Novel Diagnostic Tools

#### **Chapter 5**

## Nanomaterials-Based Biosensors against *Aspergillus* and Aspergillosis: Control and Diagnostic Perspectives

*Xiaodong Guo, Mengke Zhang, Mengzhi Wang, Jiaqi Wang and Marie-Laure Fauconnier*

#### **Abstract**

Aspergillosis is the name given to the spectrum of diseases caused by the genus *Aspergillus*. Research on aspergillosis has shown a progressive expansion over the past decades, largely due to the rise in the number of immunocompromised individuals who are at risk for the infection. Nanotechnology provides innovative tools in the medicine, diagnosis, and treatment. The unique properties of nanomaterials like small size in the nanoscale have attracted researchers to explore their potential, especially in medical diagnostics. Aptamers, considered as chemical antibody, are short, singlestranded oligonucleotide molecules with high affinity and specificity to interact with target molecules even superior to antibody. Accordingly, development of nanomaterials-based biosensors technology such as immunosensors and aptasensors against *Aspergillus* and Aspergillosis is of great significance and urgency. In this book chapter, we comprehensively introduce and analyze the recent progress of nanomaterials-based biosensors against *Aspergillus* and Aspergillosis. In addition, we reveal the challenges and provide our opinion in future opportunities for such sensing platform development. Ultimately, conclusion and future prospects are highlighted and summarized.

**Keywords:** nanomaterials, biosensor, *Aspergillus*, Aspergillosis, biomarker

#### **1. Introduction**

Biosensors are integrated analytical devices that are capable of transferring the binding events between bioreceptor and target into detectable optical and electric signals. Therefore, bioreceptors, regarded as the recognition elements, play a crucial role for constructing advanced biosensors. Conventional bioreceptors like antibodies have been extensively prepared and developed for immunosensors' establishment. The barriers such as high cost, complicated procedures, and lack of stability limited the wide applications of such sensing approaches [1–3]. Fortunately, nucleic acid aptamers are short and single-stranded oligonucleotides, selected and identified by

SELEX (Systematic Evolution of Ligands Exponential Enrichment) *in vitro* selection process [4, 5]. Aptamer, considered as chemical antibody and even superior to antibody, can recognize the target molecule to form unique 3D configuration with high affinity and selectivity [6, 7]. Accordingly, aptasensors have attracted increasing attention in recent years toward a variety of target molecules including proteins, cells, viruses, bacteria, metal ions, as well as the disease biomarkers [8, 9].

It is worth noting that the current biosensors generally suffer from the concerns such as lack of biocompatibility, low stability, as well as the poor detection sensitivity. Various advanced nanomaterials can be integrated into the sensing systems for signal transduction and improved analytical performance [10–12]. The commonly used nanomaterials for biosensors mainly include fluorophores, quantum dots (QDs), graphene oxide (GO), gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), metal-organic frameworks (MOFs), upconversion nanoparticles (UCNPs), zinc oxide (ZnO) and other semiconducting nanomaterials, and so on [13–15]. Fascinatingly, bioreceptors can be universally designed and modified with these nanomaterials and are available for optical and electrochemical signal transduction strategies, which further are measured by portable detectors such as fluorimeter, naked eyes, strip readout, as well as electrochemical detectors [16, 17]. For instance, in our recent

#### **Figure 1.**

*Comprehensive overview of aptamer- and antibody-based biosensors toward* Aspergillus *and Aspergillosis based on advanced nanomaterials.*

#### *Nanomaterials-Based Biosensors against* Aspergillus *and Aspergillosis: Control and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.111725*

study, we have introduced a fluorescent aptasensor toward mycotoxin fumonisin B1 (FB1) based on the aptamer recognition. In this effort, GO was embedded for fluorescent quenching and acted as a protectant of the specific aptamer from nuclease cleavage. The target cycling was then triggered for signal amplification and improved detection sensitivity [9].

At the present time, great advances have been achieved for immunosensors and aptasensors in numerous hazard control. Nevertheless, in this book chapter, we only outlined and highlighted the recent progress toward *Aspergillus* and Aspergillosis (**Figure 1**). *Aspergillus* is an extraordinary fungus that occurs naturally all around the world. Sixteen of the 200 known species of *Aspergillus* are isolated and identified due to their severe hazards to animals and humans. Prolonged exposure to high levels of *Aspergillus* can induce allergic symptoms, toxic symptoms, and infection. In particular, for instance, *Aspergillus fumigatus* is the most common and predominate one that can cause invasive aspergillosis (IA) [18, 19]. Conventional approaches for the detection of *Aspergillus* detection like biopsies of cerebral lesions and extraction of cerebrospinal fluid are usually not appropriate for immunocompromised patients. Therefore, simple, rapid, and accurate analytical techniques for *Aspergillus* and Aspergillosis diagnosis are of great importance for human health.

Immunosensors and aptasensors have witnessed a remarkable progress over the past decade. To the best of our knowledge, the comprehensive discussion on immunosensors and aptasensors toward *Aspergillus* and Aspergillosis diagnosis has not yet been reported. Inspired by this status, we proposed an overview of the biosensor construction and their improved performance (**Figure 1**). Moreover, we highlighted the challenges and new opportunities of advanced biosensors for early diagnosis of Aspergillosis infection.

#### **2. Recent advances of biosensors**

In the past three decades, antibody-based immunoassays have been well established from scientists in medical and biotechnology fields, including biosensing, therapy, environment, and so on, and gradually extend to disease diagnosis for various targets and biomarker identification and determination. Classic immunoassays like ELISA (enzyme-linked immunosorbent assays) and LFIA (lateral flow immunoassay) are frequently realized for screening purposes and commercial application in the market [20–22]. In particular, ELISA kit, one of the most widely available products, is suitable for qualitative and quantitative assessment of target biomolecule [23]. The ELISA-based rapid screening methods exhibit desirable sensitivity and selectivity, as well as ease of operation [24, 25]. However, high cost in antibody production and antibody stability issue in complicated environment restrict their extended applications [26, 27]. More importantly, antibody preparation against small molecule remains a rigorous challenge since its non-immunogenic and antibody generation are not significant in the process of animal immune [28–30].

Fortunately, nucleic acid aptamer, considered as "chemical antibody," is short ssDNA or RNA that shows excellent affinity and specificity against its target biomolecule even superior to antibody. Noteworthy, the aptamer selection is performed *in vitro* by SELEX technique instead of the complicated animal experiments *in vivo* [10]. Therefore, compared to antibody, the aptamer possesses obvious advantages like convenient production, low cost, high stability, non-immunogenicity, ease of modification, as well as various targets (proteins, cell, tissue, even small molecules) [31, 32]. Correspondingly, the past decade witnessed a remarkable progress on the development of novel aptasensors against *Aspergillus* and Aspergillosis and mycotoxin contamination. However, it is worth noting that there were few studies focusing on the comparison of analytical performance between antibody and aptamer. Stimulated by this status, we noticed that previous works on aptasensors demonstrated the more excellent analytical performance than that of immunosensors [33, 34]. Therefore, the discovery of specific aptamer and its further research opened up a new horizon for rapid and accurate determination of various hazards with high sensitivity and selectivity.

