Introduction

Aspergillus species are commonly found in soil, decaying biological materials, and restrained places, which cause a serious fungal infection known as aspergillosis.1 Infections caused by these ubiquitous species often challenging to diagnose, and the mortality rate can be as high as 30%–95%.2 In recent years, the incidence of invasive aspergillosis has increased significantly due to the use of various immunosuppressive agents, the development of organ transplantation, and the increase of AIDS. Among these, Aspergillus fumigatus is the most common species responsible for about 70–80% of human aspergillosis cases. Infections from other species such as A. niger, A. flavus, and A. terreus are also increasing, particularly in individuals with weakened immune systems.3

The problem of aspergillosis is increasing, provoking a significant risk to global healthcare system.4 However, the incidence of aspergillosis in China is not well documented, with only a few studies conducted in specific regions, such as the southeast.5 A 2017 study estimated that there are 3 million global cases of chronic pulmonary aspergillosis and 2.5 million of invasive aspergillosis.6 Aspergillosis is more likely to affect people with weakened immune systems, such as those with blood cancers, organ transplants, and chronic lung conditions, as well as those who come into contact with environmental factors like dust and mold.7 One study showed that mortality increased by 60% in COVID-19-infected patients and by 20% in patients with solid organ transplants.8,9

The common treatment approach for aspergillosis involves the use of triazole antifungal drugs, such as isavuconazole (ICA), itraconazole (ITR), posaconazole (POS), and voriconazole (VRC).10 In cases where isolates are resistant to azoles, significant antifungal agents like amphotericin B (AMB) and echinocandins come into play.11 Echinocandins are typically administered as part of a multi-drug antifungal treatment in conjunction with azoles, rather than as a standalone treatment therapy.12

Many factors, such as the type, severity, and susceptibility profile of the infecting Aspergillus isolate, influence the selection of a specific antifungal agent.13 Anti-fungal susceptibility testing is crucial for selecting suitable antifungal treatment, as it helps to identify wild-type and non-wild-type Aspergillus isolates with respect to specific antifungal agents.14

The susceptibility of Aspergillus isolates to antifungal drugs shows variation depending on the species, geographical location, and local patterns of antifungal agent consumption.10 There has been a recent rise in non-wild-type Aspergillus isolates, particularly in relation to azole drugs, which are the mainstay of therapy for aspergillosis.14 Recent studies have identified several mutations in the cyp51A gene that are strongly associated with azole resistance in A. fumigatus. These mutations are reported to be linked to overexpression of the mdr gene, which may contribute to drug resistance in A. fumigatus.15,16 Another study reported that genes responsible for encoding proteins involved in cell wall remodeling, oxidative stress response, and energy metabolism are key factors contributing to antifungal drug resistance in Aspergillus species.17 Surveillance studies that examine antifungal susceptibility patterns in Aspergillus species play a crucial role in detecting these emergent non-wild-type pathogens.18 Such data is essential for directing the appropriate selection of antifungal agents and has a significant impact on the management and outcomes of aspergillosis.15

This study, which is being conducted as a retrospective analysis, aims to look into the epidemiology, risk factors, and antifungal susceptibilities of Aspergillus species within Huashan hospital in Shanghai, China. The results of this study are expected to provide valuable insights for healthcare officials, aiding in the effective management of aspergillosis caused by Aspergillus species. Furthermore, the findings may have suggestions for deciding suitable antifungal drugs and developing therapeutic plans specific to the region.

Materials and Methods Patient Data Collection

The current retrospective study from the five years (2018–2022) was conducted at Huashan Hospital, which is one of the top tertiary hospitals in China located in Southeast region. Demographic and clinical information of the patient host to clinical isolates of Aspergillus species was gathered from the hospital’s digital record. The collected data encompassed various patient/host characteristics, including gender, age, reported department and sample source according to recent similar study.5Aspergillus species isolates from hospital record were subjected to analysis using appropriate statistical tests.

