3

Fungal Diseases, Antifungal Resistance, and Human Health

The second panel of the workshop focused on fungal disease in humans, the rise of antifungal resistance, and implications and challenges related to effectively treating these infections. The panel was moderated by Tom Chiller, chief of the Mycotic Diseases Branch at the Centers for Disease Control and Prevention. Andrej Spec, associate professor, associate director of the Infectious Disease Clinical Research Unit, and medical director of the Invasive Mycoses Clinic at Washington University in St. Louis, described how fungal infections are contracted, their impact on human health, and current treatment options and challenges. David Denning, chief executive of Global Action for Fungal Infections and professor of Infectious Diseases in Global Health at the University of Manchester, United Kingdom, discussed invasive, chronic, allergic, and superficial aspergillosis—including barriers to accurate diagnosis and effective treatment—and provided an overview of increasing azole resistance. Brendan Jackson, medical epidemiologist and lead of the epidemiology team of the Mycotic Diseases Branch at the Centers for Disease Control and Prevention (CDC), discussed the prevalence and ramifications of increasing Candida resistance to antifungals. He described the emergence and characteristics of Candida auris (C. auris) and presented hypotheses of why resistance is increasing. Michail Lionakis, chief of the Fungal Pathogenesis Section and of the Laboratory of Clinical Immunology and Microbiology at the National Institute of Allergy and Infectious Diseases (NIAID), presented examples of how research on the host-fungal interaction is leading to the development of prophylactic and immune-based treatments.

IMPACT OF INVASIVE FUNGAL DISEASES AND ANTIFUNGAL DRUG RESISTANCE ON HUMAN HEALTH

Spec provided an overview of the medical classification of fungi and how fungal infections are contracted. He also discussed the global burden and mortality rates of fungal infections, current treatment options, and challenges related to resistance and drug development. He noted that the general public tends to associate fungi, in a medical context, with relatively benign issues such as toenail infections, despite the potential for fungal infections to cause life-threatening conditions.

Fungal Morphology and Medical Classification

Human infections can be classified according to their causative agents into the categories of bacteria, viruses, fungi, protozoa, and helminths; the latter two are often grouped under the term “parasites.” Fungi are medically classified into three groups: (1) yeast, (2) thermally monomorphic molds, and (3) thermally dimorphic molds. Yeast cells are round and similar in shape to bacteria, although they are larger (Spec et al., 2019). Hyphae form mycelia in the soil. Pseudohyphae are present in some species of fungi and share some characteristics of both yeast and hyphae. Some species create both yeast and hyphae, depending on the temperature. Yeasts are broad and varied. For example, Candida and Cryptococcus are both classified as yeasts, yet they are so widely diverged that Cryptococcus is more closely related to mushrooms than to Candida. Thermally monomorphic molds can be classified as zygomycetes, dematiaceous, dermatophytes, and hyaline hyphomycetes. Spec added that the classification of thermally monomorphic molds is largely driven by appearance under the microscope and in the patient (Spec et al., 2019).

How Fungal Infections Spread

Spec explained that fungi can be commensal or environmental. Commensals—including Candida and dermatophytes—live on humans all of the time, while all other groups of fungi come from the environment. Infections behave differently depending on whether they are commensal or environmental, thus management strategies vary dramatically. Commensals are part of the human microbiome and are common contaminants in culture. Often seen in overgrowth syndromes, commensals cause disease by overgrowing or growing in locations that become irritated. Candida commonly causes relatively innocuous and non-life-threatening diseases such as heat rash, tinea (commonly known as ringworm), vulvovaginal candidiasis, and thrush. However, Candida can be lethal if it enters the blood stream.

Environmental fungi spend the majority of their life cycle in the environment, such as in soil or trees, said Spec. Fungi form spores, or conidia, that are released into the air and can be inhaled by humans.¹ These types of infections often start with pneumonia, which may be at a subclinical level that presents as a long-lasting cold. While the typical cold lasts 3–5 days, a fungal infection may last 10–15 days or longer. The infection may or may not disseminate from the lungs to the rest of the body, with dissemination location and frequency varying widely. Some fungal infections—particularly with Cryptococcus and Coccidioides—are prone to causing meningitis. Some fungi are more prone to causing bone or skin disease and others can cause visceral disease. Histoplasma is prone to causing disease in the liver, spleen, intestines, and other organs. Often, the method of a fungus’s dissemination is highly dependent on the individual’s underlying immune system, yet fungi can travel to virtually anywhere in the body. Although the vast majority of human fungal infections are contracted through the air, rare occurrences of direct inoculation can occur when an object is driven below the skin. Spec remarked that some of the most horrific diseases he has managed were in patients with otherwise typical immune systems who had fungi forced underneath their skin through trauma. Additionally, more people are now being treated with immunosuppressant therapies for chronic or potentially fatal conditions—such as cancers—thus there is a larger pool of people at risk of contracting serious fungal infections.

Global Burden and Mortality Rates of Fungal Infections

Spec remarked that fungi receive relatively little public attention relative to bacteria and viruses, despite the likelihood that fungal infections will become more prominent during this century. Currently, fungal infections are responsible for 1.5 million annual deaths worldwide (Hagan, 2018). Many types of fungal infections are on the rise, such as those caused by Aspergillus, which experienced an annual increase in incidence of approximately 4.4 percent in France from 2001–2010 (Bitar et al., 2014). In addition, fungal infections generate significant economic cost. For instance, direct health care costs related to fungal infections in the United States totaled $7.2 billion in 2017. This figure increases to $11.5 billion when accounting for productivity and life, and to more than $48 billion if assessed using the value of statistical life rate (Benedict et al., 2022b).

Spec noted that the high cost of fungal infections, when assessed in terms of the value of statistical life, is largely due to the high mortality rates associated with those infections. For example, although many people do

___________________

¹ One example of how fungal infections disseminate in the body is available at https://www.cdc.gov/fungal/diseases/blastomycosis/causes.html (accessed July 26, 2022).

not associate Candida with invasive disease, the mortality rate of Candida bloodstream infections is approximately 43 percent (Mazi et al., 2022). The attributable mortality rate of these infections is between 10 and 25 percent. The frequency of Candida in hospitals is approximately half that of Staphylococcus aureus, but Candida’s mortality rate is almost double that of S. aureus (approximately 20–25 percent). Spec added that the attributable mortality for these two diseases is the same for many subgroups of the patient population. However, far fewer resources are dedicated to researching and addressing Candida compared to S. aureus, despite the former’s potential to cause fatal infection.