#### **3. Applications of biosensing strategies for** *Aspergillus*

*Aspergillus* has received global concern due to its hazards on food spoilage and food safety [35]. In particular, *Aspergillus flavus* contamination can produce aflatoxins and cause Aspergillosis [36]. *AflD* gene, a potential biomarker for *Aspergillus flavus* pollution, is a structural gene in aflatoxins gene cluster of *Aspergillus* species like *Aspergillus flavus* [37]. Nevertheless, the analytical strategies for *Aspergillus* control are relatively less reported. Biosensors are thus emerged as advanced techniques for the detection of DNA attributed to their portability, high sensitivity, as well as ease of operation [38]. Sedighi-Khavidak et al. firstly fabricated a novel biosensor toward *aflD* gene analyses of *Aspergillus flavus* based on impedimetric electrochemical signal detection and Au NPs [39]. The Au NPs modified with specific DNA probe were immobilized on the glassy carbon electrode. The presence of target DNA induced the formation of double-stranded DNA *via* hybridization reaction, disrupting the reduction of [Fe(CN)6] <sup>3</sup><sup>−</sup> and enhancing the electrochemical signal (**Figure 2A**). The electrochemical biosensor exhibited a dynamic response of *aflD* gene that ranged from 1 nM to 10 μM with an LOD of 0.55 nM. Furthermore, the feasibility of this sensing protocol was confirmed for the detection of *aflD* gene in real pistachio samples.

Very recently, Liang et al. have synthesized Cu-anchored PDA (polydopamine) nanomaterials, which exhibited photothermal property and catalytic ability for colorimetric signal [40]. In this regard, the nanomaterials were further embedded into LFIA for multimodal sensing of *Aspergillus flavus* based on a sandwich design (**Figure 2B**). Consequently, compared to traditional colorimetric method, the novel LFIA platform showed a significantly sensitive detection of *Aspergillus flavus* with LODs of 0.45 and 0.22 ng/mL, respectively. The feasibility of the sensing strategy was further investigated to monitor *Aspergillus flavus* in peanut and maize samples. On the other hand, apart from the detection of *Aspergillus flavus*, volatile organic compounds (VOCs), the representative metabolites of *Aspergillus flavus*, could be identified and detected for *Aspergillus flavus* contamination [43]. In order to improve the detection efficiency, Lin et al. incorporated three types of nanomaterials into nanocomposites for colorimetric sensing of VOCs [41]. MOF (metal-organic framework), PSA (poly styrene-co-acrylic acid), and PSN (porous silica nanoparticles) were employed and integrated for improved performance due to their ultra-large surface area, excellent catalytic property, and numerous binding sites (**Figure 2C**). The developed sensing method showed high sensitivity and stability for the detection of VOCs and great promise for wheat mildew monitoring. From another viewpoint, the identification and detection of VOCs is significantly correlated with food spoilage event [44]. Traditional analytical technologies like gas chromatography-ion mobility spectrometry (GC-IMS) [45] and electronic nose [46] require expensive instruments, professional personnel, and complicated procedures. Fortunately, whole-cell biosensor

*Nanomaterials-Based Biosensors against* Aspergillus *and Aspergillosis: Control and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.111725*

#### **Figure 2.**

*(A) Schematic representation of impedimetric electrochemical DNA sensor for sensitive detection of* aflD *gene of* Aspergillus flavus *based on the Au NPs modification on electrode. The illustration was recreated according to ref. [39]. Copyright 2017: Taylor & Francis Group, LLC. (B) Schematic diagram of lateral flow immunoassay for colorimetric and photothermal detection of* Aspergillus flavus via *the functionalization of Cu-anchored PDA. The illustration was recreated according to ref. [40]. Copyright 2022: Elsevier. (C) Schematic illustration of the colorimetric biosensor toward* Aspergillus flavus *attributed to the analysis of VOCs by integrating nanocomposites. The illustration was recreated according to ref. [41]. Copyright 2022: Elsevier. (D) Mechanism illustration of a novel whole-cell biosensor for identification and monitoring of VOCs and* Aspergillus *contamination by integrating machine-learning models. The illustration was recreated according to ref. [42]. Copyright 2023: Elsevier.*

possesses a great promise for rapid, portable, highly efficient, and sensitive control of VOCs [47]. Herein, Ma et al. developed a novel whole-cell biosensing platform for VOCs finding and *Aspergillus* infection monitoring by engineering machine learning

#### **Figure 3.**

*(A) Schematic illustration of the novel immunosensor for highly sensitive detection of* Aspergillus Niger *based on the functionalization of single-walled carbon nanotube (SWNT). The illustration was recreated according to ref. [51]. Copyright 2015: Royal Society of Chemistry. (B) Schematic diagram of electrochemical immunosensor for selective quantification of the* Aspergillus Niger *based on the extracellular proteins monitoring and antibody– antigen reaction. The illustration was recreated according to ref. [52]. Copyright 2021: Elsevier.*

models [42]. Three VOCs' markers were identified in peanut by *Aspergillus flavus* infection and further realized for the validation of various response modes and the construction of biosensor (**Figure 2D**). Moreover, the proposed biosensor coupled with machine-learning models exhibited excellent prediction accuracy in both infected matrices and pre-mold stages. Hence, this novel biosensing strategy opened a new avenue for *Aspergillus* infection prediction and food control.

Conventional *Aspergillus* analytical methods generally involve sampling and culturing of *Aspergillus* spores for immunoassay signal detection, followed by DNA sequencing and quantitative analysis. These protocols suffer from the drawbacks like high cost, complicated procedures, and time-consuming [48–50]. To overcome the barrier, advanced nanomaterials-based biosensors are attracting increasing attention for improved performance. For instance, Jin et al. developed a nanoscale immunosensor for real-time monitoring of *Aspergillus niger via* single-walled carbon nanotube (SWNT) and antigen-antibody recognition (**Figure 3A**) [51]. Encouragingly, the integrating of carbon nanomaterials significantly increased the antibody immobilization sites for enhanced detection signal and high sensitivity at sub-picomolar levels. On the basis of similar antigen-antibody recognition, Lee et al. proposed an electrochemical immunosensor for selectively quantification of *Aspergillus niger via* detecting the extracellular proteins (**Figure 3B**), which relied on the immobilization of the extracellular proteins and its interference of redox cycling in interdigitated electrodes [52]. The utilization of secretion promoter at the sampling stage realized a highly sensitive response by 200-fold improvement for the *Aspergillus niger* monitoring in their previous study [53], which might attribute to the specific antibody recognition rather than to the amplified oxidation oxygen reduction reaction.

More importantly, current diagnosing methods for *Aspergillus* infection are not appropriate in clinical POC testing. To solve this concern, Yu et al. developed a novel simple and rapid analytical technique for DNA amplification based on loop-mediated isothermal amplification (LAMP) [54]. The LAMP approach was employed to detect the target gene TR34, a biomarker for *Aspergillus fumigatus* infection in patients. Assisted by the primer design of LAMP, this protocol contributed to rapid and selective identification of TR34, as well as the high detection sensitivity with 10 genomic copies per reaction. It was demonstrated that the TR34-LAMP platform can be considered as a POC diagnosis strategy for the screening of clinical *Aspergillus fumigatus* infection.

#### **4. Applications of biosensing strategies for Aspergillosis**

#### **4.1 Gliotoxin-related biomarker**

Genus *Aspergillus* is a group of fungi that can induce a majority of *Aspergillus* infection from allergic reaction to invasive diseases. Invasive aspergillosis (IA), one of the most severe and devastating *Aspergillus* infections, is defined as a rapid, acute, and life-threatening invasive disease with mortality rate as high as 90%. Of the various *Aspergillus* (such as *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*), *Aspergillus fumigatus* is the most common and predominate one that causes IA. Gliotoxin, considered as the most toxic metabolites occurred by *Aspergillus fumigatus*, poses great hazards to immunosuppressed individuals. Therefore, the development of simple, rapid, sensitive, and point-of-care strategies for gliotoxin control is of high significance and urgency for early diagnosis of IA.