Isolation and Identification of Aspergillus Species

Standard clinical mycological measures were followed for the processing of the samples collected to isolate, purify and identify Aspergillus species.19 Briefly, the samples were processed for culturing through inoculating onto Sabouraud Dextrose Agar (Crmicrobio, China). The plates were then incubated at 35°C for at least 7 days. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF-MS) developed by Zybio in China was utilized to identify the Aspergillus species.20 Resistant isolates were further confirmed through molecular identification.21

In vitro Antifungal Susceptibility Testing

The susceptibility testing for antifungal drugs (AMB; Amphotericin B, CAS; Caspofungin, ITR; Itraconazole, POS; Posaconazole, VRC; Voriconazole) was conducted using the Sensitizer YeastOneTM system (Thermo Fisher Scientific, USA). In brief, purified colonies of the Aspergillus species were added to a sterilized saline solution. The conidial suspension was adjusted to 0.5 McFarland (0.6~5×106 CFU/mL), and 100 µL of this was added to 11 mL of YeastOneTM inoculum broth and mixed well. Afterwards, 100 µL of the mixture was introduced into each well of the YeastOneTM micro-dilutions 96-well plate (Thermo Fisher Scientific, USA). A. fumigatus ATCC 204305 and Candida parapsilosis ATCC 22019 strains were used as the control reference. The broth microdilution method, following the standards set by the Clinical and Laboratory Standards Institute (CLSI). The susceptibility test findings were compared to the MIC breakpoints and epidemiological cut-off values (ECVs) established by the CLSI.

CLSI breakpoints were not available for the mentioned drugs. However, CLSI (M57S-4th ed.) provides ECVs for these drugs, which were used to classify the strains as wild-type or non-wild-type. Briefly, A. fumigatus strains having MIC values greater than 1 µg/mL were classified as non-wild type for ITR. In contrast, A. fumigatus isolates were categorized as resistant, intermediate, and susceptible when the MIC for VRC was ≥2 ug/mL, 1 ug/mL, and ≤0.5 ug/mL, respectively. For A. niger, strains were categorized as non-wild type if their ITR, POS, and VRC MIC values exceeded 4 µg/mL, 2 µg/mL, and 2 µg/mL, respectively. A. flavus strains were classified as non-wild type if their ITR, POS, and VRC MIC values exceeded 1 µg/mL, 0.5 µg/mL, and 2 µg/mL, respectively. Regarding A. terreus, strains were classified as non-wild type if their MIC values for ITR, POS, and VRC exceeded 2 µg/mL, 1 µg/mL, and 2 µg/mL, respectively.22,23

Identification of cyp51A Anomaly

The cyp51A gene is well-documented as a key target in antifungal resistance, particularly against azole drugs. Mutations in this gene are directly linked to resistance mechanisms in A. fumigatus and other species.16 To uncover possible genomic mechanisms associated with antifungal resistance in the Aspergillus species that were non-wild type and non-susceptible, we sequenced both the promoter region and the coding region of the cyp51A gene. Briefly, the selected isolates were inoculated into 15 mL of GYEP medium (containing 2% glucose, 0.3% yeast extract, 1% peptone) and cultured for 48 hours at 37°C.24.

DNA extraction was carried out using a commercial kit (Tiangen, China) following the provided instructions. Three sets of primers were designed in total: one primer for sequencing the promoter region, and two primers for sequencing the cyp51A gene, targeting amplicons of 1050 bp and 500 bp, respectively (Table 1). Primers for polymerase chain reaction (PCR) and sequencing were designed based on the promoter and coding region of cyp51A (Table 1). The cycling conditions included an initial step at 95°C for 5 minutes, followed by 40 cycles of 94°C for 30 seconds, 50–57°C (as per Table 1) for 45 seconds, and 72°C for 2 minutes. The process concluded with a final extension at 72°C for 7 minutes. Purification of the PCR products was performed using the Omega E.Z.N.A.® Cycle Pure (CP) Kit (Omega Bio-Tek, Norcross GA/USA) according to the manufacturer’s instructions. Subsequently, DNA sequences were determined using a Dye Terminator Cycle Sequencing [DTCS] Quick Start Kit (Beckman-Coulter Inc, Brea CA/USA) and CEQTM 8000 capillary electrophoresis DNA sequencer (Beckman Coulter). The obtained sequences were aligned with the sequence from an azole-susceptible strain (GenBank accession no. AF338659), and mismatches were identified through ClustalW analysis.25 Along with sequencing of the cyp51A gene, identification of the Aspergillus species was reconfirmed through sequencing of the β-tubulin gene.