Infections caused by other species of fungi are also associated with high mortality rates, Spec emphasized. For instance, Cryptococcus has been estimated to have a mortality rate of approximately 15 percent in patients living with HIV or transplants (Hevey et al., 2019). This rate increases to almost 40 percent in people without HIV or transplants. He explained that infections in non-HIV, non-transplant patients are often diagnosed late and treated imperfectly. In sub-Saharan Africa—where access to drugs and supportive care is lower—the mortality of Cryptococcus increases to 40–60 percent in individuals with HIV (Tenforde et al., 2020). Spec surmised that the mortality rate of Cryptococcus is even higher among individuals who are not living with HIV.

Spec noted a large spike in the incidence of mucormycosis—a disease caused by the Mucorales fungi—during the 2021 wave of the delta variant of COVID-19 in India (Rao et al., 2021). This disease is present independent of COVID-19 and carries a mortality rate of close to 100 percent without surgical intervention. Surgeries for mucormycosis are often highly invasive, and mortality remains at around 70 percent at 1 year even with surgical care and treatment with the best available antifungals. Many survivors of the disease are left permanently disfigured, Spec added.

Fungal Infection Treatment and Resistance

Spec described how fungal infections are treated and the growing threat of treatment-resistant infections. Antifungal treatments can be classified into (1) polyenes, (2) azoles, and (3) echinocandins (Stevens, 2011). The polyenes currently include only one systemic medication, amphotericin B. This single medication has multiple formulations and carries a broad spectrum of activity and high potency. Only available as an intravenous therapy for invasive infection, amphotericin B is an extremely antifungal agent, stated Spec. Unfortunately, the drug is also highly toxic, and virtually every person who takes amphotericin B develops renal toxicity. Electrolyte wasting is also common; the loss of potassium and magnesium through urine can lead to arrythmias and death. Infusion reactions can include

severe muscle cramps, pain, and rigors, because the drug can begin to damage human muscle cells in the same way it damages fungi. Azoles, the most diverse class of antifungals, include drugs that have varying levels of broadness of activity. They are the only antifungals that can be taken orally, but treatment with azoles can be complicated. Because many of these drugs are not easily absorbed, it can be difficult to achieve and maintain effective therapeutic level in patients and may require continuous therapeutic drug monitoring. Additionally, azoles can cause challenging drug interactions, and many azoles have idiosyncratic side effects such as sunburns, high blood pressure, and even heart failure. They can also cause liver toxicity and they have a narrow therapeutic index. The newest class of antifungals, echinocandins, were developed approximately 20 years ago and are often referred to as the “good antifungal,” because they are generally well tolerated and have few side effects. However, echinocandins must be administered intravenously and have a relatively narrow spectrum of activity that does not include many fungi.

Resistance generates further challenges in fungal treatment, Spec stated. The oral treatment options for several fungi—including Candida krusei, Candida glabrata, and azole-resistant Aspergillus—are poor or nonexistent. Moreover, some fungi have intrinsic resistance to all antifungals. These include Scapulariopsis brumptii, Lomentospora prolificans, Scedosporium apiospermum, Pseudoallescheria boydii, Candida auris, and Fusarium isolates, especially Fusarium solani. Spec commented on the difficulty of seeing immunocompetent people experiencing horrible fungal infections for which no treatment options exist. Worryingly, antifungal resistance is increasing worldwide. Noting the central role echinocandins have played in addressing Candida infections, Spec emphasized that in one study, C. glabrata resistance to echinocandins increased from 4.9 percent in 2001 to 12.3 percent in 2010 (Alexander et al., 2013). Furthermore, new emergence of invasive infections due to fluconazole resistance in Candida parapsislosis has taken place in locations throughout the world (Souza et al., 2015). Spec highlighted the explosive growth of C. auris since its emergence in 2004. Over a short period of time, it has emerged in various communities—including within the United States—and now constitutes a significant portion of new cases of fungal infections in some locations (Rhodes and Fisher, 2019). A major concern is that, of the known isolates from this fungal species, 90 percent are resistant to fluconazole, 30 percent are resistant to amphotericin B, 10 percent are resistant to echinocandins, and 4 percent are resistant to all antifungals (CDC, 2020). Citing a worldwide report of significant presence of azole-resistant Aspergillus, Spec remarked that azole-resistant fungus is likely present in most places in the world (Lestrade et al., 2019b), even in settings where it has not yet been identified due to insufficient epidemiology work and analysis.

Challenges in Antifungal Development

Spec emphasized that animal cells and fungal cells are closely related; thus, many antifungals are also “antihuman.” Many of the antifungal drugs currently used in medical treatment are effective at killing fungi, but their therapeutic index is low. Spec noted the difficulty in identifying compounds that will kill fungus without the same deleterious effects on human cells. While right now there are more potential antifungal drugs in the development pipeline than at any point in the past 20 years, some of these drugs belong to the same classes and others have already had their clinical trials terminated due to lack of efficacy (Rauseo et al., 2020). Although there is reason for optimism that better antifungal compounds will ultimately be available, these drugs are harder to develop than antibacterials, noted Spec. Therefore, it is important to protect current drugs against resistance, because outcomes tend to be much worse without therapy.

GLOBAL INCIDENCE AND PREVALENCE OF ASPERGILLOSIS AND INTRODUCTION TO AZOLE RESISTANCE

Denning described four groups of aspergillosis cases: invasive, chronic, allergic, and superficial infections. He reviewed the subpopulations most affected by aspergillosis, the scope of the disease, challenges in correctly diagnosing and effectively treating Aspergillus infections, and the rise of azole resistance.

Invasive Aspergillosis

Invasive aspergillosis is a life-threatening infection that can kill a person within 7 to 20 days. Difficult to diagnose, this disease poses a particular threat to people who are immunocompromised. People living with leukemia, transplantation, late-stage HIV, immunologic disorders, chronic obstructive pulmonary disease (COPD), and inherited immunodeficiencies are at a higher risk of developing invasive aspergillosis. Furthermore, complex hospital patients, including those in intensive care units (ICUs)—particularly those in ICUs with renal dysfunction, respiratory failure, or chronic or temporarily compromised immune systems—or individuals hospitalized due to severe influenza or COVID-19 are more prone to aspergillosis. Denning noted that of these risk pools, patients with kidney transplants and stem cell transplants are at a relatively low risk (Herbrecht et al., 2012). People with heart transplants, late-stage HIV, and various leukemias are at intermediate risk. Patients with liver, lung, heart-lung, small bowel, or allogeneic stem cell transplants are among those at highest risk for aspergillosis. Denning added that aspergillosis is sometimes mistaken for a lung cancer relapse.

