Section 1 Pathogenicity

#### **Chapter 1**

## Introductory Chapter: The Genus *Aspergillus* - Pathogenicity, Mycotoxin Production and Industrial Applications

*Mehdi Razzaghi-Abyaneh and Mahendra Rai*

#### **1. Introduction**

*Aspergillus* infections in humans were firstly reported in the eighteenth century [1, 2]. *Aspergillus* was first described in 1729 by Micheli, an Italian priest and biologist, who was the first person to attempt the scientific study of fungi [3]. *A. flavus* was named and reported by Link in 1809. John Hughes Bennett (1812–1875) was the first to describe aspergillosis. In his seminal paper published in 1842, he made the very first description of *Aspergillus* growing in the lung tissue of humans [4]. Paranasal sinus mycosis in 1893 and since then numerous cases have been reported from different parts of the world. In 1926, the genus *Aspergillus* was first classified and accepted in 69 *Aspergillus* species in 11 groups. By the year 1965, the previous classification of *Aspergillus* was declared outdated, and detailed 151 species in 18 different groups were introduced. Additional research led to further refined species designations with the use of new technologies such as thin-layer chromatography of secondary metabolites and DNA hybridization.

The genus *Aspergillus* consists of numerous species gathered in a diverse group with environmental and public health importance [5, 6]. The members of this genus are cosmopolitan fungi frequently found in various natural habitats especially in soil as the main reservoir and they are responsible for food spoilage, mycotoxin contamination, and various types of human and animal mycoses [7, 8]. Moreover, they are rich sources of beneficial metabolites such as antibiotics, organic acids, enzymes, and additives. At present, there are more than 300 species which are now accepted, and new species continue to be described and added to this list. The taxonomy of species within the *Aspergillus* genus is gradually undergoing emendation with the use of molecular methods and is not yet complete. Of the known *Aspergillus* species, only 20 have been confirmed to cause human infections and three of them are consistently and regularly encountered as etiological agents of over 95% of diseases caused by members of the genus including *A. fumigatus, A. niger,* and *A. flavus* [9]. The other species of this genus related to human lesions are *A. terreus*, *A. glaucus*, *A. nidulans*, *A. oryzae,* and *A. clavatus*. Mode of infection is the inhalation of airborne conidia, exposure to contaminated water (contact with conidia during showering), and nosocomial infections (hospital fabrics and plastics may serve as important sources of *Aspergillus* species). The incubation period is between 2 days and 3 months.

Aspergillosis is a common term used to describe infections caused by different species of *Aspergillus* [10]. Aspergillosis was described as a clinical human disease under the name of bronchopulmonary *Aspergillus.* The species *A. fumigatus*, with *A. flavus* and *A. niger* are responsible for more than 90% of aspergillosis worldwide. A wide array of clinical forms from allergic reactions (allergic bronchopulmonary aspergillosis, rhinitis, Farmer's lung) to superficial and cutaneous infections, localized aspergilloma, and invasive infections have been reported. Invasive life-threatening aspergillosis occurs mainly in immunocompromised individuals who have undergone widespread antibiotics, cancers, or autoimmune underlying disorders. Invasive infections initiate by entering air-borne conidia to lungs with clinical entities such as invasive sinusitis, fever, facial pain, headache, cough, and dyspnea with subsequent spread to the central nervous system (CNS), leading to seizures or death.

#### **2. Description**

In the current book which comprises five distinct chapters, different aspects of the genus *Aspergillus* from *Aspergillus*-host interactions to the immunopathogenesis of aspergillosis, mycotoxin production, and industrial applications of the beneficial species have gained special attention.

