lowest MEC50/ MIC50 (0.007 µg/ml), followed by POS (0.125 µg/ml), VRC, ITC, ISA (0.25 µg/ml), RAV (0.5 µg/ml), and AMB (8 µg/ml). Among all the isolates, only 15.5% (7/45) were susceptible to AMB.
Conclusion. – Antifungal susceptibility pattern of the uncommon Aspergillus species is useful to improve patient management and increase knowledge concerning the local epidemiology. Moreover, this information is necessary when an outbreak dealing with drug-resistant infections occurs.

Keywords: Aspergillus terreus, Aspergillus nidulans, Antifungal susceptibility test, benA gene

1. Introduction

Pulmonary aspergillosis may range from the invasive pulmonary aspergillosis (IPA) to the chronic necrotising aspergillosis in immunocompromised patients [1, 2]. Aspergillus fumigatus and A. flavus remain the predominant agents of pulmonary aspergillosis (PA). The prevalence of PA, due to uncommon Aspergillus species such as A. terreus [3] and other rare aspergilli such as A. lentulus [1, 4] and A. nidulans [5], has increased over the past years. Aspergillus terreus has been known to be the fourth common cause of IPA [6]. Aspergillus terreus is geographically limited and has a high incidence in Austria and Spain. [7]. Because of the varied susceptibility to antifungals in different species, identification of Aspergillus clinical species is important [4, 8]. There are fourteen species in Aspergillus section Terrei which are not phenotypically distinguishable [9, 10]. However, only a few of them have been reported in human diseases such as A. terreus stricto sensu, A. citrinoterreus, A. alabamensis, A. hortai, and A. niveus [11]. Molecular studies, which mainly deal with the sequencing, have demonstrated the presence of several cryptic Aspergillus species between isolates [1, 12], such as A. lentulus [13], A. quadrilineata [14], and A. calidoustus [12] within the A. fumigatus complex, A. nidulans complex, and A. ustus complex, respectively. The sequence analyses of previous studies demonstrated that the benA gene appeared to be a good marker for Aspergillus species identification [1, 5, 11, 15]. In the present study, besides phenotypic criteria, molecular identification was performed by sequencing of the afore-mentioned gene. To the best of the researchers’ knowledge, little research has been conducted on the epidemiology and the prevalence of the Terrei and Nidulantes sections, leading to human disease in Iran [16].
Therefore, this study was performed to designate the molecular identification, analyze the genetic diversity and examine the antifungal susceptibility profile of uncommon Aspergillus species.

2. Material and methods

2.1.Isolates and patients

Aspergillus isolates from patients with probable PA were collected retrospectively over a 2year period (2017-2018) from three hospitals located in northern parts of Tehran. The collection consisted of 45 clinical isolates from a variety of specimens, containing bronchoalveolar lavage fluid (BAL) (n=34), biopsy (n=6) and tracheal secretion (n=5) (Table 1). The samples were inoculated in three points on each plate and incubated at 25 °C for 5 days. The Sabouraud Dextrose Agar (SDA) was used for the morphological characteristics observations purpose [17]. Following this procedure was the observation of the macro/microscopic features of the colonies.

2.2.Molecular identification and Phylogenetic analysis

The cultures, which were grown on SDA at 25 °C for 5 d. DNA were extracted from the cells. The extraction process was performed using the glass-bead, grinder, and phenolchloroform-isoamyl alcohol according to Mirhendi et al. [18]. All isolates were identified by DNA sequencing of the partial β-tubulin gene using primers Bt2a (5`-GGTAACCAAATCGGTGCTGCTTTC-3`) and Bt2b (5`-ACCCTCAGTGTAGTGACCCTTGGC-3`) according to Hong et al. [19].
Obtained DNA sequences were analyzed by MEGA7.0.21 software [20]. According to the database BLASTn, identification and comparison of species were embarked. The sequencing of data was manually adjusted and analyzed by Maximum parsimony (MP) as well as Maximum likelihood (ML) in MEGA7.0.21 software (K80+I model and 1000 bootstrap), A. fumigatus was assigned to the outgroup.