COPD is a disease most often related to smoking, but it can also be caused by smoke generated by indoor cooking (particularly affecting women), by occupational lung disease, and as a post-tuberculosis effect, said Denning. A southern Chinese study of approximately 300 hospitalized COPD patients found that Aspergillus spp. was present in nearly 20 percent of patients and probable invasive aspergillosis was found in 3.9 percent of patients (Xu et al., 2012). Although steroids are a risk factor for aspergillosis, only 13 percent of those who developed the disease were taking corticosteroids, he noted. In that study, 43 percent of the COPD patients with aspergillosis died (Xu et al., 2012). At the global level, the number of people with COPD worldwide is estimated at 550 million (Hammond et al., 2020). Approximately 10 percent of these individuals require hospitalization each year, with the mortality rate of hospitalized COPD patients ranging from 5 to 12 percent. Denning stated that the lower estimate of 1.3 percent of COPD patients developing invasive aspergillosis translates to 760,000 cases per year. The higher rate of 3.9 percent found in the Chinese study puts the number of invasive aspergillosis in COPD cases at approximately 2.25 million worldwide (Hammond et al., 2020).

Chronic Pulmonary Aspergillosis

The second group of aspergillosis cases is patients with chronic disease, primarily disease affecting the lungs and occasionally the sinuses, said Denning. People with lung disorders including COPD, sarcoidosis with asthma, and prior lung disease can develop aspergillosis as a complication. Aspergillosis can also set in after a person is cured of tuberculosis (TB). Chronic pulmonary aspergillosis (CPA) can be confused with TB or TB-like nontuberculous mycobacterial (NTM) infections because of similar clinical and X-ray presentations. Denning outlined three CPA challenges related to TB: (1) CPA can be present and the initial diagnosis of TB is incorrect, (2) CPA can occur as a co-infection with TB and NTM infections, and the CPA may go untreated if not fully investigated, and (3) CPA can follow TB as a sequela. In such cases, patients may be retreated with anti-TB therapy, which is unnecessary and ineffective in treating CPA. In research Denning carried out in Vietnam, he and his colleagues found that 54 percent of post-TB patients returning for care had CPA (Nguyen et al., 2021). Research in New Delhi found similar results, with 57 percent of people diagnosed with TB having CPA (Singla et al., 2021). Recent research conducted by Denning in Ghana also indicated CPA in approximately half of patients diagnosed with TB. He emphasized the similarity in how TB and CPA present on chest radiographs, with both diseases involving the upper lobes of the lungs. Severe cavitation can occur with TB and multiple cavities can present in patients with CPA. Coccidioides and Histoplasma can

also cause similar lung symptoms, although they are less common than Aspergillus infections.

Modeling of CPA and TB in India suggests that approximately 2.5 million patients present with new TB symptoms in India annually, resulting in about 500,000 deaths. However, in 2020, only about 54 percent of those cases were confirmed bacteriologically. The remainder were diagnosed based on chest radiographs and symptoms. Modeling these numbers based on recently published data, Denning predicted that approximately 200,000 of the 2.5 million new TB cases—about 10 percent—will actually be CPA misdiagnosed as TB. An additional 150,000 patients will develop aspergillosis during or immediately after TB therapy. An additional 250,000 individuals are expected to develop CPA within 2–5 years after TB therapy. He posited that nearly one-third of the 500,000 annual deaths attributed to TB in India may actually be caused by CPA (Global Action for Fungal Infections, 2022).

Allergic Aspergillosis

A third category of people prone to aspergillosis are those with fungal allergies, said Denning. Allergic bronchopulmonary aspergillosis is a severe allergy to Aspergillus that usually occurs in people with poorly controlled asthma and can cause mucus plugs in the airways. Other people are allergic to Aspergillus and molds, such as Cladosporium and Alternaria, and can develop severe asthma as a result of the fungal allergy. He noted that these groups of patients generally derive benefit from antifungal treatment, including azoles such as itraconazole and voriconazole. Allergic fungal rhinosinusitis—which causes nasal polyps and congestion—is usually not treated with antifungals. Allergic aspergillosis is more common than other types but is far less severe.

The global burden of invasive aspergillosis is estimated at approximately 850,000, compared to 1.5 to 3 million for chronic aspergillosis and 6 to 20 million for allergic aspergillosis, said Denning. The mortality rate of invasive aspergillosis is 100 percent without treatment and decreases to 30 to 85 percent with treatment. Chronic aspergillosis carries a mortality rate of approximately 75 percent without treatment and 45 percent with treatment. The mortality rate for both treated and untreated allergic aspergillosis is less than 1 percent, although some asthmatic deaths may be linked to this condition, Denning noted (Bongomin et al., 2017).

Superficial Aspergillosis

Keratitis, onychomycosis, and otitis externa are all forms of superficial aspergillosis, said Denning. Fungal keratitis is an infection of the

front of the eye. Recent modeling indicates that approximately 40 percent of cases are caused by Aspergillus and about 50 percent are due to Fusarium (Brown et al., 2021). The annual incidence of fungal keratitis is between 1 and 1.4 million people globally. More often than not, the infection causes blindness in the infected eye, and in approximately 10 percent of cases the eye must be removed or it perforates. Natamycin is the most effective treatment for this infection, which can also be treated using azole eyedrops. Onychomycosis is a fungal infection of the fingernails or toenails. The majority of the 300 million global cases of onychomycosis are due to dermatophytes, but up to 3 percent of cases are attributable to Aspergillus (Bongomin et al., 2018). This translates to approximately 10 million cases of Aspergillus-related onychomycosis, many of which are comorbid with diabetes. Acute otitis externa—commonly referred to as “swimmer’s ear”—affects 1 in 250 people annually. Chronic otitis affects 3-5 percent of the global population, totaling 200-350 million individuals. Approximately 10 percent of otitis cases are fungal in origin, most often caused by Aspergillus spp. (Wiegand et al., 2019).

Resistance in Aspergillus

Denning explained that resistance involves two groups of patients: those who develop resistance while on therapy and those who breathe in a resistant fungus. A challenge in measuring resistance is the low culture yield for Aspergillus, with standard methodology resulting in a culture rate of only 20 percent (Vergidis et al., 2020). However, utilizing high volume culture and polymerase chain reaction testing can elevate the culture rate to 76 percent. Denning noted a gap in older data on resistance due to numerous environmental and clinical surveys lacking positive cultures while utilizing conventional culture methodology. Azole resistance in A. fumigatus became evident in the late 1990s, with rates gradually increasing in the years since. (Bueid et al., 2010; Howard et al., 2009). In 2009, data from Manchester, United Kingdom, indicated a 20 percent resistance rate, which was almost entirely driven by treatment, said Denning. Patterns of resistance indicate variation, with some cases of pan-azole resistance found. Data from the Netherlands indicate rates of resistance lower than 5 percent during the 1990s, but gradually increasing in recent decades (Buil et al., 2019). Denning noted that almost all of the A. fumigatus azole resistance found in that study was driven by environmental resistance.