Table 1 Primers, Annealing Temperatures, Target Sequence and Annealing Positions

Statistical Analysis

The data is presented in Mean ± Standard Deviation where the prevalence of Aspergillus species between different hospital wards and departments was compared using the Fisher’s exact test, chi-square tests, and ANOVA. p-values of <0.05 were considered statistically significant.

Results Aspergillus Species Distribution

In the current study a total number of 253 Aspergillus species were retrospectively collected from 2018 to 2022. It was observed that six species of the Aspergillus are involved in the cause of aspergillosis. The highest number of isolates was A. fumigatus (146, 57.71%), followed by A. niger (68, 26.88%), A. flavus (27, 10.67%) and A. terreus (10, 3.95%). Only one isolate of A. clavatus and A. nidulans was reported respectively. A detailed depiction of the reported species is given in (Figure 1A). In 2018 and 2019, there were 82 recorded Aspergillus species. The number of reported cases decreased to 30 in the year 2020, 31 in the year 2021, and 28 in the year 2022. In comparison to the total number of cases recorded in 2018 and 2019, the number of reported cases in the subsequent years decreased by approximately 0.36-fold which could be attributed to the rise in cases of severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) and challenges with sample collection. A. fumigatus was responsible for the highest portion in each recorded year, followed by A. niger. Detailed results are presented in (Figure 1B).

Figure 1 (A) Aspergillus species distribution from 2018–2022. (B) Aspergillus species distribution each year.

Among the various hospital departments, the department of infectious diseases reported the highest number of cases (71, 28.06%), followed by the Respiratory and Critical Care Medicine Department with (n = 60, 23.72%) cases. The department of Dermatology reported the third-highest number of cases (16, 8.70%), while the departments of Cardiology and Endocrinology each reported just one case. Additionally, A. fumigatus was the predominant cause in the highest number of cases in each department of the hospital. Detailed results of Aspergillus species with number and percentage of cases are given in (Table 2).

Table 2 Aspergillus Species Isolates Were Reported from Different Departments of the Hospital Presenting the Occurrence of Aspergillus Species from Each Sample

The majority of isolates (204, 80.63%) among all sample types were isolated from Respiratory samples. The second highest number of isolates was recovered from Body discharges (13, 5.13%), followed by CSF (12, 4.74%) isolates. From the bodily fluids, the minimum number of isolates obtained was (5, 1.97%). A. fumigatus accounted for the greatest number of isolates in each instance. Detailed results are presented in (Figure 2).

Figure 2 Aspergillus species isolated from different biological samples. Each pie chart next to the donut chart.

Characteristics of Aspergillus Species Hosts

Table 3 shows the demographic and clinical characteristics of the Aspergillus species isolated from the hosts. A significant number of cases (73.12%) were reported in the male population (185 isolates), while the female population had only 68 isolates (26.88%). This pattern was consistent across all the species. Majority of cases occurred in senior adults, with a median age of 64 years and an interquartile range of 51~74 years. Statistically significant differences (p < 0.05) were observed in the distribution of Aspergillus species among various age groups. A. niger showed higher prevalence in the younger age group (under 18 years), while A. fumigatus was more commonly found in adults and older age groups (over 18 years). Since our hospital predominantly treats adult patients, only five cases were reported in the 2–17 age group, consisting of three cases of A. niger, one of A. fumigatus, and one of A. flavus. A. terreus consistently ranked as the least prevalent across all age groups, followed by A. flavus.

Table 3 Baseline Characteristics of Aspergillus Species

It was observed that 60.46% of patients had underlying pulmonary issues of various types, with chronic obstructive pulmonary disease (COPD) being the most prevalent (20.15%), followed by asthma (14.62%). Additionally, 60.08% of patients had other underlying conditions, among which gastrointestinal disorders were the most common (23.90%), followed by hepatic disorders (9.09%) and diabetes (8.30%). In all cases, A. fumigatus was the dominant infecting species. However, the hospital’s electronic records did not clarify whether patients with underlying pulmonary conditions also had other systemic disorders Statistically significant difference (p<0.05) was also observed among the species based on, underlying pulmonary status and underlying systemic disorder detailed results are provided in Table 3.