2.3. Antifungal susceptibility testing

Susceptibilities Aspergillus species against AMB, ITC, RAV (Sigma-Aldrich), VRC, CFG (MSD, Kenilworth, NJ), ISA (Basilea Pharmaceutica International Ltd., Basel, Switzerland), and ]. The final concentration, and ISA, from 0.007 to 8 CFG -microplates. All tests were performed in duplicate. Candida krusei(ATCC 22019) were used as quality controls. The were calculated. The MIC was defined awas inhibited by 100% for antifungals in comparison with the control culture. was calculated according to CLSI M38-A2; isolates were grouped as susceptible (MIC or MEC ≤1 μg/ml), intermediate (MIC or MEC 2 μg/ml), and resistant (MIC or MEC ≥ 4 μg/ml) [21].

3. Results

3.1.Isolates and patients

Among the 45 patients, 62.8% were male and 37.2% were female with a mean age of 49 years. 35.5% and 23.5% of patients had a history of using corticosteroids and hospitalization in ICU admission, respectively. Also, 80% of patients were recovered using the itraconazole alone, voriconazole alone or itraconazole and voriconazole while 20% of the same subject had a fatal outcome. One of the patients was treated with itraconazole and voriconazole followed by caspofungin.

3.2. Molecular identification and genotypic analysis

Comparative sequence analyses of the benA gene of the 45 Aspergillus isolates in the database BLASTn revealed the following species distribution: 33 (73.3%) isolates belonged to PCR of the benA gene of the 45 clinical isolates showed bands at 450 bp to 550 bp. The data obtained from the MP analysis of the aligned benA sequences of A. terreus sensu stricto isolates showed that out of the 530 nucleotides to be sequenced, the 22 nucleotides were variable characters and only 14 characters (2.64%) were parsimony-informative. The largest portion of the parsimony-informative site existing in A. terreus sensu stricto isolates that was found to be between nucleotide 78-105, 200-213 and 321-374 out of the 530 bp. The other species did not show any intraspecies variation. Furthermore, the phylogenic analysis of the benA gene indicated the presence of 12 distinct genotypes among the A. terreus sensu stricto isolates. Sequences have been deposited in GenBank under the access numbers MN594517-MN594521.
The Maximum likelihood phylogenetic tree based on benA sequences of Aspergillus species showed three distinct clades. The A. nidulans complex clade showed two differentiated clusters containing A. nidulans and A. latus. In the A. terreus complex clade, five clusters were found and that A. citrinoterreus isolate remained well distanced from A. terreus sensu stricto isolates. Posterior probabilities more than 50% are given for appropriate clades (Fig. 2). Statistically, a significant association was not found between the phylogenetic analysis and the anatomical source or antifungal susceptibility pattern by Fisher’s exact test.

3.3. Antifungal susceptibility testing

The MIC/MEC range, Geometric mean MIC, MIC50/MEC50, and MIC90/MEC90 for the 45 isolates for the Aspergillus species against the seven antifungals are presented in Table 2. The lowest MIC50/MEC50 for CFG was 0.007 µg/ml, compared to 0.125, 0.25, 0.25, 0.25, 0.5, 8 µg/ml for POS, ISA, VRC, ITC, RAV and AMB, respectively. Overall, AMB exhibited the highest MIC (MIC50, 16 μg/ml) for A. nidulans complex, and CFG showed the lowest MEC (MEC50, 0.007 μg/ml) for all species. Only one A. nidulans isolate caspofungin-resistant was observed. Among all isolates, only 5 (14.7%) of 34 isolates A. terreus complex, one (50%) of isolate A. latus and one (50%) of isolate A. ochraceus had sensitivity to AMB. Thirty-eight of the isolates (84.4%) including 27 isolates A. terreus sensu stricto, one isolate A. citrinoterreus, seven isolates A. nidulans, one isolate A. latus and both isolates A. ochraceus had MICs of > 4 μg/ml for at least one antifungal drug. The highest percentage of resistance to amphotericin B was observed against A. nidulans. Interestingly, resistance to VRC, POS and ISA antifungal drugs was not observed in any species.