It has been shown that host immune status and previous underlying diseases act as important determinants of clinical outcomes and disease spectra of aspergillosis which is life-threatening in the invasive form where the etiologic fungus affects lung tissue and disseminates to different organs with high morbidity and mortality. The role of influenza and COVID-19 infections in ICU patients has been noticed as the new risk factors of invasive aspergillosis. In relation to the immunopathogenesis of aspergillosis, documents demonstrated that following entry of causative *Aspergillus* species, fungal elements are affected by pulmonary host defense in order to clearance of infective conidia. In conditions of poor host immune response, where the neutrophils and macrophages fail to recognize the etiologic fungus, *Aspergillus* conidia attack and destroy airway epithelium and neutrophils play an important role in the clearance of fungal hyphae via oxidative and non-oxidative mechanisms. As an amazing topic in mycotoxin research, the relationship between mycovirus-containing *Aspergillus flavus* and acute lymphoblastic leukemia as carcinogenesis beyond mycotoxin production has been noticed. The role of aflatoxin in *Aspergillus flavus* resistance to stress conditions is a very interesting subject in the importance of members of *Aspergillus* section *Flavi.* In this context, it has been shown that *Aspergillus* employs a considerable amount of energy to synthesize aflatoxins which are not so obviously linked to an enhancement of population fitness. Another important aspect of the genus *Aspergillus* is the industrial application of nanomaterials produced by *Aspergillus* species. These fungi produce a large number of beneficial metabolites enabling the producing fungus to the successful synthesis of nanoparticles.

In conclusion, we would like to thank all authors for their invaluable contribution and hard work to make the successful endeavor on the goals of the present book. We are also grateful to the "In-Tech" Publisher personnel, especially Ms. Karmen Ðaleta, who kindly assisted us in the arrangement of the book and scheduling our activities.

*Introductory Chapter: The Genus* Aspergillus *- Pathogenicity, Mycotoxin Production… DOI: http://dx.doi.org/10.5772/intechopen.105200*

## **Author details**

Mehdi Razzaghi-Abyaneh1 \* and Mahendra Rai2

1 Department of Mycology, Pasteur Institute of Iran, Tehran, Iran

2 Biotechnology Department, SGB Amravati University, Amravati, Maharashtra, India

\*Address all correspondence to: mrab442@yahoo.com and mrab442@pasteur.ac.ir

© 2022 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.

#### **References**

[1] Lee J. Discovery of *Aspergillus* as a Human Pathogen. http://www. antimicrobe.org/hisphoto/history/ Aspergillus-Human%20Pathogens.asp

[2] Barnes EA. Short History of Invasive Aspergillosis, 1920 to 1965. The *Aspergillus* Website. (http://www. aspergillus.org.uk)

[3] Gover DW. Pier Antonio Micheli. https://www.aspergillus.org.uk/ image\_library/pier-antoniomicheli/?sfw=pass1651379036

[4] Bennett JH. On the parasitic vegetable structures found growing in living animals. Transact Royal Society of Edinburgh. 1842;**15**:277-294

[5] Sabino R. *Aspergillus* and health. Microorganisms. 2022;**10**(3):538

[6] Razzaghi-Abyaneh M, Chen Z-Y, Shams-Ghahfarokhi M, Rai M. Research efforts, challenges, and opportunities in mitigating aflatoxins in food and agricultural crops and its Global Health impacts. Frontiers in Microbiology. 2022;**13**:881858

[7] Razzaghi-Abyaneh M. Aflatoxins: Recent Advances and Future Prospects. Croatia: InTech Open; 2013

[8] Jamali M, Karimipour M, Shams-Ghahfarokhi M, Amani A, Razzaghi-Abyaneh M. Expression of aflatoxin genes aflO (omtB) and aflQ (ordA) differentiates levels of aflatoxin production by *aspergillus flavus* strains from soils of pistachio orchards. Research in Microbiology. 2013;**164**(4):293-299

[9] Sabino R, Veríssimo C. Novel clinical and laboratorial challenges in aspergillosis. Microorganisms. 2022;**10**(2):259

[10] Razzaghi-Abyaneh M, Shams-Ghahfarokhi M, Rai M. Medical Mycology: Current Trends and Future Prospects. Boca Raton, USA: CRC Press; 2015

