Epidemiological characteristics of invasive Aspergillus isolates: morphology, drug susceptibility, and mutations in azole drug targets

doi:10.21203/rs.3.rs-4715493/v1

Download PDF

Research Article

Epidemiological characteristics of invasive Aspergillus isolates: morphology, drug susceptibility, and mutations in azole drug targets

https://doi.org/10.21203/rs.3.rs-4715493/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

The global epidemiology of aspergillosis varies and is influenced by various factors. To elucidate the disease burden and identify effective control strategies, the epidemiological characteristics of Aspergillus infections have to be investigated. The aim of this study was to assess the epidemiological characteristics of various Aspergillus species, including their morphological features, species identification, and in vitro susceptibility to nine antifungal agents in a large tertiary hospital in northern China.

Methods

Ninety-five clinical isolates of Aspergillus were collected from patients. Aspergillus species identification was performed using conventional morphological methods, MALDI-TOF MS, and gene sequencing. In vitro susceptibility to nine antifungal agents was evaluated using the Sensititre YeastOne system. Target genes (cyp51A and cyp51b) of A. tubinazole were sequenced using the Sanger method.

Results

Aspergillus fumigatus, A. niger, A. flavus, A. tubingensis, and A. terreus were the most common isolated species. Rare species included A. tamarii, A. usamil, A. versicolor, A. udagawae, A. lentulus, A. sydowii, and A. quadrilineatus. Pulmonary infections accounted for 86.3% (82/95) of collected cases, and the in-hospital mortality rate was 22.1%. The median minimum inhibitory concentration (MIC) range of amphotericin B was 1.5–4 mg/L. The MIC range of triazoles against Aspergillus species, excluding A. udagawae and A. lentulus, was 0.12–0.5 mg/L. The median minimum effective concentration range of echinocandins was < 0.008–0.03 mg/L. Non-wild-type resistance to amphotericin B was observed in 29.6% (16/54) of A. fumigatus isolates, and non-wild-type resistance to voriconazole was observed in 11.1% (1/9) of A. tubingensis isolates. Moreover, cyp51A and cyp51b of A. tabinensis had 2–29 and 10–13 nucleotide mutations, respectively.

Conclusion

Patients with non-A. fumigatus infection accounted for 43.2%. The T256A amino acid substitution in cyp51A of A. tabinensis did not lead to increased azole drug MICs.

Aspergillus

epidemiology

morphology

gene mutation

drug resistance mechanism

antifungal susceptibility

sanger sequencing

Aspergillosis refers to a series of fungal infections caused by different Aspergillus species, including allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis, and invasive aspergillosis. These infections range from mild allergic reactions to serious invasive diseases, with the latter being more commonly observed in immunocompromised patients, such as those with cancer, AIDS, or those receiving immunosuppressive treatment [1]. The global epidemiology of aspergillosis varies and is influenced by factors such as geographical location, climate, population density, and medical infrastructure [2, 3]. Studying the epidemiological characteristics of Aspergillus infections is essential for understanding the disease burden and guiding effective control strategies.

Early classification strategies for Aspergillus relied on macroscopic and microscopic morphology. Over time, the methods for identifying Aspergillus species have evolved, with molecular techniques such as polymerase chain reaction (PCR) and DNA sequencing becoming increasingly utilised [4, 5]. Treatment of aspergillosis involves the use of antifungal agents, primarily azole drugs. Azoles, such as voriconazole, itraconazole, and posaconazole, are the mainstay therapeutics owing to their broad-spectrum activity against Aspergillus species. However, the emergence of antifungal resistance presents a significant challenge to successful treatment outcomes. Evaluation of the drug sensitivity of Aspergillus isolates is thus crucial for guiding appropriate antifungal therapy [6]. Susceptibility testing assists in determining the most effective drug and dosage for a specific strain, monitoring changes in drug susceptibility over time, and identifying emerging resistance patterns [2]. Resistance to azole drugs in Aspergillus species is frequently linked to mutations in the targets of azoles, particularly CYP51A, a lanosterol 14α-demethylase. Mutations, such as TR34/L98H and TR46/Y121F/T289A in CYP51A, are commonly associated with azole resistance [7].

The aim of the study was to investigate the epidemiologically relevant characteristics of invasive Aspergillus isolates, including their morphology, drug susceptibility patterns, and mutations in genes associated with resistance to azole drugs.

2.1. Sample Collection

In total, 95 clinical isolates of Aspergillus species were collected from The First Affiliated Hospital of Hebei North University between 1 January 2021 and 31 December 2022. Isolates were obtained from different clinical specimens, including sputum, bronchoalveolar lavage fluid, ear canal secretions, and tissue samples.