In the direction, assisted by the immobilization-free and GO (graphene oxide)- SELEX technique, Gao et al. proposed the pioneer work for the isolation and selection of the specific aptamer toward gliotoxin (**Figure 4A**) [55]. After the eighth selection cycle, fortunately, the ssDNA was enriched, and the aptamer APT8 was obtained and sequenced with a dissociated constant (KD) of 376 nM. Then, the APT8 was further truncated into a shorter sequence consisting of only 24 nucleotides according to the mfold structure prediction. More encouragingly, the truncated aptamer APT8T1 was confirmed to recognize the gliotoxin with higher specificity (KD = 196 nM) and could be further designed to APT8T1M with 18-fold improvement of KD value. Accordingly, to validate the feasibility and selectivity of the aptamer, a simple fluorescent aptasensor was established for the detection of gliotoxin based on base pairing between the aptamer and its complementary DNA. The specific recognition of aptamer against gliotoxin caused the release of the aptamer/gliotoxin complex from the microplate and the fluorescent signal enhancement. The fluorescent signal was observed to be in linear relationship with levels of target gliotoxin in the range of 0.1–100 nM. The LOD was estimated to be 0.05 nM, which is significantly lower than the previous instrument methods like HPLC-MS/MS. Moreover, the successful application of the fluorescent aptasensor in human serum and urine samples demonstrated that this developed aptasensing platform offered a promising value in gliotoxin control. Inspired by this pioneer finding, combining the unique superiorities of MXene (Ti3C2) and TDNs (tetrahedral DNA nanostructures), Wang et al. developed a novel electrochemical aptasensor for highly efficient detection of gliotoxin based on nanomaterial functionalization (**Figure 4B**) [56]. Ti3C2 nanosheets, exhibiting large surface area, were modified with TDNs *via* the coordination interaction. The prepared nanocomposites allowed outstanding conductivity and molecule recognition toward the target for signal amplification. After the binding events between the aptamer and target, the cDNA was released and bound to the aptamer-modified nanocomposites, leading to

#### **Figure 4.**

*(A) Working principle of the fluorescent aptasensor against gliotoxin detection for early diagnosis of invasive aspergillosis based on the specific aptamer selection. The illustration was recreated according to ref. [55]. Copyright 2018: American Chemical Society. (B) Schematic representation of electrochemical biosensor for labelfree detection of gliotoxin incorporating of tetrahedral DNA nanostructures (TDNs) and MXene nanocomposites. The illustration was recreated according to ref. [56]. Copyright 2019: Elsevier. (C) Schematic diagram of isolation and characterization of the specific aptamer against spores of three representative* Aspergillus *species. The illustration was recreated according to ref. [57]. Copyright 2021: Royal Society of Chemistry.*

the streptavidin-decorated HRP (horseradish peroxidase) catalysis and current signal generation. As a consequence, a dynamic response was achieved between the electrochemical signal and concentrations of gliotoxin ranged from 5 pM to 10 nM with an LOD of 5 pM. Accordingly, the detection sensitivity of this electrochemical aptasensor was improved by an order of magnitude than that of the previous fluorescent

*Nanomaterials-Based Biosensors against* Aspergillus *and Aspergillosis: Control and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.111725*

detection. Notably, the practicality of this method was excellent for gliotoxin detection in human serum samples. It was demonstrated that, from this viewpoint, advanced aptasensor technology possessed a significant potential against gliotoxin for early diagnosis of IA.

On the other hand, considering the obvious superiorities of aptamer-based biosensors, Seo et al. isolated and identified the specific aptamers that can specifically recognize *Aspergillus* spore by using cell-SELEX technique for the first time [57]. In this effort, the aptamers against three *Aspergillus* spores including *Aspergillus fumigatus*, *Aspergillus flavus*, and *Aspergillus niger* were selected in the spore-SELEX process (**Figure 4C**). With 12 rounds of selection, the ssDNA was successfully enriched, and the specific aptamer Asp-3 was achieved and sequenced with dissociated constants (KD) of 80.12, 35.17, and 101.19 nM versus *Aspergillus fumigatus*, *Aspergillus flavus*, and *Aspergillus niger*, respectively. It was worth noting that the above aptamer also exhibited strong affinity to 1,3 β-D-glucans (BDGs) with KD of 79.76–103.7 nM, demonstrating the excellent affinities and recognition potential toward *Aspergillus* spore surface molecules. However, the representative aptasensors have not been further developed. Hence, how to select and optimize the aptamer for *Aspergillus* spore control is of vital significance in the future. On the other hand, the biosynthesis of gliotoxin is related to gliP gene, and gliP gene-encoded enzyme further affects the occurrence of gliotoxin. In this direction, gliP gene can be considered as a potential biomarker for *Aspergillus* control, and it eventually contributed to the early diagnosis of IA. Encouragingly, Bhatnagar et al. fabricated an electrochemical biosensor toward gliP gene detection for the first time [58]. The gliP gene was immobilized onto chitosan-stabilized Au NPs on gold electrode. DNA hybridization reaction induced the formation of double-stranded DNA and its interaction with toluidine blue. The toluidine blue was acted as the electrochemical indicator for signal output. Upon the optimal conditions, the novel biosensor exhibited a dynamic response of the target in the range of 1 × 10<sup>−</sup>14–1 × 10−<sup>2</sup> M with an LOD of 0.32 × 10<sup>−</sup>14. The proposed biosensor is stable and selective for detection of glip-T, demonstrating that it is useful for *Aspergillus* analysis and clinical diagnosis.

#### **4.2 Galactomannan-related biomarker**

GM (Galactomannan), regarded as a popular biomarker *Aspergillus* infection, is a heat-stable polysaccharide consisting of a linear mannan core with side chains of galactofuran. GM is metabolized and released in the blood and bronchoalveolar fluid (BALF) and is occurred as soluble antigen mainly in the cell wall of the genera *Aspergillus*. Ab (antibody)-based immunoassays such as ELISA (enzyme-linked immunosorbent assay), LFIA (lateral flow immunoassay), and immunosensor are the most frequently used protocols for the detection of GM in early infection of IA. Raval et al. firstly proposed an ELISA method to capture and detect GM based on the conjugation of polyclonal antibody to Au NPs [59]. The developed Au NPs immunoassay possessed simple and accurate detection of GM with low LOD at picomolar level (**Figure 5A**). Based on the similar mechanism, Guo et al. introduced a sandwich chemiluminescence immunoassay toward GM detection incorporated with luminescent nanomaterials (**Figure 5B**) [60]. In this attempt, the donor consisted of photosensitizer and phthalocyanine, and the acceptor contained the chemiluminescent dye, allowing the luminescent signal output under laser irradiation. The donor and the acceptor formed immunocomplex due to the close proximity less than 200 nm. The developed chemiluminescence ELISA platform displayed a linear response of GM in

#### **Figure 5.**

*(A) Schematic diagram of the ELISA-based immunoassay for the detection of galactomannan for early diagnosis of invasive aspergillosis based on the Au NPs conjugation. The illustration was recreated according to ref. [59]. Copyright 2019: Microbiology society. (B) Working principle of the chemiluminescence immunoassay toward galactomannan detection based on the donor and the acceptor. The illustration was recreated according to ref. [60]. Copyright 2022: Elsevier. (C) Schematic illustration of the fluorescence immunosensor to monitor galactomannan based on sandwich format and ZnO nanoflowers (ZnONFs). The illustration was recreated according to ref. [61]. Copyright 2020: Elsevier.*

the range 0.05–100 ng/mL with an LOD of 0.032 ng/mL and was capable of highly efficient GM detection in serum and BALF. Besides, nanomaterials-based miniaturized biosensors possessed outstanding advantages in portable monitoring and high-throughput analytical manner [62–64]. Therefore, Piguillem et al. prepared the ZnO nanoflowers (ZnONFs), conjugated the nanomaterials to microfluidic channel, and employed the nanocomplex for antibody modification [61]. Correspondingly, a novel immunosensor was established for fluorescent detection of GM *via* the generation of fluorescent substance resorufin (**Figure 5C**). The immunoassay principle was designed by HRP-modified antibody for catalysis of 10-acetyl-3,7-dihydroxyphenoxacine oxidation to resorufin. Excitingly, the proposed immunosensor can realize a more sensitive and highly efficient detection of GM by 14-fold improvement in LOD over the commercial ELISA method.