Antifungal Susceptibility Profiles

Antifungal susceptibility assays were conducted on Aspergillus isolates, with detailed MIC/MEC values, range, and geometric mean (GM) provided in Table 4. The profile of Wild type (WT) species is visually presented in Figure 3. Aspergillus spp. exhibited remarkably low MECs for CAS (≤0.008 to 0.50 µg/mL). Regarding azoles, the majority of isolates demonstrated MICs of ≤0.5μg/mL for POS, ITR, and VRC. All Aspergillus species (100%) were susceptible to POS. In the case of A. fumigatus, ITR emerged as the most effective antifungal drug following POS, with 139 (95%) isolates showing susceptibility to ITR. A minimal number of A. fumigatus isolates (34, 23.28%) were susceptible to AMB. For A. niger, AMB was the second most effective drug, with 52 isolates (76.47%) displaying susceptibility, while VRC was the least effective, with only 12 (17.64%) isolates showing susceptibility. In the case of A. flavus, ITR proved as effective as POS, with all isolates being susceptible to ITR, while AMB was the least effective antifungal drug, with 23 (85.11%) isolates being susceptible. As in A. terreus, all tested drugs exhibited equal effectiveness except for ITR.

Table 4 Antifungal Susceptibility Patterns and Characteristics in the Five Common Aspergillus Species

Figure 3 The percentage of susceptible and Wild-type Aspergillus species isolated in the current study. AMB; Amphotericin B, CAS; Caspofungin, ITR; Itraconazole, POS; Posaconazole, VRC; Voriconazole. (A) A. terreus (B) A. fumigatus (C) A. Niger (D) A. flavus (E) A. calactus (F) A. nidulans..

Gene Mutations of cyp51A Linked with Azole Resistance

In order to delve deeper into the association between azole resistance in Aspergillus species and mutations in the cyp51A gene, the gene was subjected to sequencing and subsequently compared with the wild-type gene. It was observed that mutation is present in the promoter region of isolate S18, while it is absent in isolates S19 and S20. Point mutations were observed at M172V and E427K in insolate S18. No mutations were observed in S19 while in S20 mutations were observed at Y121F and E427Y (Table 5).

Table 5 Detail of the cyp51A Gene Mutation Along with Minimum Inhibitory Concentration (MIC) of Aspergillus Isolates Studied in the Current Study

Discussion

The present retrospective work examines the occurrence of Aspergillus species, along with the demographic and clinical characteristics linked to these species, within a tertiary care hospital located in Southeast China. Our analysis reveals a notable surge in Aspergillus cases during 2018 and 2019, yet a subsequent decline in the following three years. This is in contrast to earlier research that showed an increase in the incidence of aspergillosis.26 The decrease observed in our investigation raises questions about potential shifts in epidemiological factors influencing the prevalence of Aspergillus species infections.27 Furthermore, these findings underscore the importance of continuous surveillance studies to monitor and understand the evolving dynamics of aspergillosis, providing valuable insights for public health interventions and medical management. Our study indicates that A. fumigatus is the predominant species identified, followed by A. niger and A. flavus. These results align with previous studies that reported similar patterns, confirming consistency in the prevalence of these Aspergillus species across different studies.28 The occurrence of A. fumigatus is attributed to its ubiquity, rapid growth, and opportunistic nature as a pathogen.29 Other studies have also documented A. flavus and A. terreus as commonly occurring species. The diverse prevalence of dominant Aspergillus species may be influenced by geographical and environmental factors.30 The prevalence of A. fumigatus in our study (57.71%) aligns with reports from Europe and North America, where it is the dominant species in Aspergillus infections.31 Similarly, resistance patterns, such as reduced susceptibility to amphotericin B, are consistent with global trends.32 However, our study also highlights unique regional aspects, such as higher susceptibility to posaconazole compared to studies from South Asia, which report emerging resistance.32 These differences may be attributed to variations in antifungal usage or environmental factors.33