#### **Chapter 2**

## *Aspergillus-*Human Interactions: From the Environment to Clinical Significance

*Arsa Thammahong*

#### **Abstract**

*Aspergillus* species are ubiquitous fungi found in the environment worldwide. The most common *Aspergillus* species causing diseases in humans are *A. fumigatus*, *A. flavus*, *A. niger*, and *A. terreus.* However, species causing human infections are also depending on human immune status. Host immune status and previous underlying diseases are important factors leading to different clinical manifestations and different disease spectra of *Aspergillus* infections. The most severe form of *Aspergillus* infections is invasive aspergillosis in human tissue, especially invasive pulmonary aspergillosis (IPA), which has high morbidity and mortality in immunocompromised patients. ICU patients with influenza infections and COVID-19 infections are recently risk factors of invasive pulmonary aspergillosis. New diagnostic criteria include galactomannan antigen assays, nucleic acid amplification assays, and lateral flow assays for early and accurate diagnosis. Voriconazole and the newest azole, isavuconazole, are antifungals of choice in IPA. Nevertheless, azoleresistant *Aspergillus* strains are increasing throughout the world. The etiology and spreading of azole-resistant *Aspergillus* strains may originate from the widespread use of fungicides in agriculture, leading to the selective pressure of azole-resistant strains. Therefore, there is a necessity to screen *Aspergillus* antifungal susceptibility patterns for choosing an appropriate antifungal agent to treat these invasive infections. In addition, mutations in an ergosterol-producing enzyme, i.e., lanosterol 14-α demethylase, could lead to azole-resistant strains. As a result, the detection of these mutations would predict the resistance to azole agents. Although many novel azole agents have been developed for invasive *Aspergillus* infections, the rate of novel antifungal discovery is still limited. Therefore, better diagnostic criteria and extensive antifungal resistant *Aspergillus* screening would guide us to better manage invasive *Aspergillus* infections with our existing limited resources.

**Keywords:** *Aspergillus*, *Aspergillus-*human interactions, invasive aspergillosis, antifungal susceptibility test, azole, voriconazole, amphotericin B, influenza-associated pulmonary aspergillosis, COVID-19-associated pulmonary aspergillosis

#### **1. Introduction**

*Aspergillus* species are saprophytic ubiquitous filamentous fungi [1]. They are in Phylum Ascomycota with both sexual and asexual forms [1]. In their sexual form, they produce asci and ascospores within the appropriate environment, while they produce conidia, or asexual spores, on phialides surrounding their

vesicles at the tip of conidiophores in their asexual form [1]. *Aspergillus* conidia are different in size and shape depending on *Aspergillus* species, which affects the dispersion and infectivity properties of *Aspergillus* [1]. Their conidia can be found in the soil, decomposed piles, air, animals, and humans. They cause diseases in immunocompromised hosts, e.g., patients with acquired immunodeficiency syndrome (AIDS), allogenic hematopoietic stem cell transplant or solid organ transplant candidates, patients with immunosuppressive drugs, patients with prolonged neutropenia, and patients with other underlying diseases [2]. The common pathogenic *Aspergillus* species are *A. fumigatus, A. flavus, A. niger,* and *A. terreus* [3]. There are a wide variety of disease spectra of *Aspergillus* infections, i.e., invasive aspergillosis, chronic aspergillosis, and allergic forms of aspergillosis [1, 2]. The most severe form causing high morbidity and mortality rate, especially in immunocompromised hosts, is invasive aspergillosis (IA) [2, 4]. An increase of immunocompromised hosts would also increase patients with IA with a high mortality rate [4–14].