#### **5. Conclusions and outlook**

Over the past decade, immunosensors and aptasensors have been well established for the detection and control of *Aspergillus* and Aspergillosis. Advanced

#### *Nanomaterials-Based Biosensors against* Aspergillus *and Aspergillosis: Control and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.111725*

nanomaterials-integrated approaches possess great potential to improve the performance for early diagnosis of IA. Of them, optical and electrochemical responses (including fluorescent, colorimetric, electrochemical, and electrochemiluminescence systems) are the two major signal transduction mechanisms. Besides, several researches have also focused on the principles toward binding events between the aptamer and target, catalytic transformation of target, truncation of the known aptamer, as well as the selection of new aptamers, and so on. These efforts indicated that advanced biosensors possessed excellent dynamic response and high sensitivity for AFB1 detection, as well as the feasibility and accuracy in *Aspergillus* detection and Aspergillosis diagnosis. Accordingly, biosensors techniques should have fascinating potential in industrial applications for food safety and risk assessment in the future.

Even though nanomaterials-based biosensors toward *Aspergillus* and Aspergillosis have witnessed the remarkable achievement, there are still several vital scientific limitations and challenges that required to overcome. (i) Advanced material-integrated biosensors exhibited excellent and improved analytical performance. For instance, as mentioned in this article, combining GO with AuNCs can significantly improve the fluorescent quenching efficiency compared to single GO. Novel luminescent materials such as UCNPs, PLNPs, and AIE probes displayed unique superiorities in terms of photostability, anti-interference, and even label-free detection. Therefore, the exploration of new materials and their synergistic effects are of great significance. (ii) Numerous reports are rarely aimed at developing the novel biosensors for their detection analysis rather than at the mechanism research. It is worth noting that the configuration change, site modification, and binding kinetics are of vital importance to better understand the principles and signal response. (iii) The selectivity assay is not appropriate in some case studies; the structural analogues should be taken into consideration for the interferences. (iv) It can be seen that the novel aptamer and aptasensors were relatively less developed, which is mainly attributed to the barriers in precise selection of aptamers. The stability and folding characteristics may be influenced by the special conditions like temperature, pH, ionic strength, and so on, thereby affecting the detection performance. Thus, how to select and identify highquality aptamers that can undergo various reaction systems is a pursuing work. (v) Most established aptasensing protocols are limited in the laboratory conditions; in particular, at the present time, no commercial kits integrated by aptamers are available in the market. Enormous endeavors should be exerted to design and develop portable sensors or miniaturized devices in POC testing of *Aspergillus* and Aspergillosis.

#### **Acknowledgements**

We thank the University of Liège-Gembloux Agro-Bio Tech and more specifically the research platform AgricultureIsLife for the funding of the scientific stay in Belgium that made this paper possible.

#### **Funding**

This work was supported by the National Natural Science Foundation of China (No. 32001682, 21305158), the Special Fund for Agro-scientific Research in the Public Interest (201403071), and the Agricultural Science and Technology Research system of the PR China (ASTIP-IAS12).

### **Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### **Author details**

Xiaodong Guo1,2\*, Mengke Zhang3 , Mengzhi Wang1 , Jiaqi Wang4 and Marie-Laure Fauconnier5

1 Laboratory of Metabolic Manipulation of Herbivorous Animal Nutrition, College of Animal Science and Technology, Yangzhou University, Yangzhou, China

2 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China

3 College of Food Science and Light Industry, Nanjing University of Technology, Nanjing, China

4 Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

5 Chimie Générale et Organique, Gembloux Agro-Bio Tech, Université de Liège, Gembloux, Belgium

\*Address all correspondence to: guoxiaodong233@163.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Nanomaterials-Based Biosensors against* Aspergillus *and Aspergillosis: Control and Diagnostic… DOI: http://dx.doi.org/10.5772/intechopen.111725*

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#### **Chapter 6**

## Metagenomic Next-Generation Sequencing (mNGS) for the Diagnosis of Pulmonary Aspergillosis

*Hao Tang, Shujun Bao and Caiming Zhong*

#### **Abstract**

The diagnosis of pulmonary aspergillosis is a critical step in initiating prompt treatment and improving patients' prognosis. Currently, microbiological analysis of pulmonary aspergillosis involves fungal smear and culture, serum (1,3)-β-D-glucan (G) or galactomannan (GM) tests, and polymerase chain reaction (PCR). However, these methods have limitations. Recent studies have demonstrated that polymorphisms in pentraxin3 (PTX3), a soluble pattern recognition receptor, are associated with increased susceptibility to invasive aspergillosis. mNGS, a new microbial diagnostic method, has emerged as a promising alternative. It has high sensitivity in identifying pulmonary aspergillosis and can accurately distinguish species. Additionally, it outperforms other methods in detecting mixed infections and instructing the adjustment of antimicrobial treatments. As a result, mNGS has the potential to be adopted as the gold standard for the diagnosis of pulmonary aspergillosis.

**Keywords:** mNGS, fungi, pulmonary aspergillosis, diagnosis, other methods

#### **1. Introduction**

*Aspergillus* belongs to Ascomycetes fungi, including *Aspergillus* fumigatus, *Aspergillus* flavus, *Aspergillus* niger, and *Aspergillus* terreus, which are known to cause a wide range of diseases, such as allergic reactions, airway or lung infections, and extrapulmonary spread, especially skin infections. Lungs are the most common sites of *Aspergillus* infection in human. Meanwhile, pulmonary aspergillosis are often difficult to be diagnosed and have a high mortality rate. The clinical spectrum of pulmonary aspergillosis can present in various forms, including chronic pulmonary aspergillosis (CPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA), and invasive pulmonary aspergillosis (IPA). The histopathology of the lesion can be understood as a phenotype that reflects the interaction between decreased host defense mechanisms and increased fungal virulence. In the case of *Aspergillus* infection, tissue reactions to the fungus are diminished, but the reasons and degree of the decrease in defense mechanisms vary among cases. Pulmonary aspergillosis presents


#### **Table 1.**

*The main patient population and crucial diagnostic methods of different types of pulmonary aspergillosis.*

in various forms in the disease spectrum depending on the interaction between *Aspergillus* and the host, and there may be an overlap between different clinical phenotypes. Moreover, the key diagnostic methods for different types of pulmonary aspergillosis vary. The main patient population and crucial diagnostic methods of different types of pulmonary aspergillosis are shown in **Table 1**.

In recent years, several new rapid diagnostic methods for pulmonary aspergillosis have emerged, in addition to traditional pathological and cultural diagnostic methods. These include the detection of antigens, antibodies, novel molecular markers, and genes. Among them, the second-generation sequencing technology for genetic testing has gained attention since 2014 [1], which has high sensitivity and a short detection cycle [2]. It can detect various samples such as sputum, bronchoalveolar lavage fluid (BALF), blood, and cerebrospinal fluid, making it a promising microbial identification technology. Which means that mNGS can classify a wide range of pathogens in several infection sites, including the respiratory tract [3], blood [4], central nervous system [5], and focal sites [6]. It is especially useful in detecting special and rare pathogens, as well as analyzing drug-resistance genes and virulence factors related to pathogens. The popularization of mNGS has proven to be of great value in diagnosing pulmonary aspergillosis.

