Epidemiology of Systemic Mycoses in the COVID-19 Pandemic

The physiopathologic characteristics of COVID-19 (high levels of inflammatory cytokines and T-cell reduction) promote fungal colonization and infection, which can go unnoticed because the symptoms in both diseases are very similar. The objective of this work was to study the current epidemiology of systemic mycosis in COVID-19 times. A literature search on the subject (January 2020–February 2021) was performed in PubMed, Embase, Cochrane Library, and LILACS without language restrictions. Demographic data, etiological agent, risk factors, diagnostic methods, antifungal treatment, and fatality rate were considered. Eighty nine publications were found on co-infection by COVID-19 and pneumocystosis, candidiasis, aspergillosis, mucormycosis, coccidioidomycosis, or histoplasmosis. In general, the co-infections occurred in males over the age of 40 with immunosuppression caused by various conditions. Several species were identified in candidiasis and aspergillosis co-infections. For diagnosis, diverse methods were used, from microbiological to molecular. Most patients received antifungals; however, the fatality rates were 11–100%. The latter may result because the clinical picture is usually attributed exclusively to SARS-CoV-2, preventing a clinical suspicion for mycosis. Diagnostic tests also have limitations beginning with sampling. Therefore, in the remainder of the pandemic, these diagnostic limitations must be overcome to achieve a better patient prognosis.


Introduction
SARS-CoV-2 is a betacoronavirus that causes the "2019 coronavirus disease" . Since the World Health Organization (WHO) declared a pandemic in March 2020, the world has been facing a health crisis that has involved significant challenges in diagnosing, treating, and preventing COVID-19 and its complications. As of 21 March 2021, it has affected 122,524,424 people in 222 countries resulting in the deaths of 2,703,620 individuals, showing an overall fatality rate of 2.2% [1]. The clinical course and progression of SARS-CoV-2 infection is variable and can lead to respiratory failure [2]. Clinical presentations of COVID-19 include fever (≥38 • C), cough, shortness of breath, loss of smell, chills, headache, and joint pain, among the most frequently reported. In most cases, the disease is asymptomatic or with mild to moderate symptomatology (80-85%) [3]. However, the disease can evolve to more severe clinical pictures in some patients, putting their lives at risk.
Reports indicate that the overall host state and the presence of comorbidities facilitate the spread of the virus and tropism of target organs with angiotensin-converting enzyme 2 (ACE2) receptors and increased production of interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF)-α in severe cases [2]. In the case of the disease requiring hospitalization, it is characterized by pneumonia, lymphopenia, and cytokine release syndrome (CRS), which trigger an exaggerated immune response that causes damage at the local and systemic level. The previous occurs because, after the virus enters the cells and releases its genetic material (RNA), it is recognized by innate immunity receptors located intracellularly. Receptors such as the Toll 7 (TLR7), retinoic acid-inducible gene I (RIG-1), and melanoma differentiationassociated protein 5 (MDA-5), activate a signaling cascade, leading to the expression of interferon (IFN) type I (α and β), the objective of which is to interfere with viral replication. On the other hand, viral antigens can be processed by antigen-presenting cells through their major histicompatibility complex (MHC)-I to T cell receptor (TCR) of T CD8+ lymphocyte, which involves releasing their proteolytic enzymes (cytotoxicity). At the same time, the cytokine storm begins (an increase of IL-1B, IL-6, IL-7, IL-8, IL-9, IL-10, and TNF-α [2][3][4]. In mild pneumonia, patients may be asymptomatic or have at least one of the following symptoms: fever (constant or intermittent), dry cough, sore throat, headache, myalgia, tiredness, or diarrhea. However, they do not present dyspnea nor hypoxia [3]. Although in most patients (81%), the course of the disease is mild, some develop severe pneumonia, characterized by the acute respiratory distress syndrome (ARDS), refractory hypoxemia, and dyspnea. More than 50% experience lung damage radiologically characterized by the presence of bilateral ground-glass opacities. In some cases, patients also develop damage and dysfunction of the extrapulmonary system, such as alterations in the hematological and digestive system, risk of sepsis, and septic shock, with a high mortality rate [4]. Some of these patients present patchy bilateral opacities in the lungs, leukopenia or lymphopenia, and increased serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase myocardial band (CK-MB), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) [5].
Approximately 5 to 30% of COVID-19 patients have been reported to become critically ill, require mechanical ventilation, and admission to the Intensive Care Unit (ICU) [2,3], which constitutes a risk factor for the development of co-infections with other bacterial, viral, or fungal pathogens. SARS-CoV-2 infection is characterized by a cytokine storm involving increased inflammatory proteins (TNF-α, IL-6, IL-2R) and decreased anti-inflammatory proteins. The latter contributes to lung pathology and lymphopenia development (drastic decrease in T cells, CD4+ and CD8+) in patients admitted to the ICU [5]. Lymphopenia in patients with severe COVID-19 is a clinical situation that increases the risk of severe fungal infections by different fungal genera, such as Pneumocystis, Candida, and Aspergillus, as T cells are the second most crucial line of defense against mycoses [6,7].
Fungal co-infections in COVID-19 patients is not a surprising situation. There are precedents that viral pneumonia, such as H1N1 and H7N9 influenza, can have co-infections caused by fungi and bacteria, and even other viruses [8][9][10][11][12][13][14][15][16][17]. During the outbreak of severe acute respiratory syndrome (SARS) detected in Guangdong, China, in November 2002, that subsequently spread to more than 30 countries, a series of autopsy cases were reported, which evidenced invasive fungal infection compatible with invasive aspergillosis in 10% of cases [18]. Interestingly, all infected patients were treated with high doses of corticosteroids, possibly resulting in immunosuppression that facilitated the establishment of mycoses. Another autopsy study of individuals who died of SARS confirmed fungal co-infection with the isolation of Aspergillus sp. and Mucor sp. in the upper respiratory tract and lungs. A case with multiple Aspergillus abscesses in different organs was also reported [19]. With this background, the co-infection scenario is expected to be similar in SARS-CoV-2 pneumonia.
This work analyzes the epidemiology of fungal co-infections in COVID-19 patients based on the review of publications on the subject during the first year of the pandemic.