2.2. Morphological Identification

The collected isolates were cultured on Sabouraud dextrose agar (SDA, Tianjin Jinzhang Technology Development Co., Ltd, Tianjin, China) at 28°C for 3–5 days. Morphological characteristics of the colonies, including colour, texture, and growth rate, were observed and recorded. A drop of lactophenol cotton blue stain was added to the slide. Transparent adhesive tape was used to collect the mycelium and spores from the centre of the colony. Another drop of lactophenol cotton blue stain was then added, and the slide was covered with a cover slip. Finally, its morphology was observed under a microscope at 400 × magnification (Olympus Corporation, BX53, Tokyo, Japan).

2.3. MALDI-TOF MS Identification and Sequencing of Housekeeping Genes

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS; Autobio, Autof ms1000, Zhengzhou City, Henan Province, China) was used for the species identification of Aspergillus isolates. Protein extracts were prepared from each isolate using the formic acid extraction method (Supplementary Materials). Protein extracts were spotted onto a MALDI target plate and analysed using a MALDI-TOF MS system. The protein fingerprints of tested strains were compared to standard fingerprints in the database using a similarity-based scoring system (0–10). Supplementary Table 1 presents the grading criteria, which categorise the identification results into 'Species level confidence,' 'Genus level confidence,' and 'Poor' based on their respective scores. A. fumigatus ATCC®MYA-3627TM was utilised as a quality control and reference strain. To improve the accuracy of Aspergillus identification, ITS, Beta-tubulin, and Calmodulin gene sequencing were performed on samples identified as ‘poor’ via MALDI-TOF MS. Supplementary Table S2 shows the gene amplification primers used.

2.4. Antifungal Susceptibility Testing

Antifungal drug sensitivity experiments were performed according to the manufacturer’s instructions (Sensititre YeastOne, Thermo Fisher Scientific, Waltham, MA, USA). Amphotericin B, anidulafungin, caspofungin, itraconazole, micafungin, posaconazole, voriconazole, and fluconazole were tested. The epidemiological cutoff values provided in the CLSI supplemental document M59 were used to determine the wild-type and non-wild-type isolates of Aspergillus [8]. Candida parapsilosis ATCC22019, Candida krusei ATCC6258, and A. fumigatus ATCC®MYA-3627TM were used as quality control and reference strains.

2.5. Sequencing of Azole Resistance Genes

The genomic DNA of Aspergillus tubingensis was extracted using the specific method described in the Supplementary Materials. PCR was employed to amplify the genes encoding azole drug targets (CYP51A and CYP51B; primer sequences are shown in Supplementary Table 2). In a centrifuge tube, 12.5 µL of Phanta Max Master Mix (Nanjing Novozan Biotechnology Co., Ltd., Nanjing, China), 1 µL of genomic DNA, and 1 µL each of forward and reverse primers were added, and the reaction system was supplemented with deionised water to a total volume of 25 µL. The tube was then placed in a PCR thermal cycler for pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. Subsequently, 5 µL of the amplification product was subjected to agarose gel electrophoresis (1%) (100 V, 40 min) to confirm successful gene amplification. The amplified products were purified and subjected to Sanger sequencing (ABI 3730XL). The gene sequences were imported into the CLC Sequence Viewer 7 software for further analysis.

2.6. Data Analysis

The collected data, including patient age, average length of hospitalisation, and mass spectrometry identification score, were analysed using the appropriate statistical methods. Descriptive statistics, such as frequencies and percentages, were used to summarise the data. The Kruskal–Wallis Test or Dunn’s test was used to analyse the association between different variables. Statistical analysis was performed using R software (ggplot2[3.3.6], stats[4.2.1], and car[3.1-0]). Statistical significance was set at p < 0.05.

3.1. Identification of Aspergillus Species

MALDI-TOF MS was performed to identify the clinical isolates of Aspergillus species. Aspergillus fumigatus was the most prevalent among all isolates, accounting for over half of all isolates (54/95, 56.8%). The second most common species was the Aspergillus niger complex (19/95, 20%), which included Aspergillus niger (10/95, 10.5%) and Aspergillus tubingensis (9/95, 9.5%). Aspergillus flavus (10/95, 10.5%) and Aspergillus terreus (4/95, 4.2%) were the third and fourth most common species, respectively. In addition to these commonly encountered invasive fungal pathogens, several clinically rare Aspergillus species were isolated in the current study, including Aspergillus tamarii (2/95, 2.1%), Aspergillus usamil (1/95, 1.1%), Aspergillus versicolor (1/95, 1.1%), Aspergillus udagawae (1/95, 1.1%), Aspergillus lentulus (1/95, 1.1%), Aspergillus sydowii (1/95, 1.1%), and Aspergillus quadrilineatus (1/95, 1.1%).