#### **2. The diagnostic value of mNGS in pulmonary aspergillosis**

#### **2.1 Principles and methods of mNGS**

In recent years, the clinical application of mNGS has become an important method for diagnosing pulmonary fungal infections, which is of significant clinical value. Unlike traditional methods, mNGS technology does not require the culture of clinical samples. Instead, it extracts nucleic acid from all microorganisms in samples, constructs a standard sequencing library, and conducts high-throughput sequencing. Finally, the bioinformatics analysis confirms genome sequence comparison of each species, allowing for identification of microorganisms and calculation of various

*Metagenomic Next-Generation Sequencing (mNGS) for the Diagnosis of Pulmonary Aspergillosis DOI: http://dx.doi.org/10.5772/intechopen.111827*

**Figure 1.** *Detection workflow of mNGS.*

parameters. Please refer to **Figure 1**. mNGS has been utilized to identify the etiology of various clinical samples, significantly improving the diagnostic rate of pulmonary fungal infections. Several studies have demonstrated that mNGS is more sensitive than traditional tests, such as pathology, culture, serological tests, and PCR in fungal detection. Clinical studies have further confirmed the efficacy of mNGS in diagnosing common fungi and its ability to improve the sensitivity of pathogenic diagnosis of pneumonia fungi, making it an effective supplement to traditional microbiological detection methods for fungal infections.

#### **2.2 Sample source for mNGS**

Several studies have employed mNGS to detect microorganisms in lung biopsy specimens, respiratory secretions, and pleural effusions. For instance, mNGS was performed on formalin-fixed paraffin-embedded (FFPE) lung tissue samples from patients with granulomatous lesions and unclear diagnoses. Results showed that mNGS had a detection rate of 87.8% for total fungi and mycobacteria, which was 68.3% higher than histopathology. Moreover, pathogenic bacteria could be identified at the species level [7]. Other studies utilized mNGS to detect pathogenic microorganisms in computer tomography (CT)-guided lung biopsies of patients with lung diseases. Results showed that the specificity and positive predictive value of mNGS for detecting fungi were 100%, which was higher than histopathological methods [8].

Furthermore, the sensitivity of mNGS in detecting pathogenic bacteria and fungi in respiratory tract samples (such as nasopharyngeal swabs, sputum, and BALF) from patients with pulmonary infection was higher than traditional methods, such as respiratory virus culture and PCR analysis [9]. Other studies analyzed the diagnosis of pulmonary invasive fungal infections (IFIs) using mNGS and found that the fungal species detected by mNGS in BALF were higher than those detected by standard culture methods [10].

Some studies also tested patients with pulmonary infection combined with pleural effusion for mNGS, and the positive rate was higher than that of the culture method. Candida and Pneumospora were the most common fungal infections detected [11].

Bronchoscopic lung biopsy and BALF samples were also analyzed to investigate the differences in the detection of mNGS. Results showed that mNGS had a wider range of pathogen detection than traditional tests, especially for pulmonary fungal infection. The proportion of fungi detected and identified by mNGS was significantly higher than that by conventional tests [12]. Moreover, the sensitivity of mNGS in

diagnosing pulmonary fungal infection was significantly higher than conventional tests, such as pathology, GM test, and culture. However, there was no difference in sensitivity and specificity between lung biopsy-mNGS and BALF-mNGS [13]. In other studies, transbronchial lung biopsy (TBLB), BALF, and bronchoalveolar needle brush (BB) specimens were tested for mNGS to detect suspected pulmonary infectious diseases. Results showed that the sensitivity of mNGS in detecting fungi in the three specimens was higher than that of conventional culture, but there was no difference in the sensitivity of mNGS among different specimen types [14].

According to the above researches, we can conclude that compared to the sensitivity of mNGS in the lung biopsy specimens, there is no obvious difference in that in respiratory secretions. Therefore, BALF/BB can be alternative samples in the detection of mNGS.

#### **2.3 Fungi that can be detected by mNGS**

The incidence of pulmonary fungal infections in clinical settings is attributable to a diverse range of pathogens such as *Aspergillus*, Mucor, Pneumospora [15], Cryptococcus, among others. Conversely, Candida is a relatively infrequent cause of pulmonary infections.

Lung infections associated with other pathogenic fungi such as Histoplasma capsulatum, Talaromyces marneffei, and Exophiala dermatitidis are even less common. mNGS has shown significant value in identifying rare fungi. It has been reported that a 27-year-old Chinese male with chronic progressive lung disease was asymptomatic for over a year until the disease progressed to the epiglottis, causing progressive pharyngeal pain. Despite negative results from BALF and epiglottic tissue cultures, as well as epiglottic and pulmonary pathology, mNGS was able to detect Histoplasma capsulatum in both epiglottic organizations and BALF and determined the cause after itraconazole treatment was successful [16]. In another case, mNGS successfully identified Talaromyces marneffei infection in a non-human immunodeficiency virus (HIV) patient, which is the first reported case in North China [17]. In the third case, a 52-year-old man with cough, sputum, and hemoptysis showed multiple lesions on both sides according to chest CT. Although the pathogen could not be identified after three biopsies, the subsequent mNGS results and therapeutic response confirmed that the pathogenic pathogen was Exophiala dermatitidis, and the diagnosis was Exophiala dermatitidis pneumonia [18].

Additionally, pulmonary fungal infections were frequently combined with bacterial infections, and immune deficiency and the presence of pulmonary bulba were risk factors associated with fungal and bacterial co-infections [19]. Furthermore, mNGS has been found to be able to distinguish colonization and infection in certain fungi, the colonization group and infection group of Pneumocystis yersii were differentiated by BALF-mNGS, and the fungal load was significantly different between the two groups [20].

#### **2.4 The clinical application of mNGS in pulmonary aspergillosis**

There are limited clinical studies on the diagnosis of pulmonary aspergillosis using mNGS. Although the studies are mostly limited to case reports, and the number of cases included are small, the available studies have demonstrated the good diagnostic performance of mNGS.

#### *Metagenomic Next-Generation Sequencing (mNGS) for the Diagnosis of Pulmonary Aspergillosis DOI: http://dx.doi.org/10.5772/intechopen.111827*

mNGS plays a key role in the diagnosis of pulmonary aspergillosis, either alone or in combination with other diagnostic methods. See **Figure 2** for details.

Patients with *Aspergillus* that can be detected by mNGS tend to have an incompetent immune function. One study reported that five patients' blood samples were positive for *Aspergillus* using mNGS, the five patients had various underlying diseases, including myelodysplastic syndrome (two cases), acute myeloid leukemia (two cases), and kidney transplantation (one case), which suggests that mNGS has guiding value in the diagnosis of IPA in immunocompromised patients [21]. In other studies, mNGS was used to detect plasma for the diagnosis of Corona Virus Disease 2019 (COVID-19)-associated pulmonary aspergillosis, suggesting that mNGS has good diagnostic performance for COVID-19-associated pulmonary aspergillosis when detecting plasma, and is highly specific [22]. Additionally, researchers detected mNGS in BALF samples and described three cases of IPA (respectively with chronic obstructive pulmonary diseases (COPD) and asthma history, and one case without underlying disease). *Aspergillus* fumigatus gene was found in all mNGS in BALF, indicating the diagnostic value of mNGS in non-neutropenic IPA patients [23]. Further, a study was conducted to analyze the accuracy of using mNGS to diagnose IPA in patients with different immune status. The study found that the consistency rate of IPA diagnosis in immunocompromised patients was significantly higher (82.1%) than in non-immunocompromised patients (52.9%). Therefore, mNGS detection combined with pulmonary CT imaging can be used for IPA diagnosis in patients with immunocompromised function. However, the diagnosis of IPA based on positive mNGS results in non-immunocompromised patients should be approached with caution [24].