Invasive aspergillosis (IA) is recently increasing in patients with allogenic hematopoietic stem cell transplantation (HSCT) and solid organ transplantation [5, 8, 13, 15–22]. Underlying conditions of patients with IA are hematological malignancies, e.g., leukemia or lymphoma, bone marrow transplant, and solid-organ transplant patients [5, 8, 13, 15–22]. Recently, not only neutropenic patients are at risk for IA, but non-neutropenic patients with immunosuppressive agents, e.g., biologics, small-molecule kinase inhibitors (SMKIs), Chimeric Antigen Receptor (CAR) T cells, are also at risk [23–28]. In developing countries, poor-controlled diabetes mellitus is one of the critical risk factors of IA [10, 12]. Therefore, risk factors of IA are now patients with malignancy, autoimmune, inflammatory diseases, complex immune-metabolic diseases from aging, immunosuppressive treatment, previous septic conditions, novel biologic treatment, including patients with hematological malignancies receiving SMKIs, patients in ICU, patients with a cytokine storm syndrome from CAR-T cells treated with high-dose corticosteroids, patients in ICU with severe influenza or other viral infections [23–36]. In an era of Coronavirus Disease 2019 (COVID-19) infections, IA was recognized as a severe complication of patients with COVID-19 infections in ICU [37–46].

#### **2. Pathogenesis of** *Aspergillus* **and its virulence factors**

Among thousands of *Aspergillus* species, only less than twenty species could cause diseases in humans [47]. The pathogenic species usually possess virulence factors that help them survive and cause infections inside hosts. *Aspergillus fumigatus* was utilized as a model to study virulence factors in many studies (**Table 1**) [1].

To survive inside the host environment, *Aspergillus* species need to adapt to heat and hypoxic conditions inside hosts. For the heat stress, the trehalose pathway was shown to have a role in heat tolerance and virulence of *A. fumigatus* [47]. Heat shock proteins (HSPs), especially Hsp90, are chaperone proteins associated with stress tolerance, not only for heat [48–50]. In mammalians, HIF1α, as a common transcription factor, controls cellular homeostasis in hypoxic conditions [51]. In fungi, a homolog of HIF1α, called the sterol regulatory element-binding protein (SREBP) or SrbA in *A. fumigatus,* is induced by hypoxia and iron starvation conditions [52–56]. SrbA protein is also associated with the virulence of *A. fumigatus in vivo* [52–54].

*A. fumigatus* possesses enzymes to protect itself against host reactive oxygen species (ROS), e.g., catalase, superoxide dismutases, thioredoxin, glutathione, including mitochondrial electron transport chain [57–62]. In some animal

Aspergillus-*Human Interactions: From the Environment to Clinical Significance DOI: http://dx.doi.org/10.5772/intechopen.98509*


#### **Table 1.**

*Essential virulence factors in Aspergillus fumigatus requiring for causing infections inside humans [1].*

models, e.g., an eye infection model, demonstrated that these fungal enzymes were essential for fungal virulence [63]. Secondary metabolites are also playing a role in fungal virulence [64–66]. *A. fumigatus* secondary metabolites are gliotoxin, fumigaclavine, trypacidin, helvolic acid, fumitremorgin, fumagillin, and pseurotin, associated with host cellular toxicity [67–71]. However, the mechanisms behind this toxicity is still unclear and need to be further investigated *in vivo* [71]. *A. flavus* produces aflatoxins, which are important carcinogenic secondary metabolites, and other secondary metabolites, called Velvet complex, as environmental response mechanisms [72, 73]. Circadian rhythms or light response, which were studied thoroughly in the *Neurospora* model system, are essential to react with the environment [74]. Light-induced mycelial pigmentation and germination acted as a stress signaling pathway in *A. fumigatus* via transcription factor LreA and FphA, respectively [75–77].