Upon analyzing the incidence of Aspergillus species based on hospital departments, it was noted that over one-third of the total cases (28.03%) originated from the Department of Infectious Disease. The justification for increased reporting from this department lies in its dedicated role in monitoring and managing of infectious diseases.34 The department with the second-highest incidence of reported aspergillosis isolates, particularly A. fumigatus, in the current study is Respiratory and Critical Care Medicine, contributing to nearly one-third (23.4%) of the total cases. This may be attributed to the pulmonary infective nature of Aspergillus species, coupled with the weakened immune systems of patients in critical care unit, prolonged usage of corticosteroids, and immune-suppressive drugs.10 To address this issue, it is crucial to implement timely detection, quick anti-fungal therapy, rigorous infection surveillance measures, regulation of immunity, and continuous education and surveillance.35 According to a recent study, prophylaxis is needed in patients who are at risk of invasive aspergillosis due to changing medical conditions.23 In our study, we do not have specific information on these groups, but patients in the departments of ICU, respiratory and critical care medicine, and nephrology are likely to be immunocompromised and may need antifungal prophylaxis.

In the analysis of Aspergillus species incidence based on samples, it was observed that most of the isolates (80.63%) come from Respiratory samples sputum, followed by Body discharge fluids and CSF with 5.13% and 4.74% of total cases, respectively, with A. fumigatus being the most prominent species. These findings align with a prior study covering a 20-year period, which reported the incidence of Aspergillus in China. The study noted that A. fumigatus is an endemic respiratory pathogen frequently isolated from sputum and associated fluids.36 The isolation of Aspergillus species from Biliary secretion through T-Tube, Fibroscope, and Catheter indicates contamination of these equipments and suggests a systemic nature of the infection.37

Upon examining the incidence of Aspergillus species based on gender, it was discovered that the majority of cases (73.12%) are documented in the male population. This pattern persists across each reported species. Our findings align with a recent study that also observed a higher prevalence of Aspergillus species in the male population. The study additionally proposed that smoking and occupational exposure could be contributing factors to the elevated incidence among males.5 However, further investigation is required to pinpoint the underlying causes. In terms of age, the majority of infections were documented in the senior adult age group, with a median age of 64 years (IQR; 51–74). This study reveals that each documented species shows a heightened prevalence among individuals aged above 50 years. Furthermore, these species are mostly associated with cases of pulmonary aspergillosis, mainly in individuals within the respiratory and critical care unit who have substantial pulmonary and systematic disorders. The increased susceptibility of older individuals to aspergillosis could be linked to their compromised immune systems and the presence of substantial associated complications.38 Amongst children (age group 2–17 years), only five cases were reported where three cases were of A. niger, one was of A. flavus and one was of A. fumigatus. The high prevalence of A. niger is similar to a recent published study, where A. niger was highly prevalent in the children.5 However, it is yet to be explored what causes the incidence of Aspergillus in children and the prevalence of any one species in particular.

When the cases were analyzed on the basis on underlying pulmonary status and underlying systemic disorders, it was observed that 51 (20.15%) patients are suffering from chronic obstructive pulmonary disease, 37 (14.62%) are suffering from Asthma and 34 (13.44%) are suffering from Bronchiectasis. A recent investigation has revealed that obstructive pulmonary aspergillosis occurs as a result of a relapse of acute respiratory aspergillosis, with asthma and bronchiectasis identified as associated factors.39 This underscores the critical significance of early detection and appropriate treatment of aspergillosis. It was noted that most patients did not exhibit underlying disorders. However, among those with disorders, gastrointestinal issues were the most prevalent, observed in 23.90% of the cases. Research has indicated that the use of antibiotics and medications may contribute to the development of gastric problems.40