The rise of azole resistance has led experts to question whether azoles can be retained for clinical practice, said Denning (Verweij et al., 2016). Jennifer Shelton, doctoral student at Imperial College London, conducted a community science project throughout the United Kingdom and found that 14 percent of isolates sampled were resistant to tebuconazole (see

Chapter 4) (Shelton et al., 2022). This constitutes a higher rate than earlier research, indicating a gradual increase in resistance. Research conducted in the Mekong Delta in Vietnam used a screening assay to identify azole resistance in 95 percent of environmental isolates, with persistent azole residues detected in the soil (Duong et al., 2021). Research conducted in China suggests resistance rates of approximately 80 percent in some agricultural areas (Zhou et al., 2021). Denning stated that given the need for azoles, these rates of resistance suggest a crisis situation.

THE EMERGING THREAT OF ANTIFUNGAL-RESISTANT CANDIDA SPECIES

Jackson provided an overview of the prevalence and risk factors for Candida infections. He discussed increasing Candida resistance to antifungals, the emergence and characteristics of C. auris, challenges of addressing resistant fungi, and hypotheses about why resistance is increasing.

Candida Infection Prevalence and Risk Factors

Candida species are a leading cause of health care-associated bloodstream infections in the United States and likely worldwide, said Jackson, who presented a hypothetical case to illustrate the threat Candida can pose to hospitalized patients (see Box 3-1). The COVID-19 pandemic further increased these infections with the associated rise in patients requiring ICU and central line treatment. He emphasized that Candida infections are not rare pathogens. A study found that in 2020, 28 percent of central line-associated bloodstream infections (CLABSIs) in U.S. ICUs were caused by Candida (Weiner-Lastinger et al., 2022). Additionally, Candida caused 13 percent of CLABSIs in adult hospital wards. This fungus was more common than any single type of bacteria in both ICU and adult ward settings (Magill et al., 2018; Weiner-Lastinger et al., 2022).

Data from an ongoing 10-site surveillance study suggests that Candida bloodstream infections are associated with a mortality rate of 25-30 percent, a significant portion of which is attributable mortality (Toda et al., 2019). Risk factors include broad spectrum antibiotic use—due to the associated disruption of the microbiomes—as well as central lines, immunocompromisation, prolonged ICU stay, and abdominal surgery. Candida infections can occur when the intestinal barrier is disrupted. In such cases, conventional wisdom largely holds that Candida infection stems from auto-infection when the host flora enters the bloodstream. Deep-seated Candida bloodstream infections can affect specific internal organs. However, the frequency of these deep-seated infections is unknown due to the difficulty in detecting and monitoring them.

Jackson stated that non-invasive candidiasis should not be ignored. Types of non-invasive candidiasis include vulvovaginal infections (“yeast infections”), oral infections (“thrush”), esophageal infections, and skin infections. Esophageal Candida infections can be associated with morbidity. Although most cases of non-invasive candidiasis are not lethal, they require substantial medical care. In 2017, more than 3.6 million outpatient visits were due to these infections, creating over $2 billion in direct medical costs (Benedict et al., 2019). Furthermore, these infections likely account for the vast majority of systemic azole therapy, said Jackson (Benedict et al., 2022a).

Antifungal Resistance Among Candida Species

Jackson explained that Candida albicans was once the dominant Candida species for both invasive and non-invasive infections, accounting for approximately 90 percent of invasive human infections. Currently,

two-thirds of invasive Candida infections are non-albicans (CDC, 2021c). In the United States, C. glabrata is almost as common as C. albicans, and C. parapsilosis, C. tropicalis, and C. krusei cause a significant portion of infections. In some countries, C. parapsilosis and C. tropicalis have become more common than C. glabrata. Jackson remarked that the rise of other Candida species is significant due to antifungal resistance rates being higher in non-albicans species. To this point, CDC labeled drug-resistant Candida species as a serious threat in 2019 (CDC, 2019).

The CDC Emerging Infections Program conducts sentinel surveillance in the United States, said Jackson. In testing bloodstream samples, they found that 6–10 percent of C. glabrata isolates were resistant to azoles, 1–4 percent were resistant to echinocandins, and less than 1 percent were resistant to polyenes (CDC, 2021c). He noted that efforts are made to avoid treating Candida with polyenes, given the complications outlined by Spec in his presentation. Increasing resistance can be found in other species of Candida, including C. parapsilosis and C. tropicalis, which have up to 10 percent and 6 percent fluconazole resistance, respectively, in the United States (Toda et al., 2019). Moreover, increasing emergence of resistant clones has been found worldwide for both species (Fan et al., 2017; Govender et al., 2016; Pristov and Ghannoum, 2019). Jackson stated that some strains are resistant to both fluconazole and echinocandins, and C. krusei—the fifth most common species—is typically intrinsically resistant to fluconazole.

Increasing resistance to fluconazole contributed to the Infectious Diseases Society of America recommending echinocandins as first-line treatment for most forms of candidiasis in 2016, said Jackson (Pappas et al., 2016). However, the guidelines have yet to be fully adopted in clinical practice; a recent study found that up to 30 percent of patients were receiving fluconazole as initial treatment (Gold et al., 2021). Furthermore, 56 percent of patients were found to have a non-albicans species, reducing the likelihood that fluconazole would be the most effective treatment. Growing concern regarding antifungal resistance prompted the World Health Organization (WHO) to include Candida in their Global Antimicrobial Resistance and Use Surveillance System, which should help increase the volume of data collected worldwide on this type of resistance.²

The Rise of Candida auris

C. auris was basically unknown in the scientific community before 2009, when it was detected in an ear specimen and was thus named “auris,” Jackson

___________________

² More information on the Global Antimicrobial Resistance and Use Surveillance System (GLASS) is available here: https://www.who.int/initiatives/glass (accessed February 15, 2023).

explained. By the early 2010s, global reports of invasive C. auris infections began to emerge. In 2015 and 2016, outbreaks occurred in the United Kingdom and cases were detected in the United States, garnering media attention (Richtel and Jacobs, 2019; Smothers, 2016). Jackson described C. auris as behaving like multidrug-resistant bacteria, sharing similarities with Staphlococcus aureus, Clostridioides difficile, and Acinetobacter. This yeast can spread rapidly in health care facilities, and long-term care facilities are fertile ground for outbreaks in the United States. C. auris colonizes patients, especially skin, and appears to exploit a disrupted microbiome. Jackson noted that health care employees do not tend to be colonized for very long, if at all, indicating that a disrupted microbiome may be key in the colonization process. Moreover, C. auris causes invasive infections in 5–10 percent of people colonized with the yeast (Southwick et al., 2018). The fungus survives disinfectants, particularly quaternary ammonium compounds, used to decontaminate health care facilities. Special disinfectants are often required to kill C. auris, Jackson added.