For nutrient acquisition, exoenzymes or proteases are major enzymes produced by *A. fumigatus,* especially the alkaline protease Alp1 and the metalloprotease Mep1 [1, 78]. In *A. fumigatus,* a transcriptional repressor called CreA has a vital role in carbon catabolite repression. *Af*CreA regulates growth on different nitrogen, carbon, and lipid sources and has a role in amino acid transportation, nitrogen, and carbon assimilation, including glycogen and trehalose metabolism [79, 80]. Although CreA is not required for virulence, it is required for disease progression in invasive pulmonary aspergillosis (IPA) mouse models [79–81]. For nitrogen utilization, *Af*RhbA, a Ras-related protein in a nitrogen-regulated signaling pathway, and *Af*AreA, a GATA transcription factor requiring the expression of genes involving nitrogen utilization, are related to virulence in *A. fumigatus* [82–84]*. A. fumigatus* still needs divalent cations, i.e., iron, copper, magnesium, zinc, calcium, for its growth and virulence inside hosts via siderophores, calmodulin, calcineurin, specific importers, and exporters [85, 86].

Additionally, cell wall components of *Aspergillus fumigatus* are also essential virulence factors for fungal survival inside hosts and are important for host immune response [87–92]. Cell wall components consist of β-1,3-glucan, chitin, galactomannan, α-1,3-glucan, and melanin depending on different stages of *A. fumigatus*, i.e., conidial, or hyphal stage [91–95]. β-1,3-glucan, a central component of *Aspergillus* cell wall polysaccharide, is a pathogen-associated molecular

pattern (PAMP) recognized by host pattern recognition receptors (PRR), e.g., dectin-1 [88]. During its conidial stage, rodlet, or hydrophobins, and dihydroxynapthalene (DHN) melanin are present to protect fungal conidia against host immune response by evading host pathogen-associated molecular patterns (PAMPs) recognition, including protecting fungi from unfavorable stress conditions [93–97]. Furthermore, in its hyphal stage, galactosaminogalactan (GAG), which is a water-insoluble polymer consisting of a pyranose-form galactose, galactosamine, and N-acetylgalactosamine (GalNAc), is present as an extracellular matrix on an outer layer of the cell wall [98]. GAG is associated with biofilm formation and immunosuppression properties by masking PAMP exposure and resisting neutrophil killing via neutrophil extracellular traps (NETs) [99–102]. The linkage between cell wall components and metabolic pathways is still unclear. Nevertheless, these components share the same building blocks, e.g., UDP-glucose, glucose 6-phosphate, with specific metabolic pathways, e.g., glycolysis, trehalose biosynthesis pathway [81, 103–105]. It is possible that the homeostasis of cell wall biosynthesis is involved with some metabolic pathways, e.g., the trehalose biosynthesis pathway. Disruption of one of these trehalose enzymes or building blocks would result in decreased virulence due to changes in cell wall compositions [81, 103–105]. Understanding this homeostasis would lead to the discovery of novel antifungal targets in the future.

#### **3. Diagnosis of invasive** *Aspergillus* **infections: challenge in the field**

*Aspergillus* infections are associated closely with host immune status [106, 107]. Severe asthma with fungal sensitization and allergic bronchial pulmonary aspergillosis (ABPA) are found in immunocompetent hosts with hypersensitivity, while aspergilloma and chronic pulmonary aspergillosis are found in immunocompetent hosts with previous structural diseases, such as lung cavity from previous tuberculosis infections [108]. In immunocompromised hosts, invasive aspergillosis is common and severe, causing high morbidity and mortality in patients [108, 109].