Additionally, severe pneumonia caused by any type of pathogenic bacteria may be accompanied by *Aspergillus* infection. Therefore, some studies have used mNGS for early secondary infection screening, and secondary *Aspergillus* infection was found in a case of severe pneumonia caused by Legionella infection, leading to early precise treatment [25]. Another retrospective study found that mNGS performed well in the detection of common pathogens in *Aspergillus* infection, the most common bacteria were Klebsiella pneumoniae and Acinetobacter baumannii. Furthermore, 91.7% of pulmonary aspergillosis patients changed antibacterial therapy based on mNGS

#### **Figure 2.**

*The value of mNGS in the diagnosis of pulmonary aspergillosis.*


**Table 2.**

*Comparing the sensitivity and specificity of different diagnostic methods of pulmonary aspergillosis.*

results [26]. In summary, compared to other diagnostic methods, mNGS has higher sensitivity in the diagnosis of pulmonary aspergillosis. Please refer to **Table 2** for details.

mNGS can also be used as a supplement for routine microbial detection. A comparison of community-acquired pneumonia (CAP) patients diagnosed with IPA found that the sensitivity of GM to detect *Aspergillus* was the highest, followed by mNGS, culture, and smear. However, the specificity of mNGS, culture, and smear was 100%. The specificity of GM detection was 92.9% [27]. In patients with neutropenic, *Aspergillus* was detected by pathology and mNGS and diagnosed as IPA [28]. In patients with normal immune function, *Aspergillus* fumigatus was identified through endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) tissue with mNGS. Histological analysis of mediastinal biopsy and tissue fungal culture also indicated *Aspergillus* fumigatus infection, confirming the detection of mNGS [29]. In an infant patient initially suspected of having a lung tumor, mNGS identified *Aspergillus* fumigatus in BALF as the causative agent. The patient recovered quickly and was discharged after receiving appropriate antifungal therapy [30].

#### **2.5 Precautions of mNGS report in the diagnosis of pulmonary aspergillosis**

The interpretation of the mNGS report cannot disregard the value of a few or a dozen *Aspergillus* readings in the genus. This may occur when the thick cell wall of fungi makes nucleic acid extraction challenging. Additionally, after antifungal treatment, the number of fungi may decrease, resulting in low read lengths. In such cases, the sequenced samples may contain only free nucleic acid fragments that were lysed after the death of pathogenic bacteria. Moreover, the original pathogen load in the sample may have been low, leading to a low reading length. Consequently, some clinical studies fail to reflect the excellent diagnostic value of mNGS. In severe immune dysfunction patients suspected of pneumonia, the diagnostic accuracy of BALF's mNGS and conventional microbiological tests (CMTs) in detecting fungal infections was much lower. The low sensitivity to IPA was mainly responsible for this finding [31]. Furthermore, in rheumatic patients with suspected pneumonia and acute respiratory failure, the sensitivity of BALF sample culture combined with the GM test was superior to that of the mNGS test in detecting *Aspergillus* [32].

When low sequence numbers of microorganisms detected by mNGS are difficult to interpret clinically and cannot be verified by routine laboratory methods for clinical microorganisms, PCR detection may be used for confirmation. Combining mNGS with fungal PCR methods [33] can effectively detect fungi in clinical settings

#### *Metagenomic Next-Generation Sequencing (mNGS) for the Diagnosis of Pulmonary Aspergillosis DOI: http://dx.doi.org/10.5772/intechopen.111827*

and reduce the rate of missed diagnoses. A small amount of *Aspergillus* detected by BALF-mNGS can be confirmed by BALF-GM and/or *Aspergillus*-specific IgG antibody. While histopathology is an objective criterion for detecting fungus, it cannot identify the species and should not be considered the ultimate criterion. At present, the best diagnostic method for pulmonary aspergillosis is a combination of lesion morphology with culture or sequencing.

If pulmonary fungal infection is detected only by mNGS, it could lead to significant confusion in clinical diagnosis. *Aspergillus* is commonly present in the environment and can colonize the respiratory tract of healthy individuals. A positive culture or molecular test does not necessarily indicate an infection, as *Aspergillus* nucleic acid sequences have been found in nearly 40% of samples from non-pulmonary fungal-infected individuals. Consequently, microbial sequences detected by mNGS may originate from pathogenic microorganisms, normal flora, transient colonization, sample contamination, or even incorrect database comparison.

#### **3. Other new rapid diagnostic methods for pulmonary aspergillosis**

The new rapid diagnostic methods for pulmonary aspergillosis include antigen and antibody detection, novel molecular markers, and gene detection.

Antigen detection methods mainly include the G test and GM test. The G test detects (1,3)-β-D-glucan, a specific component in fungal cell wall, as a pan-fungal detection biomarker. GM is a foreign antigen released by *Aspergillus* mycelium when invading host tissue, and it is a specific polysaccharide in cell wall of *Aspergillus*. The GM test is used for early *Aspergillus* infection diagnosis through an enzymatic-linked immunosorbent assay. The blood-GM test is relatively simple and easy to operate, but its sensitivity is low in non-neutropenic patients such as COPD [34]. Additionally, it is affected by antibacterial drugs [35], intravenous immunoglobulin [36], and multiple myeloma [37]. The BALF-GM test has better diagnostic value than serum-GM test does [34]. European and American guidelines recommend that non-neutropenic patients choose the BALF-GM test to improve the detection rate [38, 39]. However, its sensitivity and specificity of the BALF-GM test depend on the population included in the study, the defined cut-off point, the detection platform, and the time node, and whether *Aspergillus* covering drug therapy is performed. There is no universal diagnostic threshold.

Antibody detection includes IgG and IgE detection. IgG antibody detection is the most sensitive microbial test for the diagnosis of CPA [40]. The increase of specific IgE of *Aspergillus* and total IgE levels can confirm the diagnosis of ABPA.

Furthermore, the level of plasma PTX3 in IPA patients was significantly increased, which could be used as a novel molecular marker for diagnosis [41]. Additionally, PCR is a sensitive and rapid gene detection method, but has certain false positive and high negative predictive value. Studies have confirmed that BALF-PCR detection has a high diagnostic value in the diagnosis of invasive aspergillosis with immunocompromised function [42].

#### **4. Conclusions**

The mNGS technique has considerable diagnostic value in pulmonary aspergillosis, however, it should be closely integrated with clinical comprehensive judgment to achieve accurate diagnosis.

#### **Acknowledgements**

The authors would like to thank all patients for participating in this study. The authors also thank the BGI (Shanghai, China) for their helpful technical support. The research was sponsored by "Shuguang Program" supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (20SG38), Shanghai Municipal Science and Technology Committee of Shanghai Outstanding Academic Leaders Plan (20XD1423300), and General Program of National Nature Science Foundation of China (No. 82070036).