4. Discussion

Lung is the most common site of the infection as influenced by the Aspergillus species. This involvement with IPA presentation is caused by different Aspergillus species. As an example in case, A. terreus and A. nidulans often cause disseminated infection with increased lethality due to higher MIC to AMB [1, 7, 11]. Because of the similarity and overlapped morphological features of the Aspergillus species, complex morphology-based identification methods are not reliable, therefore, molecular approaches are necessary for accurate identification of Aspergillus to species level. [1, 5].
Little is known about the prevalence of Aspergillus sections Terrei and Nidulantes in Tehran clinical samples and antifungal susceptibility. This study allowed assessment of the uncommon Aspergillus species and antifungal susceptibility testing in patients with PA of this geographical area. Genetic diversity of A. terreus sensu stricto species were analyzed by benA gene which revealed high genetic diversity among this species. Results of the previous studies on RAPDPCR and AFLP [3, 22, 23] are in line with the ones found in the current study.
The species A. terreus sensu stricto and A. nidulans were the most represented groups among these isolates, with 33 (73.3%) and 7 (15.5%) strains, respectively. Also, A. citrinoterreus was isolated from a BAL sample of a 66-year-old woman with respiratory symptoms. In the Imbert et al [11] and Vaezi et al [16] studies, A. terreus sensu stricto were the dominant species in A. terreus complex, which is consistent with the results of the current study.
In the current study, similar to the one conducted by Mouas et al. [24] and Kontoyiannis et al. [25], A. nidulans species are resistant to AMB. Rosen-Wolff et al. indicated that a considerable amount of species of A. nidulans were resistant to most antifungal so far [26]. Since amphotericin B is considered to be the main treatment for severe fungal infections [27], identification of Aspergillus isolates at the species level and antifungal susceptibility pattern should be performed.
Results of the present work showed that MIC50 of VRC against A. nidulans complex was 0.125µg/ml. Also, novel antifungal drugs such as ISA and POS are effective. Considering that van’t Hek et al. introduced voriconazole as a drug possible for the treatment of A. nidulans [28].
According to the results of this study, it seems ISA and POS are as effective in treating A. nidulans as voriconazole. It is recommended to use ISA and POS in patients resistant to voriconazole. Further studies seem to be needed. Findings also manifested that A. nidulans isolates, which showed resistance to ITC and RAV, were resistant to AMB. Aspergillus latus species were sensitive to ITC, VRC, POS, ISA, CFG, and RAV.
In the study undertaken by Oakley et al., which is similar to the current study, no cross-resistance was observed between VRC and ITC [29]. Of the nine A. terreus isolates resistant to ITC under examination, 88.8 % were cross-resistant to AMB and 11.1 % were cross-resistant to RAV.
Among A. nidulans isolates resistant to AMB, only one isolate was resistant to ITC. Two of thirty-four A. terreus isolates were resistant to RAV. As far as the researchers perceive, this is the first report so far, showing that A. terreus isolates are resistant to RAV, the cause of pulmonary Aspergillosis in Iran. Only one of two isolates aforementioned was resistant to AMB. Interestingly, all A. terreus isolates resistant to ITC were also resistant to AMB. According to the study led by Patterson et al., VRC is the main drug and POS is an alternative when it comes to the treatment of IA [30].
Outcomes of the current research, as reported by Vaezi et al. [16], showed caspofungin (echinocandins) had the lowest MEC50/MIC50 (0.007 µg/ml) compared to the other drugs. Therefore, CFG could be considered as an alternative for the treatment of IA in some resistance cases. A number of previous studies have shown that A. terreus species are resistant to AMB [3134], while in this study, AFST showed that15.2% (5/33) of A. terreus isolates were sensitive to AMB (MICs < 4 μg/ml). The study put forth by Vaezi et al. confirms the results of the current experiment [16]. Such outcomes may be due to the genetic diversity of different region species for which the present study was performed. However, no significant differences were observed between A. terreus sensu stricto and A. citrinoterreus of A. terreus complex and A. nidulans and A, latus of A. nidulans complex in the seven antifungals.

5. Conclusions

The obtained results in this study were in accordance with the in vitro tests while it seems that in vivo trials should be performed for more reliable and precise results. In the current experiment, similar to the ones conducted by Tortorano et al. and Neal et al. [35, 36], no relationship was found between the phylogenetic analysis and AMB susceptibility. The results may be attributable to the low number of samples. Overall, identifying Aspergillus at the species level especially uncommon Aspergillus species and cryptic Aspergillus species as well as antifungal susceptibility testing of these clinical samples seem useful to improve patient management and increase the knowledge of local epidemiology. Furthermore, AmB may not be suitable antifungal for treatment of uncommon Aspergillus species.

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