Among the five common Aspergillus species (Aspergillus fumigatus, Aspergillus niger, Aspergillus tamarii, Aspergillus flavus, and Aspergillus terreus), the mass spectrometry identification scores (mean ± SD) ranked from highest to lowest were as follows: Aspergillus terreus (8.9 ± 1.6), Aspergillus tamarii (8.6 ± 1.2), Aspergillus fumigatus (7.6 ± 1.7), Aspergillus flavus (7.2 ± 1.4), and Aspergillus niger (5.9 ± 1.4). The within-group comparison yielded a p-value of 0.013. The average identification score for all Aspergillus species was relatively low (7.3 ± 1.7) (Table 1)

Table 1

Clinical characteristics of 95 patients with *Aspergillus* infection.
Species	Number (n/%)	Msis (mean ± SD)	Patient characteristics
	Number (n/%)	Msis (mean ± SD)	Male (n/%)	Age(Median[IQR])	Pulmonary infection (n/%)	In-hospital mortality(n/%)	LOS (days), median[IQR]
A. fumigatus	54/56.8	7.6 ± 1.7	37/65	70[14]	54/100	19/35.2	14[12]
A. niger	10/10.5	5.9 ± 1.4	5/50	73[11.25]	5/50	1/10	11.5[10.3]
A. tubingensis	9/9.5	8.6 ± 1.2	5/55	70.5[11.25]	8/88.9	0/0	10.5[10.3]
A. usamil	1/1.1	6.7	1/100	56	1/100	0/0	21
A. flavus	10/10.5	7.2 ± 1.4	6/60	68[17.75]	8/80	0/0	9.5[11]
A. tamarii	2/2.1	6.7	1/50	74.5	2/100	1/50	13.5
A. terreus	4/4.2	8.9 ± 1.6	2/50	69[18]	1/25	0/0	7.5[9]
A. versicolor	1/1.1	6.7	1/100	56	0/0	0/0	6
A. udagawae	1/1.1	6.5	1/100	71	1/100	0/0	9
A. lentulus	1/1.1	6.2	1/100	87	1/100	0/0	10
A. sydowii	1/1.1	6.8	0/0	59	0/0	0/0	5
A. quadrilineatus	1/1.1	7.8	1/100	92	1/100	0/0	19
Total	95/100	7.3 ± 1.7	61/64	70[14]	82/86.3	21/22.1	13[11.5]
Msis, Mass spectrometry identification score; A., Aspergillus; LOS, length of hospital stay; IQR, interquartile range; NEUT, neutrophil percentage; APP of VOR, Application of Voriconazole

Gene sequencing results for A. lentulus and A. sydowii were aligned with those obtained through MALDI-TOF MS identification. However, discrepancies were observed in the gene sequencing results compared to MALDI-TOF MS identification for certain rare Aspergillus species, including A. quadrilineatus, A. tamarii, A. udagawae, and A. usamil (Supplementary Material Table S3).

3.2. Morphological Characteristics of Clinical Aspergillus Isolates

The morphological characteristics of Aspergillus isolates, including colony, conidial, and hyphal morphologies, were recorded. The colony morphology of Aspergillus isolates varied, with colours ranging from white to green or black and textures ranging from fluffy to powdery. The conidial morphology showed typical features of Aspergillus, such as the presence of conidiophores and chains of conidia. The hyphal morphology revealed septate hyphae with branching patterns, which are characteristic of Aspergillus species.

The colony of Aspergillus niger was fluffy in texture, occupying one-third of the plate after 4 days of growth. The centre was black, the middle was yellow, and the periphery was white (Fig. <link rid="fig1">1</link>A-1). The conidial heads were spherical and radiated in a black-brown colour. The conidial stalks had a diameter of 15–20 µm and were smooth (Fig. 1A-2).

The colony of Aspergillus tubingensis was fluffy in texture, occupying one-third of the plate after 4 days of growth. The periphery was white and fluffy, whereas the interior was black and radiated like a wheel (Fig. <link rid="fig1">1</link>B-1). The conidial heads were spherical, and the double-layered conidial stalks were covered with vesicles (Fig. 1B-2).

The colony of Aspergillus flavus had a yellow, cotton-like centre and a white, fluffy periphery. There were visible yellow, coarse, granular conidial heads (Fig. <link rid="fig1">1</link>C-1). The conidial heads were loosely radiating, with single- or double-layered small stalks, and the conidia were spherical with a rough surface (Fig. 1C-2).

The colony of Aspergillus tamarii was yellowish-brown, with an irregular shape and velvety texture (Fig. <link rid="fig1">1</link>D-1). The conidial heads ranged from spherical to loosely radiating, and the conidial stalks had rough surfaces. The conidia were larger and spherical or elliptical (Fig. 1D-2).