#### **Conflict of interest**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **Notes**

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

#### **Nomenclature and abbreviations**


*Metagenomic Next-Generation Sequencing (mNGS) for the Diagnosis of Pulmonary Aspergillosis DOI: http://dx.doi.org/10.5772/intechopen.111827*

#### **Author details**

Hao Tang\*, Shujun Bao and Caiming Zhong Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Naval Medical University, Shanghai, China

\*Address all correspondence to: tanghao\_0921@126.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 4

## Drug Development and Resistance

#### **Chapter 7**

## Aspergilosis: Resistance and Future Impacts

*Amanda Junior Jorge*

#### **Abstract**

Fungal infections have been increasingly reported in routine, especially opportunistic ones such as aspergillosis, which represents a serious challenge for health professionals. The use of itraconazole, for a long time, was effective for a good clinical response, but factors associated with the advancement of medicine, length of stay, diagnostic errors, incorrect doses, and wrong choice of antifungal classes favored the appearance of resistance mechanisms. Thus, new research, together with the development of new molecules, is being carried out in order to reduce the advance of resistance, increasing patient survival.

**Keywords:** *Aspergillus*, therapeutics, ineffectiveness, antifungal, fungal infection

#### **1. Introduction**

Microorganisms of the genus *Aspergillus* are filamentous fungi, ubiquitous saprobes, and possess biological characteristics that allow their survival in temperature changes and extreme conditions, such as desert areas and polar regions. Its spores are much more resistant due to the development of thermotolerance, being able to survive up to 50°C. They have the ability to produce mycotoxins, such as glycotoxin, which have immunosuppressive activity, and the melanin pigmentation on the surface of the spores helps protect against UV rays [1].

Airway is the main form of infection (**Figure 1**), it occurs through inhalation of spores that are easily dispersed through the air, allowing their distribution over wide areas, such as open and closed environments, including hospitals. *Aspergillus* spores can be inhaled between 100 and 1000/day and reach the lung alveoli because of their reduced size (about 2–3 μm) [2]. However, they may not cause illness if inhaled by healthy individuals with competent immune systems. In turn, in immunocompromised individuals [3], the fungus lodges and causes lesions through the synthesis of enzymes (hemolysins, proteases, and peptidases) and toxins (fumagillin and gliotoxin) [4].

The proteases of some *Aspergillus* damage the protective barrier of the respiratory epithelium, inducing an inflammatory reaction to allow greater penetration of fungal antigens. They can also stimulate the release of pro-inflammatory cytokines (IL-8) and growth factors, causing bronchiectasis [4].

**Figure 1.** *Infectious cycle of* Aspergillus *[2].*

One of the most severe forms is Invasive Pulmonary Aspergillosis, responsible for significant morbidity and mortality rates ranging from 80 to 100% in immunodeficient patients, with *A. fumigatus* being the main etiological agent. Some criteria for acquiring the disease are chronic obstructive pulmonary disease, asthma, hematologic malignancies such as acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), acquired immunodeficiency syndrome (AIDS), recipients of solid organ transplants and cirrhosis [5]. COPD is the main factor for the occurrence of API in 50% of cases, followed by solid organ transplants, because generally, in these patients, colonization with *Aspergillus* is 16.3 in every 1000 hospital admissions [6].

#### **2. Discussion**

As medical care has progressed, the number of patients at risk for invasive aspergillosis has increased. Bacterial treatments, intubation, and advances in medicine to prevent respiratory failure have also caused aspergillosis to evolve in a degree of pathogenicity [7]. In addition, the genus *Aspergillus* is much more frequent in solid organs; therefore, in transplanted individuals, caution must be exercised, and this is where we will most often find prophylactic therapy [8]. In general, to treat *Aspergillus*-related diseases are used polyenes, azoles, and echinocandins (**Table 1**) [9, 10].

The polyenes will bind to the ergosterol of the cytoplasmic membrane of the fungus, where they will change the permeability of the membrane, forming pores. These pores will allow the exit of proteins, carbohydrates, and cations, which will end up causing the death of the fungus, being, for this reason, a fungicidal drug [10].

The azoles represent an important class of chemicals for the management of fungal diseases in plants, animals, and humans and for the preservation of materials [11]. They are the first option for the treatment of aspergillosis [12] because of their azole ring that prevents the growth of fungi [13].

The mechanism of azoles is by blocking the cytochrome P-450-dependent enzyme lanosterol demethylase, affectin the ergosterol, a component of fungal plasma membranes [14]. Exposure to azoles in *A. fumigatus* decreases ergosterol levels, changing the membrane shape and structure, reducing the absorption of nutrients, chitin synthesis, and fungal growth [15, 16].

*Aspergilosis: Resistance and Future Impacts DOI: http://dx.doi.org/10.5772/intechopen.112755*


#### **Table 1.**

*The main classes of clinically-used antifungal agents for the treatment of invasive fungal infections [9].*

Triazole is for long-term treatment, and it is the only anti-*Aspergillus* that can be administered orally [17]. For the treatment of noninvasive aspergillosis, itraconazole is used [18], although voriconazole is the first-line treatment for invasive aspergillosis [15, 16, 19].

Echinocandins is a new class of antifungal drugs, with the first example, caspofungin, entering clinical use a decade ago [20]. They are lipopeptide compounds that inhibit the enzyme [21, 22] beta-D-glucan synthase, which produces glucan, the main building block of fungal cell walls. It is indicated for patients with aspergillosis unresponsive to or intolerant of other treatments [20].

Regarding agriculture, plants are often attacked by various pathogenic fungi that cause a variety of diseases, such as leaf spot, blast, downy, etc. The stability of azoles is an important feature because even with small changes in their chemical composition, many azoles function for many days in agricultural habitats (soil and water). The azoles are effective against several fungal plant diseases [23]. Several foods contain azole residues; therefore, there is evidence that large amounts of antifungal residues, especially azoles, can remain in the environment [24].

Clinical resistance occurs when the maximum concentration of the drug is no longer efficient to eliminate the infection [25]. The emergence of the HIV pandemic, the increasing inappropriate use of drugs and illicit substances, transplant surgeries, abusive use of antifungals as prophylaxis for long periods in patients with immunosuppression, diagnostic failures, prolonged use in plantations, and factors inherent to the care units; intensive care such as mechanical ventilation, surgical interventions, total parenteral nutrition and prolonged treatment with antibiotics are some of the predisposing conditions for increased fungal infections by *Aspergillus* [26].

Increased resistance to azole therapy in patients with *Aspergillus* infections. *A. fumigatus*, which causes about 80% of invasive infections, has the highest resistance to azoles [27]. Infections with resistant *Aspergillus* strains cause ineffectiveness of azole antifungals, resulting in high mortality rates. In the United States, in the 1990s, strains resistant to *Aspergillus* had already been reported [28, 29], and since then, virtually all European nations have reported cases of azole resistance, including Germany, Ireland, Italy, Austria, Denmark, France, Sweden, Portugal, Spain, and Turkey [30, 31]. Studies began to be designed to analyze the resistance of *Aspergillus*, mainly *A. fumigatus*, which one carried out in the United Kingdom concluded that the resistance to azoles in *A. fumigatus* increased by 1,77% in 1998–2011 and 2015–2017 [32]. Another multicenter study in Taiwan found a high prevalence rate (4%) for azole-resistant *A. fumigatus* [33]. In addition, this study raised significant concerns about the use of azole antifungal drugs to treat invasive aspergillosis in the future.

Triazoles have been shown to have increased resistance due to an *Aspergillus* strain resistant to azoles after prolonged treatment in which mutations occur in the cyp51A codons [34]. Several mechanisms associated with cyp51A have been identified in patients on azole therapy [35]. Human-to-human transmission is therefore highly unlikely, and spread of resistance is very rare.

Soil is known to provide a natural habitat for a number of fungi that can be harmful including *Aspergillus*, *Coccidioides*, *Histoplasma*, and *Cryptococcus* [36]. Fungicides used repeatedly over a long period of time can create persistent selection pressure and lead to the development of resistant *Aspergillus* species. As a result, the environment contains *Aspergillus* species that are resistant to azoles. When susceptible individuals inhale these conidia, the *Aspergillus* species become resistant to the triazoles used for treatment. Several cases of triazole-resistant aspergillosis in humans and animals without prior triazole treatment have been reported worldwide [37, 38].