For invasive pulmonary aspergillosis, early diagnosis and prompt treatment are the keys to decrease the disease burden. Differentiation between *Aspergillus* colonization and invasive infections is still challenging [25, 92, 93]. Recently, the revised EORTC guideline for diagnosis of invasive fungal infections, including *Aspergillus* infections, recommended the diagnostic criteria including host factors, clinical, radiological, and microbiological criteria with new diagnostic methods (**Table 2**) [109]. Proven invasive aspergillosis is confirmed with histopathologic, cytopathologic, microscopic analysis, or nucleic acid analysis of sterile specimens or tissue or formalin-fixed paraffin-embedded tissue (FFPE), including culture recovered from sterile sites [109]. Species of common *Aspergillus* recovered from cultures are differentiated using macroscopic and microscopic morphology, but the nucleic acid analysis is necessary for the species complex (**Table 3**) [110]. For probable and possible invasive aspergillosis, host factors, clinical features, and mycological evidence are including for the diagnosis of invasive aspergillosis. Host factors include the history of neutropenia, which is less than 500 neutrophils/mm3 , for more than ten days, hematological malignancy, allogenic stem cell transplantation, solid organ transplantation, therapeutic-dose corticosteroids at not less than 0.3 mg/kg for not less than three weeks during the previous 60 days, treatment with T-cell or B-cell immunosuppressants, inherited immunodeficiency, or acute graft-versus-host disease grade III or IV [109]. For clinical evidence of pulmonary aspergillosis, a chest high-resolution CT scan is recommended to observe any halo


#### **Table 2.**

*Diagnosis of invasive aspergillus infections from revised EORTC/MSG criteria 2020 (BAL: bronchoalveolar lavage; CT: computed tomography; CSF: cerebrospinal fluid; GVHD: graft versus host disease; PCR: polymerase chain reaction) [109].*

sign, air-crescent sign, cavity, or wedge-shaped and segmental or lobar consolidation [109, 111]. Probable invasive aspergillosis still needs at least one mycological evidence to support the diagnosis. Mycological evidence is including cultures recovered from sputum, bronchoalveolar lavage (BAL), bronchial brush, or


#### **Table 3.**

*Macroscopic and microscopic features of clinical-relevant Aspergillus species (colony on Czapek Dox agar at 30°C) [110].*

aspirate [109]. *Aspergillus* galactomannan antigen assays with different thresholds depending on specimens, including serum, BAL fluid, plasma, and cerebrospinal fluid (CSF), support the diagnosis of invasive aspergillosis [112–115]. However, decreased sensitivity of galactomannan antigen assay is observed in patients with anti-mold therapy [115]. In addition, *Aspergillus* PCR from blood and BAL fluid is introduced to confirm the diagnosis and identify specific *Aspergillus* species with certain mutations related to triazole resistance [109, 116–124].

Nonetheless, revised EORTC/MSG criteria for diagnosing invasive fungal infections may be applied mainly for neutropenic patients or immunocompromised patients. Therefore, specific guidelines for the diagnosis of invasive aspergillosis in non-neutropenic patients in ICU (Invasive pulmonary aspergillosis in ICU, AspICU) or patients with influenza (Influenza-associated pulmonary aspergillosis, IAPA) or Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) (COVID-19 associated pulmonary aspergillosis, CAPA) co-infections were developed and published for early and accurate diagnosis (**Table 4**) [31, 125–127].

#### **4. Treatment of** *Aspergillus* **infections**

IA also includes the infections of the lower respiratory system, sinuses, and skin as entry routes. In addition, the cardiovascular system, central nervous system, and other tissues could be infected from hematogenous dissemination or direct extension from adjacent infected tissues [2]. Infectious Diseases Society of America (IDSA, 2016) and ESCMID-ECMM-ERS (2017) recommended voriconazole (6 mg/ kg, intravenous route every 12 hours for one day, and then 4 mg/kg every 12 hours; 200–300 mg every 12-hour, oral route) as a first-line treatment for invasive pulmonary aspergillosis (IPA) [2, 128]. For alternative treatment, liposomal amphotericin B (3–5 mg/kg/day, intravenous route) and isavuconazole (200 mg every 8 hours for three days and then 200 mg daily) [2]. For other invasive aspergillosis syndromes, i.e., invasive sinus aspergillosis, tracheobronchial aspergillosis, invasive aspergillosis of the central nervous system or cardiovascular system, *Aspergillus* osteomyelitis,


#### Aspergillus-*Human Interactions: From the Environment to Clinical Significance DOI: http://dx.doi.org/10.5772/intechopen.98509*