Aspergillus fumigatus grew rapidly, with a dark smoky green fluffy or cotton-like colony (Fig. <link rid="fig1">1</link>E-1). The conidial heads were columnar and formed chains of conidia, while the conidial stalks were short (Fig. 1E-2).

The colony of Aspergillus quadrilineatus was greenish-blue, with raised centres and peripheries (Fig. <link rid="fig1">1</link>F-1). The hyphae were unbranched, and the conidial stalks had swollen tips, occupying half of the vesicles (Fig. 1F-2).

The colony of Aspergillus lentulus was white and fluffy, with a soft and fluffy texture (Fig. <link rid="fig1">1</link>G-1). The conidial heads resembled fans and produced fewer conidia. The conidial stalks were slender and unbranched (Fig. 1G-2).

The colony of Aspergillus udagawae was white and raised, resembling an inverted plate (Fig. <link rid="fig1">1</link>H-1). Conidiation began on the third day, with conidial chains growing on the conidial heads. The conidial stalks were short (Fig. 1H-2).

The colony of Aspergillus terreus grew slowly, with a diameter of approximately 1 cm after 5 days of cultivation. It was a hard, tan-coloured colony (Fig. <link rid="fig1">1</link>I-1). The conidial stalks were single-layered and arranged in clusters, and the conidia were spherical (Fig. 1I-2).

Aspergillus sydowii grew slowly, with a diameter of approximately 0.5 cm after 3 days of cultivation. Its colony was hard, smoky, and green-coloured (Fig. <link rid="fig1">1</link>J-1). The conidial stalks covered the entire vesicle and were smooth (Fig. 1J-2).

3.3. Clinical Characteristics of Patients with Aspergillus Infection

Among all patients with Aspergillus infections, the male-to-female ratio was 1.8:1 (61:34), with the highest proportion of males being infected with Aspergillus fumigatus (37/54, 64.9%). The median age [IQR] was 70 [14], with Aspergillus niger infections having the highest median age among the five common Aspergillus infections (73 [11.25] years) and Aspergillus flavus having the lowest (68 [17.75] years). Within-group comparisons did not reveal significant differences (p > 0.05). Pulmonary infections were the most common (86.3%, 82/95). Approximately 5.3% (5/95) of the patients had cancer, belonging to the immunocompromised population. The hospital mortality rate of patients with fungal infections was 22.1% (21/95). Patients infected with Aspergillus fumigatus had a high mortality rate of 35.2% (19/54). The average hospital stay length for patients was 13 [11.5] days (Table 1).

Table 2 shows the underlying diseases of patients infected with the four common disease-causing Aspergillus species (A. fumigatus, A. niger, A. tubingensis, and A. flavus). The prevalence rates of hypertension and coronary heart disease were 31.3% (26/83) and 15.7% (13/83), respectively. The patient’s infection was confirmed using four inflammatory markers (NEUT%, CRP, PCT, IL-6 [interleukin-6]). Approximately 25% (20/83) of patients were treated with voriconazole (Table 2).

Table 2

Basic diseases and inflammatory indicators in patients with common clinical *Aspergillus* infections
Diagnosis and treatment	A.fumigatus (n = 54)	A. niger (n = 10)	A. tubingensis (n = 9)	A.flavus (n = 10)	All (n = 83)
COPD(n/%)	6/11.1	0/0	1/11.1	1/10.0	8(9.6)
Bronchial Asthma(n/%)	2/3.7	0/0	0/0	0/0	2(2.4)
CHD(n/%)	12/22.2	0/0	0/0	1/10	13(15.7)
Hypertension(n/%)	17/31.5	5/50.0	2/22.2	2/20.0	26(31.3)
Diabetes(n/%)	9/16.7	0/0	0/0	2/20.0	11(13.3)
Cancer(n/%)	3/5.6	0/0	2/22.2	0/0	5(6.0)
Autoimmune Disease(n/%)	6/11.1	1/10.0	1/11.1	0/0	8(9.6)
NEUT%(Median[IQR)	79.7[19.6]	75.8[28.1]	74.6[25.5]	73.2[14.6]	77.9[19.5]
CRP (Median[IQR)	30.5(130.3)	13.9(48.7)	19.2(72.6)	1.5(5.2)	18.7(104.8)
Procalcitonin (Median[IQR)	0.3(5.2)	0.11(2.9)	0.06(0.1)	0.14(0.3)	0.23(1.2)
Interleukin-6 (Median[IQR)	19.4(62.4)	50.5(74.0)	85.2(46.0)	2.2(12.2)	34.4(79.3)
Application of Voriconazole(n/%)	19/35.2	0/0	0/0	1/50	20(24.1)
COPD, Chronic Obstructive Pulmonary Disease; CHD, coronary artery heart disease; NEUT, neutrophil percentage; CRP, C-reactive protein; A., Aspergillus

3.4. Antifungal Susceptibility Testing of Clinical Aspergillus Isolates

Except for Aspergillus udagawae, which had a higher minimum inhibitory concentration (MIC) (2 mg/L) for posaconazole, the MICs of the other three azole drugs were relatively low (0.06–1 mg/L) for all Aspergillus species. The three echinocandins showed high antifungal activity against Aspergillus species, with an MEC range of 0.008–0.03 mg/L. Amphotericin B had a MIC of 1.5–4 mg/L against Aspergillus species (Table S4).