In comparison with C. glabrata, C. auris resistance to antifungals is more prevalent, said Jackson. Approximately 90 percent of C. auris isolates are resistant to azoles and 33 percent are resistant to polyenes. Echinocandin-resistance is lower, at less than 5 percent, but cases of resistance have occurred during echinocandin treatment. Pan-resistant C. auris—resistant to all three drug classes of antifungals—has also emerged (Lyman et al., 2021). Furthermore, resistance is not only developing in patients receiving echinocandin therapy, but also in patients in health care facilities who never received antifungal therapy and contracted transmitted strains that are already resistant. If the type of outbreaks that have occurred in Texas and in Washington, DC, begin to occur more frequently, echinocandins may become far less useful, creating a lack of treatment options.

Jackson stated that C. auris is perplexing in that at least four, and possibly five, clades developed in different parts of the world at approximately the same time (Chow et al., 2018). These clades were first detected in South Asia, southern Africa, east Asia, and South America. Within the United States, the clade that first appeared in South Asia emerged in New York and New Jersey and spread to other northeastern states. The clade that was first detected in South America emerged in Chicago, and the clade that was first detected in Africa appeared in Indiana. In some cases, CDC was able to contact trace the spread to an incoming traveler from a country where a clade of C. auris was present, but in other cases, the initial introduction remains unknown. He described fungal taxonomy as complicated and in flux. According to this taxonomy, C. glabrata and C. albicans—the two most common species of Candida—are as different from each other as humans are from fish (Gabaldón and Carreté, 2016). Jackson added that the Candida taxonomy includes Saccharomyces, the

yeast used in making bread and beer, which was not named Candida due to its common uses. The species of Candida not yet discovered likely number in the hundreds of thousands or even millions.

Although C. auris was discovered relatively recently, it is not new and has had lengthy time to evolve, said Jackson. Improved detection methods may have played a role in its discovery, but a variety of causes may be responsible for its emergence and proliferation in human health care settings. Specifically, modern health care settings have facilitated its spread and it is probable that multiple spillover events from the environment have occurred. Relatives of C. auris have recently been isolated from the environment, and people have collected samples of it from flowers in Southeast Asia, rubber tree sap, bugs, fish, and dolphins. In 2021, C. auris was located for the first time outside of health care and clinical settings in the Indian Ocean (Cunningham, 2021). Multiple theories for the rise of C. auris have been proposed, including (1) effects of climate change, (2) intrusion of humans into its natural habitat via deforestation, (3) human activities that may amplify the pathogen’s reservoir, such as shrimp aquaculture in which antibiotics and fungal probiotics are added to water, (4) the use of environmental fungicides and antifungals, and (5) changes to the human host’s microbiome (Casadevall et al., 2019; Jackson et al., 2019; Steffen et al., 2015). Jackson noted increasing evidence that Candida, including C. glabrata and C. albicans, are common colonizers that can occupy trees and migratory birds that then play a role in circulating the fungi (Al-Yasiri et al., 2016; Bensasson et al., 2019).

The extent of the linkages between Candida and the environment are not yet known, added Jackson. However, there has been a marked increase in the use of azole fungicides over the last decade (Toda et al., 2021). This increase of fungicides has taken place in different parts of the world at different rates; its use in the United States is driven by corn, wheat, and soy. Research on links between Candida resistance and agricultural fungicides is in the early stages (Brilhante et al., 2019). Jackson cautioned that the full effect of azole fungicides on the yeast species remains to be seen. Because the environment appears to play a stronger role in the emergence of resistance than once thought, resistance stewardship will be a key pillar in addressing Candida.

CURRENT RESEARCH ON PATHOGENESIS AND HOST IMMUNITY TOWARD TREATMENT OPTIONS

Lionakis discussed how understanding interactions between the host and fungi at the research level may enable hematologists and oncologists to develop targeted immune-based therapies to treat severe fungal infectious diseases. Robust immune factors protect humans from the

commensals—such as Candida—and fungi that they are exposed to on a daily basis. The occurrence of immune perturbations increases the risk of fungal infections. Therefore, a deeper understanding of these events could facilitate efforts to boost immune responses, cultivate a more thorough knowledge of patients at risk, and develop personalized therapies.

Pathways for Fungal Infections in Immunocompromised Patients

Some individuals have genetic or acquired iatrogenic risk factors that increase their likelihood of developing fungal infections, said Lionakis. Susceptibility to infection can emerge when patients are given certain medications or when congenital genetic diseases are inadvertently triggered or exacerbated in the hospital setting. Understanding these pathways could enable diseases to be researched in mouse models to discern what helps the immune system control fungal disease, which in turn would inform the development of treatments. Research in mice thus far has demonstrated the ability of neutrophils and macrophages to attack and destroy fungi. However, despite medical therapy, Candida and Aspergillus cause over 20,000 deaths in the United States each year. This mortality is greater than that of Staphylococcal infections or HIV/AIDS and highlights the need for new treatments.

Lionakis recalled that until the introduction of imatinib in 2001, no personalized approach to treat oncologic patients was available outside of toxic chemotherapy. The use of imatinib ushered in a wave of biologics that have transformed the treatment of cancers (Wu et al., 2016). In 2018, Jim Allison won the Nobel Prize for Physiology or Medicine in response to his discovery of the immune checkpoint inhibitor, which utilizes the immune system to combat cancer. Lionakis posited that fungal treatment can follow a similar trajectory to that taken by cancer treatment. Transplantation, HIV, and chemotherapy are conventional risk factors for fungal infection related to the immune system. He predicted that the number of patients prescribed biologics will expand into a significant subset of individuals at risk of developing fungal infections. Furthermore, physicians prescribing biologics are not always aware of this risk.³

___________________

³ Warnings of increased risk of serious and life-threatening meningococcal infections are included in the medication guides for some biologics. An example is available at https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125166Orig1s427s428MedGuide.pdf (accessed July 30, 2022).

