Aspergilosis: Resistance and Future Impacts

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.

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].

Figure 1.

Infectious cycle of Aspergillus [2].

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].

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].

Antifungal class	Mechanism of action	Biological effect	Spectrum of action
Polyenes	Target ergosterol and extract sterols from fungal cell membranes	Fungicidal	Broad spectrum antifungal in treatment of invasive fungal infections; resistance is rare
Flucytosine	Inhibits DNA and RNA synthesis	Fungicidal against Cryptococcus spp.	Almost exclusively used for cryptococcal meningitis, but resistance is extremely common so never used in monotherapy
Azoles	Inhibit 14-α-lanosterol demethylase thereby inhibiting ergosterol synthesis	Mostly fungistatic	As a class they display broad spectrum against yeasts and filamentous fungi, although some species display intrinsic resistance to commonly used derivatives; secondary resistance can often develop during treatment
Echinocandins	Target 1,3-β-d-glucan synthase, thus preventing production of cell wall 1,3-β-d-glucan	Fungicidal against Candida spp., but fungistatic against Aspergillus spp.	First line of defense for candidiasis and used in aspergillosis when refractory to other treatments; resistance is emerging

Table 1.

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

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].

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].

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].

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].

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-d-glucan 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.