Approximately 29.6% (16/54) of Aspergillus fumigatus strains exhibited non-wild-type susceptibility to amphotericin B, whereas 11.1% (1/9) of Aspergillus tubingensis strains exhibited non-wild-type susceptibility to voriconazole (Table 3).

Table 3

Determining the antifungal susceptibility of *Aspergillus* to amphotericin B, triazoles, and echinocandins based on epidemiological cutoff values.
Species (n)	Antifungal agents
	AMB		Triazoles						Echinocandins
			IZ		PZ		VOR		CAS
	WT	NWT	WT	NWT	WT	NWT	WT	NWT	WT	NWT
A. fumigatus (54)	38	16*	54	0	ND	ND	54	0	54	0
A. niger (10)	10	0	10	0	10	0	10	0	10	0
A. tubingensis (9)	9	0	9	0	9	0	8	1*	9	0
A. usamil (1)	1	0	1	0	1	0	1	0	1	0
A. flavus (10)^#	10	0	10	0	10	0	10	0	10	0
A. tamarii (2)	2	0	2	0	2	0	2	0	2	0
ND, No epidemiological cutoff values data, unable to determine;
AMB, amphotericin B; AND, anidulafungin; CAS, caspofungin; ITR, itraconazole; MF, micafungin; PZ, posaconazole; VOR, voriconazole; FZ, Fluconazole.
*The only “non wild-type” strain is highlighted in bold

3.5. Correlation between CYP51A and CYP51B Mutations in Aspergillus tubingensis and the MIC of Azole Drugs

One strain of Aspergillus tabinensis displayed a non-wild-type response to voriconazole. The genes encoding azole targets CYP51A and CYP51B were sequenced. The results revealed that CYP51A was expressed from three allelic genes. Among the samples, 44.4% (4/9) harboured four nucleotide mutations (G6A, C174T, A789G, and T1218C). Furthermore, 11.1% (1/9) of the samples harboured 29 nucleotide mutations, one of which (1/29, T256AA766G) resulted in an amino acid substitution. Additionally, 11.1% (1/9) of the samples harboured 3 nucleotide mutations (A42C, C429T, and A789G). CYP51B comprises two allelic genes. Among the samples, 11.1% (1/9) displayed 13 nucleotide mutations (G33A, T69C, C408T, G546A, G588A, C652A, C756G, C759T, T795C, G927A, G951A, G993C, and G1005A), while 44.4% (4/9) exhibited 10 nucleotide mutations (G33A, C152T, G588A, C756G, C759T, T795C, G927A, G951A, G993C, G1005A) (Table 4, Supplementary Table 5).

Table 4

Target gene mutations of *Aspergillus tabinensis* and minimum inhibitory concentration of azole drugs
Isolates ID	Gene synonymous and missense mutations			Minimum inhibitory concentration
	CYP51A		CYP51B	PZ	VOR	IZ	FZ
	Number of SNPs	Amino acid changes	Number of SNPs
21BB035	2			0.5	8	1	> 256
22BB080	2			0.25	0.5	0.5	256
21BB018	2			0.5	2	1	256
22BB101	2		13	0.25	0.5	0.5	256
21BB004	29	T256A(A766G)		0.25	0.5	0.5	64
22BB152			10	0.25	0.5	0.5	256
21BB028			10	0.25	0.5	0.5	128
22BB130			10	0.5	1	0.5	256
21BB007	3		10	0.5	0.5	0.5	> 256
SNP, Single Nucleotide Polymorphism;PZ, posaconazole; VOR, voriconazole; FZ, Fluconazole

Invasive aspergillosis is a severe complication of chronic obstructive pulmonary disease (COPD), with an annual incidence of 1.3–3.9% worldwide [9]. Aspergillus fumigatus remains the most common species, causing invasive aspergillosis [10]. Our study revealed that Aspergillus fumigatus was the most prevalent species among clinical isolates, which is consistent with the results of previous studies conducted in China [11]. However, regional variations in species distribution and antifungal resistance patterns have been reported [12, 13]. These regional differences in Aspergillus species distribution may be attributed to variations in climate, environmental factors, and patient populations. These international comparisons highlight the importance of regional surveillance and tailored treatment strategies based on local epidemiological data.