Complement Component 5 Pathway

Lionakis provided examples of how biologics used in an expanding population of already-immunocompromised patients can create pathways for severe fungal infections. Complement is an ancient protein network utilized within the immune system to defend against bacterial disease. The advent of medical treatment that neutralizes complement component 5 (C5) led to an eruption of fungal disease. In response, the U.S. Food and Drug Administration (FDA) updated the drug’s package insert to caution that patients on the drug may develop fungal disease. People on complement C5 inhibitors are at an increased risk of developing infections from Aspergillus or Candida, with mortality rates that can exceed 70–80 percent. Lionakis described that this information is being used to develop predictors for poor outcomes in patients. Researchers have found that mice who lack complement C5a die, whereas their wild-type counterparts do not (Mullick et al., 2004). With ICU patients, measuring low activation of the C5a pathway due to the introduction of biologics enables the development of independent predictors of patient outcomes.

Although invasive candidiasis has become a common disease in acutely ill patients in the ICU, both disease severity and mortality vary among this group of patients. Research has examined the relationship of genetic variation in certain genes with resistance and immunopathology levels (Collar et al., 2018; Kumar et al., 2014; Lionakis et al., 2012, 2017a; Smeekens et al., 2013; Swamydas et al., 2016). Certain studies indicated that some genetic variations will not make a person prone to disease until they are in a hospital setting, at which point they can be up to 20 times more likely to develop fungal infections (Kumar et al., 2014). Lionakis stated that this has important implications for envisioning the future of developing treatments and personalized approaches to patient care.

SYKCARD9 Pathway

Lionakis presented another example of the relationship between biologic drugs and fungal infections. Humans have a pathway of C-type lectin receptors that utilizes spleen tyrosine kinase (SYK) and caspase recruitment domain family member 9 (CARD9) protein-coding genes to eliminate fungi. Deficiencies in the SYK-CARD9 pathway increase the likelihood of severe fungal infections (Drummond et al., 2019). In 2018, a drug called fostamatinib gained FDA approval for treatment of chronic immune thrombocytopenia (FDA, 2018). Fostamatinib is an SYK-inhibitor, yet many physicians who prescribe it are unaware that the drug has the capacity to predispose patients to fungal disease, said Lionakis.

Providing a case example, Lionakis described a Colombian child who

came under his care due to infection by Corynespora, a plant pathogen that is responsible for target leaf spot on cucumbers and tomatoes and is presumed not to infect humans. Moreover, it is not supposed to grow in temperatures over 30 degrees Celsius, he added. However, in a susceptible human host, Corynespora has developed the ability to grow at 37 degrees. Lionakis remarked that this demonstrates the ability of fungi to evolve and to develop azole resistance within the human brain to the point of killing the patient despite bone marrow transplant. Ten percent of patients treated with fostamatinib have developed severe fungal disease, indicating that expansive populations of patients are susceptible (Zarakas et al., 2019). Lionakis added that this susceptibility puts individuals at risk of developing resistance or capturing fungi with intrinsic resistance during the process of receiving medical treatment.

Ibrutinib and Fungal Infections

Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is an example of a small molecule drug that transformed the treatment of hematological malignancies, said Lionakis. Ibrutinib treatment may be associated with the later development of fungal infections (Ahn et al., 2016; Lionakis et al., 2017b; Messina et al., 2017). He described a series of patients seen at the National Institutes of Health (NIH) who had refractory central nervous system lymphoma that historically caused death within 2 to 3 months. Ibrutinib was effective in significantly reducing the size of brain tumors (Lionakis et al., 2017b). However, while the lymphoma moved into remission, the incidence rate of Aspergillus infections increased to approximately 30 percent among these patients. This high rate of fungal infection is in the context of a treatment that caused years of almost complete remission for nearly 70 percent of patients, who otherwise would have died within months (Roschewski et al., 2018). These simultaneous high rates of infection and near-remission led researchers to examine how to provide patients with life-extending therapy without the high risk of fungal disease. In this case, targeted prophylaxis with isavuconazole enabled over 100 patients to receive ibrutinib treatment without developing invasive aspergillosis.⁴ Lionakis noted that this example demonstrates how understanding immunity can inform a targeted prophylactic approach to safely delivering a potentially toxic therapy. Exploring host-pathogen interactions can lead to identification of potential prognostic biomarkers, which in turn inform patient risk stratification. This understanding can also create awareness in clinicians regarding the infections their patients on biologics are prone to contracting.

___________________

⁴ More information about this ongoing study is available at https://clinicaltrials.gov/ct2/show/NCT02203526 (accessed July 30, 2022).

Developing Immune-Based Treatment Options

Lionakis also shared that research could potentially lead to the development of immune-based therapies that co-opt the immune system to boost the effect of antifungal drugs. NIAID research on ibrutinib found that the drug hinders the ability of neutrophils to fight Aspergillus within 3 days of initial administration. Healthy neutrophils produce oxidative burst products that kill Aspergillus by damaging the hyphae of fungi. Ibrutinib disrupts this process, inducing a reactive oxygen species (ROS) defect in neutrophils. By gaining an understanding of the molecular underpinnings of that process, researchers found that utilization of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF)—an FDA-approved drug—restores the ability of neutrophils to generate oxidative bursts. Researchers have successfully overcome the BTK-inhibition-induced ROS defect by using GM-CSF in the laboratory setting, and GM-CSF also rescues the ROS defect in individuals who are BTK-deficient. GM-CSF could potentially be used in patients receiving ibrutinib treatment in the future to restore their ability to fight Aspergillus. He noted that GM-CSF actually boosts the immune system’s ability to kill Aspergillus above normal levels.

Antibiotics are a common risk factor for systemic candidiasis in both humans and mice, said Lionakis. Mice models indicate that in addition to the role of microbiota, antibiotics cripple the immune system’s ability to kill fungi through lymphocytes and GM-CSF mechanisms (Drummond et al., 2022). Part of this susceptibility can be rescued by giving mice GM-CSF. Furthermore, examination of a large dataset of over 10,000 U.S. patients with candidemia suggests that this observation may translate into humans. This is another example of the potential for future immune-based interventions that can be added to antifungals in treating patients prone to fungal infections. In the 1990s, interferon gamma was the first (and remains the only) FDA-approved immune-based therapy for infectious disease (International Chronic Granulomatous Disease Cooperative Study, 1991). Twenty years ago, no FDA-approved targeted biologics for hematology oncology were available, Lionakis noted. Just as immune therapies have advanced for infectious diseases and oncology, further developing the fungal-host interaction knowledge base will increase the ability to treat difficult fungal disease in patients with immune defects and in patients with resistant fungal infections.