The mechanism of mutations induced in the environment in resistant *Aspergillus* comes from ergosterol (**Figure 2**), which is the component in a greater quantity of the cell membrane of fungi and is essential for the bioregulation of fluidity, asymmetry, and integrity of the cell membrane [10]. The cytochrome P450 enzyme, also called sterol-14α-demethylase, converts lanosterol to ergosterol. cyp51A is a gene that encodes the cytochrome P450 enzyme. The ergosterol biosynthetic pathway is the general target of azole antifungals. Triazoles prevent the cytochrome P450 enzyme from playing its role in converting lanosterol in the ergosterol biosynthetic pathway and cause ergosterol depletion and deleterious lanosterol accumulation [40]. Azole resistance is caused by mutations in the cyp51A gene that alter the cyp51A gene, protein structure, and reduce the enzymes' affinity for azole therapies [40].

Another factor associated with greater difficulty in achieving therapeutic success against aspergillosis is biofilm formation. Like biofilms on bacteria and yeast, *A. fumigatus* biofilms provide protection against antifungal and host immune defenses [41, 42].

Biofilms are formed by cells that adhere to abiotic and biotic surfaces and are surrounded by an extracellular matrix composed of polysaccharides. They act as a protective layer, aiding adhesion, surface integration, and cell propagation for subsequent invasion. This protection becomes less sensitive to treatments with antifungal drugs and attacks immune cells, making them more difficult to fight [43].

The *A. fumigatus* biofilm is highly resistant to all current classes of antifungal drugs, including azoles, echinocandins, and polyenes. The antifungal resistance associated with the *A. fumigatus* biofilm is thought to be a consequence of several interrelated factors, including elevated efflux pump activity, extracellular matrix production, and altered metabolic states [44].

#### **Figure 2.**

*Azole resistance mechanisms (a) wild-type fungi in the presence of azole drug unable to make ergosterol. (b) Mutations in the cyp51A region alter the structural modifications of the enzyme leading to reduce azole affinity. (c) Insertion of 34 and 46 bases pair in the promoter region along with point mutation in the cyp51A region causes overexpression of the gene. (d) Overexpression of efflux pump genes causes a reduced intracellular accumulation of azole drug [39].*

When we compare the number of new antimicrobials with bacterial action, it can be seen that the development of antifungals faces challenges, as fungi present cellular similarities to the host, both are eukaryotic, and substances that will be toxic to the pathogen should not cause harm to the patient. Thus, the reduced number of antifungals currently used target structures belonging only to fungi [45].

Future therapeutic options aim to circumvent the existing limitations of current antifungal agents, and investigations have been carried out looking for targets different from those currently on the market, namely at the level of ergosterol, 1,3-β-d-glucan, and DNA. Such new approaches are favorable insofar as the toxicities and interactions may not be evidenced, as well as the resistances verified with other antifungal classes. New targets under development must be unique in addition to allowing cell viability [46].

A new option is the biosynthesis of glycosylphosphatidylinositol (GPI) AX001 (Amplyx Pharmaceuticals, San Diego, CA, USA), which consists of a new agent that, from the inhibition of inositol acyltransferase, mediated by the conserved fungal enzyme Gwt1, prevents the maturation of proteins linked to the GPI. Allowing agents to adhere to mucous membranes and epithelial surfaces, biofilm formation, and hyphal growth are crucial for colonization/infection. The main advantage of this molecule is its action only in fungal cells, having no activity in the acylation of human cells [45].

Its spectrum of action is quite broad, which allows it to act against *Candida*, *Aspergillus*, *Fusarium*, and *Scedosoporium*. It has no activity against *C. krusei* and *Mucorales* but has demonstrated *in vivo* efficacy against *Candida* species resistant to echinocandins and azoles. This molecule is undergoing a phase 1 trial of oral and intravenous formulations, seeking to assess its safety and tolerability. It has also been designated an orphan drug and Qualified Infectious Disease Product (QIDP) by the FDA [47].

The agent F901318, which is part of the orotomid class, has the ability to inhibit an oxidoreductase enzyme, dihydroorotate dehydrogenase, which interferes with the biosynthesis of pyrimidine. The inhibition by olorofim may affect the fungal cell wall and result in cell lysis. An *in vitro* study reported that exposure of A. fumigatus hyphae to olorofim (0.1 μg/mL for 24 h) led to significant reductions in 1,3-β-dglucan at the hyphal tips and at the periphery of the mycelium [47].

Siderophores are iron chelators, so they manage to eliminate the available iron in various organisms such as plants, fungi, and bacteria. It is known that iron is essential for the viability of microorganisms, and if it is assimilated, it is no longer available for pathogens, constituting a good strategy. In this way, they manage to eliminate the iron present in the hosts that the agents may be infecting. VT-2397, formerly designated ASP2397, is isolated from Acremonium and permits aluminum chelation and was developed by Vical Pharmaceuticals (San Diego, CA, USA), demonstrating activity against azole-resistant *A. fumigatus* [48].

New molecules have also been developed based on the same targets: Azoles, with the elaboration of two molecules (VT-1161 and VT-1129) that are metalloenzymes and similar to azoles inhibit 14-α-demethylase, being directed to the treatment of infections by *Candida* and cryptococcal meningitis, respectively. What differentiates them from current azoles is the better selectivity, not binding to human CYP5, due to the fact that they have a tetrazole fraction in their structure, instead of triazole or imidazole, present in the agents available on the market [47].

Current echinocandins are only available in IV formulation. The SCY-078 molecule derived from enfumafungin has the advantage of being available in oral formulation, showing activity against several species of Candida and species resistant to fluconazole and in isolates with mutation at the level of the FKS1/FKS2 genes, which confer resistance to echinocandins. It demonstrated a spectrum of action comparable to commercial echinocandins, with emphasis on *C. glabrata* where it was eight times more effective. Recent studies have demonstrated activity against the new species *C. auris* [47, 48].

The CD101 molecule is also part of the echinocandins and has better solubility and less toxicity due to a modification at the choline level. It also demonstrates a much longer half-life and can be administered more widely – activity against *Candida* and *Aspergillus* species [47].

Regarding polyenes, the MAT2203 molecule (Matina BioPharma Holdings, Inc., USA) is a version of amphotericin B carried by nanoparticles that allows an oral formulation. In August 2015, it was approved by the FDA for the treatment of invasive candidiasis and aspergillosis. In phase 1 trial, it demonstrated a positive safety and tolerability profile [47].

#### **3. Conclusion**

Therefore, it was concluded that drugs for the treatment of fungal infections have undergone great advances in relation to pharmacodynamic properties, pharmacokinetics, spectrum of action, toxicity, and side effects. However, some factors contributed to an increase in the resistance profile and reduced therapeutic efficacy, and that is why researchers are developing new molecules based on the same targets as the available antifungal agents or new ones to circumvent clinical resistance. It is important to point out that the destination for antifungal effectiveness will not depend only on the synthesis of new drugs; it is also necessary to improve diagnoses, consider the benefits and harms of immunosuppressive therapies, and especially the choice of appropriate antifungal.

*Aspergilosis: Resistance and Future Impacts DOI: http://dx.doi.org/10.5772/intechopen.112755*

#### **Author details**

Amanda Junior Jorge Rua José Augusto Tourinho Dantas, Praia do Flamengo, Salvador, BA, Brazil

\*Address all correspondence to: amandajjorge@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 5