In the present study, the isolation rate for the Aspergillus niger complex was second only to that for Aspergillus fumigatus (20% vs. 56.8%). Approximately 70% of the Aspergillus niger complex (Aspergillus niger and Aspergillus tubingensis) isolates caused pulmonary infections (68.4%, 13/19). Aspergillus niger complex was previously reported as responsible for 69.6% (48/69) of ear canal fungal infections in northern China [14]. This highlights the significance of Aspergillus niger complex as a pathogen causing pulmonary and auditory system infections. In addition, we identified certain Aspergillus species, such as Aspergillus usamil, Aspergillus tamarii, Aspergillus versicolor, Aspergillus udagawae, Aspergillus lentulus, Aspergillus sydowii, and Aspergillus quadrilineatus, which rarely cause human infections. This is particularly true for Aspergillus udagawae and Aspergillus quadrilineatus, which have only been reported in isolated cases of infection [15–19]. The present work is the first to identify Aspergillus udagawae and Aspergillus quadrilineatus as causative agents of pulmonary infections among patients in China[20]. The drug sensitivities of these two Aspergillus species were also reported.

The morphological identification of Aspergillus species is an essential step in epidemiological studies and clinical diagnosis. Our findings revealed the diverse distribution of Aspergillus species, with A. fumigatus, A. flavus, A. niger, and A. terreus being the most commonly isolated species. The identification of these species is consistent with previous epidemiological reports, highlighting their clinical significance and impact on patient outcomes. Furthermore, the observation of distinct morphological features among different species underscores the importance of accurate species-level identification for guiding appropriate antifungal therapy.

Owing to the similarity in morphology between Aspergillus subspecies, these could hardly be distinguished based on morphology. Therefore, the use of mass spectrometry-based identification enhanced the accuracy and reliability of our findings. MALDI-TOF MS can differentiate between two subspecies of Aspergillus, namely Aspergillus niger and Aspergillus tubingensis, within the Aspergillus niger complex. MALDI-TOF MS can also identify rare Aspergillus species, such as Aspergillus quadrilineatus and Aspergillus udagawae, offering a significant advantage over morphological identification. The MALDI-TOF MS Autof ms1000 system employs a proprietary database containing reference spectra of various microorganisms, including Aspergillus species. These reference spectra were obtained from well-characterised strains of known species. However, MALDI-TOF MS may sometimes yield low identification scores for Aspergillus species, which could be attributed to limitations in the instrument’s database and the quality of protein extraction from fungal strains.

The European Society of Microbiology recommends itraconazole and voriconazole as primary treatments for invasive aspergillosis while advocating for the cautious use of liposomal amphotericin B [21]. Antifungal susceptibility testing is crucial for guiding the selection of appropriate antifungal agents and predicting treatment outcomes. Notably, we observed differences in susceptibility patterns among different Aspergillus species, emphasising the need for species-specific susceptibility testing to optimise treatment strategies. Previous in vitro drug susceptibility tests on Aspergillus strains isolated from healthcare and other environmental settings revealed that 14.5% (19/131) of Aspergillus niger complex strains were resistant to azole drugs [7]. Our findings demonstrated that 29.6% (16/54) of Aspergillus fumigatus exhibited a non-wild-type response to amphotericin B, whereas 11.1% (1/9) of Aspergillus tubingensis showed a non-wild-type response to voriconazole. Other Aspergillus species demonstrated wild-type characteristics when exposed to azoles and echinocandins. The emergence of azole-resistant Aspergillus fumigatus strains represents a growing concern in several countries [22–24]. Notably, the emergence of antifungal resistance poses a significant challenge in the management of Aspergillus infections. Therefore, the continuous surveillance of antifungal resistance is crucial to guide appropriate treatment strategies and prevent the spread of resistant strains.

Azole resistance in Aspergillus species is primarily attributed to alterations in the drug targets, particularly CYP51A and CYP51B. The CYP51 demethylase is the most widely distributed member of the cytochrome P450 family, which plays a crucial role in sterol biosynthesis and serves as a target of antifungal drugs. The CYP51 gene family consists of CYP51, CYP51A, CYP51B, and CYP51C [25]. In this study, we identified the presence of CYP51A and CYP51B genes in Aspergillus tubingensis. However, the missense mutation in CYP51A did not result in a higher MIC for azole drugs. In contrast, no missense mutations were detected in the CYP51A and CYP51B genes of strains exhibiting a non-wild-type response to voriconazole. Variations in CYP51 expression are associated with azole resistance in Aspergillus fumigatus [26]. In contrast, the T256A^A766G amino acid substitution appeared to have decreased the MIC of fluconazole against Aspergillus tubingensis strains by a factor of 2–4 when compared to other strains (Table 4). It may be a silent mutation unrelated to drug resistance.