We analyzed the isolates for susceptibility to amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole. The observed 100% susceptibility of Aspergillus species is consistent with earlier research findings that have documented the effectiveness of POS against aspergillosis.41,42 POS is recognized as a broad-spectrum antifungal drug and is frequently recommended as the initial choice for fungal treatment.43 Our research revealed that ITR ranks as the second most effective drug against the identified fungal pathogens, particularly A. fumigatus, with 95% of the isolates demonstrating susceptibility to ITR. These results align with recent publications that identified ITR as highly effective against A. fumigatus isolates.14 Nevertheless, the limited efficacy of AMB underscores the necessity for alternative antifungal agents when dealing with pathogens resistant to AMB.44 Regarding A. niger, our findings suggest that AMB emerges as the most effective drug, with 74.47% of A. niger isolates showing susceptibility to AMB. These results align with the well-established understanding that AMB is the most efficacious antifungal drug against A. niger.45 Additionally, our study reveals that A. niger exhibits the least susceptibility to VRC, a trend consistent with recent reports indicating resistance of A. niger to VAR.46 For A. flavus, our investigation indicates that 85% of the isolates exhibited susceptibility to AMB, while for other drugs, this percentage ranged from 96% to 100%. These findings align with recently published data, which also reported the susceptibility of A. flavus to several tested antifungal drugs.36 In our investigation of A. terreus, it was observed that ITR displayed the lowest effectiveness among the tested drugs. Given A. terreus’s inherent resistance to amphotericin B, these results underscore the significance of exploring alternative antifungal agents in the management of A. terreus infections.43

We also sequenced cyp51A gene and its promoter, for the azole resistant A. fumigatus strains. Mutations were observed in the gene as well as in the promoter region. Recent studies have also reported mutation in the promotor region of the cyp51A gene.47 Our finding of mutations at Y121F and E247K aligns with a recently published study that identified similar point mutations in azole-resistant Aspergillus isolates at these specific positions.10 Studies have shown that these mutations may alter the binding site of the CYP51 enzyme, impairing the efficacy of azoles. These observations underscore the need for continuous surveillance of such mutations to guide antifungal therapy effectively.48,49

The retrospective nature of this study introduces certain limitations, as some data may be unavailable for analysis. The present study is confined to a single center, with a comparatively low sample size, thereby restricting the generalizability of the results to other regions in China. Additionally, information on antifungal treatment earlier the isolation of Aspergillus species for the included patients was not accessible, impeding an investigation into the impact of early antifungal therapy on resistance occurrence. Despite these limitations, the findings of this study may provide valuable insights for health officials managing aspergillosis in Southeast China. To address these limitations and enhance our understanding, future studies should focus on epidemiology and antifungal resistance approaches across multiple centers at the molecular level. Furthermore, it is essential to establish the association between previous antifungal treatment and the progression of resistance in fungal isolates.

Conclusion

The current investigation documented 253 isolates of Aspergillus species, with a significant majority attributed to A. fumigatus. Among non-fumigatus cases, A. niger featured prominently, followed by A. flavus. The Department of Infectious Disease reported a substantial number of isolates, underscoring its pivotal role in infection management. POS emerged as the most potent antifungal agent against all Aspergillus species. The prevalence of A. fumigatus in our study consistently aligns with reports from Europe and North America. Similarly, resistance patterns, such as reduced susceptibility to AMB, are consistent with global trends. However, our study shows higher susceptibility to POS compared to studies from South Asia, which report emerging resistance. It is essential to conduct extensive molecular-based surveillance studies with large and diverse populations, within the framework of One Health approaches, to continuously monitor Aspergillus species and facilitate effective management of aspergillosis.

Data Sharing Statement

Data used in this paper is included in the paper.

Ethics Approval and Informed Consent

The study was authorized by the Ethics Committee of Huashan Hospital, Fudan University (permission number: KY2014-219 R) and carried out in accordance with the Declaration of Helsinki’s principles. Every participant in the research gave their informed permission.

Acknowledgment

This paper/The abstract of this paper was presented at the 8th Congress of Asia-Pacific Society for Medical Mycology (APSMM 2024) Osaka Japan as conference talk with interim findings on November 6, 2024.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This research work was supported by the National Key Research and Development Program of China (2021YFC2300400), the Science and Technology Commission of Shanghai Municipality (22Y11905600) and National Natural Science Foundation of China (82273544).

Disclosure

The authors declare no conflicts of interest.

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