Therapeutic strategies that dampen the immune system to combat fungal disease are also being explored, said Lionakis. COVID-19 illustrated how some infections can trigger an excess immune response, leading doctors to treat COVID-19 patients with steroids and Janus kinase (JAK) inhibitors. Chronic mucocutaneous candidiasis is a severe, non-lethal infection that can cause significant morbidity and resistance. Approximately 60 percent of

these patients develop resistant Candida. Researchers have discovered that susceptibility to Candida is not necessarily due to an inability of the immune system to address the fungi; it can also be attributed to an exacerbated immune response known as autoimmune regulator (AIRE) deficiency (Break et al., 2021). They found that by inhibiting the interferon gamma immune response in mice with AIRE deficiency and uncontrolled Candida, the mice became able to control the infection. A phase II clinical trial at NIH is currently testing this intervention, and Lionakis shared anecdotal evidence based on a study (manuscript in preparation) of patients with multidrug-resistant Candida and candidiasis going into remission within 1 week of treatment with an immune modulator such as a JAK inhibitor. He added that this type of research can inform approaches to more common diseases, including HIV, in which cluster of differentiation 4 (CD4) decline is unlikely to be solely responsible for Candida infections, as other patients with CD4 deficiency such as those with idiopathic CD4 lymphocytopenia are not at risk for developing mucosal candidiasis. A better understanding of host-fungal interactions could fuel the development of personalized risk stratification and prognostication strategies, targeted prophylaxis strategies, and immune-based adjunct therapies for treating patients with resistant and difficult-to-treat infections. Given that the development of multiple new classes of antifungals is unlikely, this area of research holds particular promise in improving patient outcomes.

DISCUSSION

Diagnostic and Data Challenges

Noting challenges in fungal infection diagnosis and data on disease burden and resistance, Chiller asked about the factors that contribute to these challenges and their connection to stewardship efforts. Denning replied that antigen and antibody tests are the most sensitive and rapid tests for fungal disease not occurring on the skin, but a culture is required for identifying resistance. However, culture tests are relatively insensitive for fungal infections: the sensitivities of Candida blood culture and of Aspergillus sputum culture are approximately 40 percent and 30 percent, respectively. This low sensitivity poses a problem for diagnosis. Moreover, in low- and middle-income countries, diagnostics are often unavailable. For instance, many countries in Africa only have microscopy and simple skin culture diagnostics. Thus, global health issues contribute to the challenges that the insensitivity of diagnostics present. Spec added that fungi are ubiquitous, which makes it difficult to differentiate between fungal infection, colonization, contamination, and presence from a culture. The combination of this ubiquity and the insensitivity of cultures makes accurate diagnosis, and especially detection of resistance, challenging.

Aspergillosis Incubation and Dosing

Chiller asked about the route of transmission and infectious dose range for aspergillosis. Denning stated that the incubation period is variable, from 2 to 90 days. Lungs are not sterile, and autopsy research has demonstrated that Aspergillus can grow in human lungs (Lass-Florl et al., 1999). Macrophages, neutrophils, and epithelia in the lungs play an important role in eradicating Aspergillus, but they are not fully effective, leading to ongoing colonization. Denning added that the infective dose for Aspergillus has not yet been fully determined, although more progress has been made on determining the infective dose for Histoplasma.

Cornyespora Infections and Resistance Types

Given that the young patient suffering with Corynespora had an azole-resistant fungal isolate, Chiller asked whether the resistant type at play was inherent or acquired due to the medical use of azoles, and—if the latter—whether the resistance mechanisms were investigated. Lionakis replied that the child arrived at NIAID with a fully susceptible strain for azoles. During the 15 months of treatment, the brain isolate became resistant, while the skin isolate did not. He posited that the lower levels of azoles within the brain tissue may have facilitated the development of secondary resistance. The strains are currently in the process of whole genome sequencing; thus, the mechanism of acquired resistance has not yet been determined. Cases have occurred in which patients are susceptible and happen to contract an intrinsically resistant strain. Lionakis stated that as more individuals become immunocompromised due to medical treatment, both patterns will occur. Some patients will contract inherently resistant strains; other patients who require intensive therapy for difficult-to-treat infection sites will have strains that acquire resistance during treatment.

The discussion continued at the end of the day. Marin Brewer, associate professor of Mycology and Plant Pathology at the University of Georgia, co-moderated this portion of the discussion with Chiller. Spec stated that a fascinating aspect of this case is that Corynespora is a plant pathogen. Historically, plant pathogens have rarely caused human disease. However, in recent years, he has seen several cases at Washington University involving plant pathogens, including Thyronectria austroamericana, a pathogen of honey locust trees. These trees have long thorns that can cause injuries leading to infections. He co-authored a case study about a patient in Missouri who developed Thyronectria austroamericana septic arthritis, and shortly after publication he was contacted by a physician in Kentucky who developed a tenosynovitis after receiving a wound from a honey locust tree (Rutjanawech et al., 2021). Spec emphasized that this pathogen had never before been

described as causing human disease, yet in 2 years, two cases of human infection from the pathogen were identified within the same ecological area. Recently, he has seen a case of mycorrhizal fungi that caused brain disease in a patient who was exposed via his work on tractors and tilling equipment. Spec remarked that it is not yet known whether these cases can be attributed to improved detection efforts or if new pathogens are now infecting humans.

Antifungal Drug Development

Chiller asked whether pharmaceutical companies are investing in antifungal therapies for medical use. Spec replied that an uptick in the development of antifungals has taken place in recent years. No new major class of drugs has been developed for invasive mycoses since the echinocandins in 2002. Currently, several antifungal drugs are in clinical trials with the possibility of being marketed in the next few years. For instance, phase 3 clinical trials for ibrexafungerp was approved with a limited indication for vulvovaginal candidiasis, but it has not been approved for invasive disease. Olorofim is another new antifungal drug in phase 3 clinical trials and belongs to the orotomide class of antifungals. Spec remarked that the number of clinically available antibiotics far outnumber the number of antifungal drugs. He highlighted systematic disinvestment in antifungals for financial purposes, stating that the lack of financial incentives plays a large role in the limited antifungal options available. Denning commented that small biotechnology companies are often responsible for the development of antifungal drugs, and once a drug looks promising, large pharmaceutical companies become involved to make marketing deals. Antifungal research is often funded by nondilutive grants followed by venture capital, which tends to invest more heavily in antibacterials than in antifungals. Thus, a lack of funding persists in both the nondilutive funding space and with large pharmaceutical companies.

Denning added that three companies are developing inhaled azole antifungals for the management of patients with severe asthma, cystic fibrosis, and for prophylactic use in patient with leukemia and transplants. These forthcoming drugs could be rendered ineffective should rates of resistance become high. Thus, minimizing azole resistance in the environment plays a role in maintaining the effectiveness of existing and newly developed antifungal drugs.