While our study provides valuable insights into the epidemiological characteristics of invasive Aspergillus isolates, it has several limitations. The retrospective nature of the study limited the availability of detailed clinical data, which could have provided additional context for interpreting the observed epidemiological trends. Moreover, the study was conducted in a specific geographic region, and the generalisability of the findings to other settings may be limited by regional variations in Aspergillus species distribution and antifungal resistance patterns.

In conclusion, our comprehensive analysis of invasive Aspergillus isolates provides insights into the morphological diversity, drug susceptibility patterns, and mutations in the target genes of azole drugs. These findings contribute to a deeper understanding of the epidemiology of invasive aspergillosis and hold implications for guiding clinical management and future research. Continued surveillance and research endeavours are essential to address the evolving challenges posed by invasive Aspergillus infections and optimise patient outcomes.

Chronic obstructive pulmonary disease (COPD)

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)

Minimum inhibitory concentration (MIC)

Ethics approval and consent to participate

The research study has been reviewed and approved by the Ethics Committee of Hebei North University Affiliated First Hospital (Ethical approval number: K2019147). Informed consent forms have been signed by all patients or their relatives. The study follows the principles outlined in the Helsinki Declaration, ensuring the protection of patient privacy and other related matters. Informed consent was obtained from all subjects involved in the study.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work received support from the Key Research and Development Program of Zhangjiakou City (2121098D), Natural Science Foundation of Hebei Province (C2022405023), the Research Fund Project of the Health Commission of Hebei Province (20210702), and the 2024 Government Funded Clinical Medicine Excellent Talent Training Project (ZF2024224).

Author contributions

W.Z. and H.Z. handled the methodology. W.Z. managed the software tasks. X.X. conducted formal analysis and investigation. W.Z. provided resources. H.Z. curated the data. M.H. drafted the original manuscript. J.R. contributed to manuscript writing and editing. X.W. oversaw project administration. Z.Z. secured funding. All authors have read and approved the final version of the manuscript.

Acknowledgements

We would like to extend our heartfelt appreciation to all healthcare professionals and patients who contributed to this project. Their dedicated efforts laid the groundwork for the data collection in this study. We would like to thank Editage (www.editage.cn) for English language editing.

Cadena J, Thompson GR 3rd, Patterson TF. Aspergillosis: Epidemiology, Diagnosis, and Treatment. Infect Dis Clin North Am. 2021;35(2):415–34.
Franconi I, Rizzato C, Ghelardi E, Lupetti A. Hospital distribution, seasonality, time trends and antifungal susceptibility profiles of all Aspergillus species isolated from clinical samples from 2015 to 2022 in a tertiary care hospital. BMC Microbiol. 2024;24(1):111.
Mahmoud DE, Herivaux A, Morio F, Briard B, Vigneau C, Desoubeaux G, Bouchara JP, Gangneux JP, Nevez G, Gal SL et al. The epidemiology of fungal infections in transplant patients. Biomed J 2024:100719.
Pandey M, Xess I, Sachdev J, Sharad N, Gupta S, Singh G, Yadav RK, Rana B, Raj S, Ahmad MN, et al. Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections. J Infect. 2024;88(5):106147.
van Grootveld R, van Paassen J, Claas ECJ, Heerdink L, Kuijper EJ, de Boer MGJ, van der Beek MT. Prospective and systematic screening for invasive aspergillosis in the ICU during the COVID-19 pandemic, a proof of principle for future pandemics. Med Mycol 2024.
Peng D, Li A, Kong M, Mao C, Sun Y, Shen M. Pathogenic Aspergillus Strains Identification and Antifungal Susceptibility Analysis of 452 Cases with Otomycosis in Jingzhou, China. Mycopathologia. 2024;189(2):30.
Kofoed VC, Campion C, Rasmussen PU, Møller SA, Eskildsen M, Nielsen JL, Madsen AM. Exposure to resistant fungi across working environments and time. Sci Total Environ. 2024;923:171189.
CLSI. Epidemiological CutoffValues for Antifungal Susceptibility Testing. CLSI supplement M59 2018, 2nd ed.
Hammond EE, McDonald CS, Vestbo J, Denning DW. The global impact of Aspergillus infection on COPD. BMC Pulm Med. 2020;20(1):241.
Toyotome T. Resistance in the Environmental Pathogenic Fungus Aspergillus fumigatus. Med Mycol J. 2019;60(3):61–3.
Chen H, Zhang X, Zhu L, An N, Jiang Q, Yang Y, Ma D, Yang L, Zhu R. Clinical and immunological characteristics of Aspergillus fumigatus-sensitized asthma and allergic bronchopulmonary aspergillosis. Front Immunol. 2022;13:939127.
Deng S, Zhang L, Ji Y, Verweij PE, Tsui KM, Hagen F, Houbraken J, Meis JF, Abliz P, Wang X, et al. Triazole phenotypes and genotypic characterization of clinical Aspergillus fumigatus isolates in China. Emerg Microbes Infect. 2017;6(12):e109.
Song G, Zhang M, Liu W, Liang G. Epidemiology of Onychomycosis in Chinese Mainland: A 30-year Retrospective Study. Mycopathologia. 2022;187(4):323–31.
Jing R, Yang WH, Xiao M, Li Y, Zou GL, Wang CY, Li XW, Xu YC, Hsueh PR. Species identification and antifungal susceptibility testing of Aspergillus strains isolated from patients with otomycosis in northern China. J Microbiol Immunol Infect. 2022;55(2):282–90.
Gyotoku H, Izumikawa K, Ikeda H, Takazono T, Morinaga Y, Nakamura S, Imamura Y, Nishino T, Miyazaki T, Kakeya H, et al. A case of bronchial aspergillosis caused by Aspergillus udagawae and its mycological features. Med Mycol. 2012;50(6):631–6.
Kano R, Shibahashi A, Fujino Y, Sakai H, Mori T, Tsujimoto H, Yanai T, Hasegawa A. Two cases of feline orbital aspergillosis due to Aspergillus udagawae and A. viridinutans. J Vet Med Sci. 2013;75(1):7–10.
Kuzume A, Yuda J, Abe M, Yamaguchi T, Hisano M, Yamauchi N, Nakamura H, Nagata A, Song-Gi C, Kaku E, et al. [Disseminated aspergillosis due to Aspergillus udagawae during immunosuppressive treatment for myelodysplastic syndrome]. Rinsho Ketsueki. 2021;62(1):51–4.
Sabo MC, Blain M, McCulloch D, Glasgow HL, Sengupta DJ, Le T, Cookson BT, Pottinger PS, Liles WC, Graham SM. Back Pain in a 23-Year-Old Male With X-Linked Chronic Granulomatous Disease. Open Forum Infect Dis. 2019;6(11):ofz449.
Seki A, Yoshida A, Matsuda Y, Kawata M, Nishimura T, Tanaka J, Misawa Y, Nakano Y, Asami R, Chida K, et al. Fatal fungal endocarditis by Aspergillus udagawae: an emerging cause of invasive aspergillosis. Cardiovasc Pathol. 2017;28:14–7.
Zhang H, Zhang W, Li M, Wang B, Zhang Z. A case of Aspergillus quadrilineatus pulmonary infection in China. Heliyon. 2024;10(12):e33000.
Ullmann AJ, Aguado JM, Arikan-Akdagli S, Denning DW, Groll AH, Lagrou K, Lass-Flörl C, Lewis RE, Munoz P, Verweij PE, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect. 2018;24(Suppl 1):e1–38.
Rivelli Zea SM, Toyotome T. Azole-resistant Aspergillus fumigatus as an emerging worldwide pathogen. Microbiol Immunol. 2022;66(3):135–44.
Jeanvoine A, Rocchi S, Bellanger AP, Reboux G, Millon L. Azole-resistant Aspergillus fumigatus: A global phenomenon originating in the environment? Med Mal Infect. 2020;50(5):389–95.
Burks C, Darby A, Gómez Londoño L, Momany M, Brewer MT. Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance. PLoS Pathog. 2021;17(7):e1009711.
Celia-Sanchez BN, Mangum B, Brewer M, Momany M. Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi. G3 (Bethesda) 2022, 12(11).
Pfaller MA, Carvalhaes CG, Rhomberg PR, Desphande LM, Castanheira M. Trends in the activity of mold-active azole agents against Aspergillus fumigatus clinical isolates with and without cyp51 alterations from Europe and North America (2017–2021). J Clin Microbiol. 2024;62(2):e0114123.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Epidemiological characteristics of invasive Aspergillus isolates: morphology, drug susceptibility, and mutations in azole drug targets

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

1. Background

2. Methods

2.1. Sample Collection

2.2. Morphological Identification

2.3. MALDI-TOF MS Identification and Sequencing of Housekeeping Genes

2.4. Antifungal Susceptibility Testing

2.5. Sequencing of Azole Resistance Genes

2.6. Data Analysis

3. Results

3.1. Identification of Aspergillus Species

3.2. Morphological Characteristics of Clinical Aspergillus Isolates

3.3. Clinical Characteristics of Patients with Aspergillus Infection

3.4. Antifungal Susceptibility Testing of Clinical Aspergillus Isolates

3.5. Correlation between CYP51A and CYP51B Mutations in Aspergillus tubingensis and the MIC of Azole Drugs

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1