Aspergillus flavus

Noting that Aspergillus flavus (A. flavus) is less relevant from a clinical perspective than A. fumigatus, Brewer asked whether azole resistance is also present in A. flavus. Denning stated that low levels of A. flavus resistance to

voriconazole as a sole compound have been found, but the mechanisms for this resistance have not yet been determined. The researchers that looked for A. fumigatus in Vietnam did find high rates of A. flavus with resistance in the environment, at approximately 50 percent (Duong et al., 2020). A. flavus is a bigger issue in some countries that in others—for instance, it is particularly common in India. A. flavus is more related to cutaneous infection, superficial infection, and sinus infection for reasons not fully understood, said Denning.

Preventative Measures for People at Risk

Chiller asked whether any preventative measures related to lifestyle factors such as diet, travel, or employment can be taken to decrease the likelihood of fungal infections. Lionakis replied that keeping one’s body healthy can help to some extent; he highlighted the value of patients with neutropenia controlling diabetes and staying vigilant in their health and lifestyle decisions. However, fungi are ubiquitous and mold outbreaks can occur in most locations. Denning added that immunocompromised patients should avoid any significant amount of gardening and composting. For instance, pruning flowers may be safe, but heavy garden work is not advised. Wood chips and mulch contain huge quantities of spores, and therefore these immunocompromised people should avoid mulching entirely. Additionally, people with compromised immune systems should avoid any kitchens or bathrooms that are in the process of being remodeled and should not sort through dusty old photographs or papers, said Denning. He noted that this guidance is a bit vague and acknowledged that although immunocompromised people are advised to avoid large fungal inocula, the effectiveness of these preventative measures is unknown. Spec echoed that although the inoculum levels are not known, highly susceptible individuals should avoid situations known to expose them to a large inocula. Mulching poses a documented risk of pneumonitis from fungi for patients with inherent immunodeficiencies but quantifying the exact amount of outdoor exposure that poses a significant health risk is difficult. Spec recalled that during a recent trip to Houston, he saw a construction site and imagined that the combination of humid weather and activity that potentially disperses more fungi spores into the environment could synergize to increase the risk of infection. However, quantifying such scenarios into real risk for patients is complicated.

Risk Assessment for Fungicide Resistance

Fungicides are evaluated for their potential to develop resistance in plant pathogens, and A. fumigatus is not a plant pathogen, Chiller noted (see Chapter 4 for further detail). Given that antifungal resistance in

A. fumigatus is a downstream effect of fungicide use in the environment that ultimately affects human health, he asked how the evaluation or risk assessment process can help assess the potential for resistant fungi. Denning stated that this question is difficult to answer from a clinical perspective. One method involves sampling of the environment for resistance. European guidelines on Aspergillus recommend annual sampling of at least 100 strains from a locality’s environment to determine the likelihood of a resistant strain (Ullmann et al., 2018). In cases where 10 percent or more of the captured fungal isolates exhibit resistance, patients who are critically ill or severely immune-compromised should receive combination dual antifungal therapy rather than monotherapy, which is the standard recommendation. Denning noted that these guidelines do not specify the strains or quantity to sample, nor the location from which to collect the sample. Collaborative generation of better data on rates of environmental resistance could inform guidance on a standard approach and sampling methods and locations (e.g., whether to test only for A. fumigatus, itraconazole, or tebuconazole in a certain scenario). Developing the knowledge base to provide a more specific set of guidelines would be a valuable joint exercise, said Denning.

Infection by Fungi Ingestion

Chiller asked whether it is possible to become infected by ingested fungi spores via the gastrointestinal tract. Denning replied that this can occur in leukemia patients with profound neutropenia. He has seen cases of intestinal Aspergillus, and he stated near certainty that this is contracted through the intestines. Although such cases of infection via ingestion are exceedingly rare, food preparation can serve as a transmission route. For example, during the grinding and sprinkling processes, pepper mills can aerosolize Aspergillus that are then inhaled. Similarly, loose tea can release spores into the air that can be inhaled and lead to infection.

Differentiating Colonization and Infection

Chiller asked whether colonization can be differentiated from infection in patients with COPD. Additionally, he queried whether susceptible hosts are treated for all fungi. Denning stated that this question goes to the heart of the difficulty of distinguishing an infection from a colonizing organism. In the context of Candida, the presence of the fungi in the respiratory tract is rarely treated because Candida pneumonia and bronchitis are uncommon. Aspergillus is more complicated in that its yield is often higher in COPD patients than in the general population. This is due to the depression of the ability of epithelial cells and macrophages to kill Aspergillus in people with COPD. Furthermore, inhaled steroids are often prescribed

to people with COPD, further depressing their ability to kill Aspergillus in the airways. Denning remarked that these factors increase the likelihood that colonization, rather than infection, is taking place, although diagnosis is difficult. He added that traditionally, Aspergillus was regularly dismissed as colonization in patients who did not have leukemia or transplants. Awareness of allergic aspergillosis and Aspergillus bronchitis has increased, and clinicians need to be better educated about factors affecting susceptible hosts in determining treatment, said Denning.

Variations in Azole Resistance

Given that azoles have been used in agriculture since the 1970s, but resistance with A. fumigatus did not appear until the twenty-first century, Brewer asked whether evidence indicates that some azoles are more inclined to promote resistance than others. Denning replied that in the agriculture environment, five of the more modern azoles, such as the triazoles, seem to have produced a slightly higher rate of resistance than older azoles, such as the imidazoles. In the clinical environment, itraconazole likely has a slightly higher rate of resistance than voriconazole and posaconazole. He added that not much is yet known about isavuconazole. Lionakis stated that fungi do not have a uniform evolutionary response to azoles; different fungi can have varied responses. He emphasized the importance of examining how humans are changing the microbial community of the environment. In addition to concerns about resistant A. fumigatus, the killing of some types of fungi potentially creates opportunities for other fungi to grow, such as Lomentospora or Scopulariopsis, which Lionakis described as “nightmare fungi.” The use of azoles that increase the relative fitness of fungi in the environment could be increasing the likelihood of a fungus that is ubiquitous, spreads through the air, and cannot be treated.

Standardized Assays to determine resistance emergence

During the discussion at the end of the second day of the workshop, Denning was asked whether having a standardized assay to measure the effect of exposure to sublethal concentrations and minimum inhibitory concentrations (MIC) on the potential for bystander species to develop resistance would be beneficial. He agreed that this type of assay is beneficial, explaining that the current process to develop antifungal includes repetitively exposing a set of known fungal strains to MIC and sub-MIC doses over multiple generations to measure the emergence and levels of resistance. Establishing this assay is essential because a drug for which resistance emerges in the fungus after two or three generations would not be fit for clinical practice. Furthermore, this research is relatively inexpensive.