Materials and Methods
A thorough search of publications on the subject was performed from January 1st, 2020, to 28 February 2021. The search was conducted on PubMed, Embase, Cochrane Library, and LILACS. The search words used were "fungi" OR "fungus" OR "fungal infection" OR "invasive fungal diseases" OR "pneumocystis" OR "candidiasis" OR "aspergillosis" OR "mucor" OR "histoplasmosis" OR "coccidioidomycosis" OR "co-infection" OR "secondary infection" AND "COVID-19" OR "SARS-CoV-2" OR "2019-nCoV" OR "2019 novel coronavirus" without language restrictions. Cases caused by Cryptococcus, Fusarium, Saccharomyces, and other fungi were excluded from the study due to the low number of reports. The review was performed based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) (Figure 1).
The fatality rate in cases of Aspergillus co-infection with SARS-CoV-2 was between 13% and 100% [23,56,. The affected patients were mainly men between 42 and 87 years of age [23,56,. The reported predisposing risk factors for the mycosis progression were diverse, including treatment with corticosteroids or immunosuppressants, hospitalization in the ICU, diabetes mellitus, hypertension, obesity, invasive mechanical ventilation, neoplasms, chronic obstructive pulmonary disease (COPD), asthma, and kidney disease [23,56,.

Endemic Mycoses and COVID-19
Information on endemic fungal co-infections with SARS-CoV-2 is scarce. Only five cases have been published so far, two of COVID-19 and coccidioidomycosis in California, United States [102,103], and three of COVID-19 and histoplasmosis in Rio Grande, Southern Brazil, and Buenos Aires, Argentina [104][105][106] (Table 5). Coccidioidomycosis co-infections were caused by C. immitis [102,103]. They occurred in a man and a woman, both 48 years of age, with the main predisposing factor of residing in endemic areas [102,103]. The diagnosis was established with serological test results, and fluconazole was administered as an antifungal treatment. Both patients showed clinical improvement [102,103].

Future Challenges of Systemic Mycoses Co-Infections and COVID-19
To date, there is no specific treatment for patients with COVID-19. However, in severe cases, high-dose systemic glucocorticoids are administered as it has been observed that they improve patient survival. Broad-spectrum antibiotics are also used [96]. The latter, in addition to the specific physiopathologic characteristics of COVID-19, such as the cytokine storm and reduced T-cell levels, favor fungal co-infections [94,95,101,107]. However, reports of fungal co-infections in COVID-19 patients are scarce, probably because fungal lung infections, such as pneumocystosis, aspergillosis, histoplasmosis, and coccidioidomycosis, can be mistaken for SARS-CoV-2 infection.
Furthermore, they might be unnoticed because the symptoms in both diseases are very similar, including fever, dry cough, dyspnea, myalgia, and headache [7,103]. There are no pathognomonic radiographic or tomographic findings that can differentiate a COVID-19 pneumopathy from a fungal infection [108]. These facts undoubtedly limit proper patient therapeutic management, which can lead to fatal outcomes, as shown by the few reports we present in this work, where the fatality rate ranges from 11 to 100% (Tables 1-5). This situation poses three new challenges related to the diagnosis of fungal infections that will have to be overcome in the remainder of the pandemic.
In the first challenge, it is crucial that, in the case of evidence of fungal co-infection in COVID-19 patients, the clinical picture is not attributed exclusively to SARS-CoV-2 infection, particularly in severe cases. While many of the secondary infections in these patients are caused by bacteria or viruses, it is vital to not forget about fungi, particularly those with clinical pictures virtually indistinguishable from SARS-CoV-2 pneumonia, like P. jirovecii. In this sense, healthcare workers must consider the medical history and epidemiological data, as they may be key to establishing clinical suspicion and directing diagnosis by selecting appropriate laboratory or cabinet tests.
The second challenge is related to diagnostic tests. The conventional tests that identify a fungal pathogen and confirm a mycosis diagnosis have different limitations, such as the time required to obtain a result or the impossibility of determining the fungus at the species level. The latter is utterly important, especially in mycosis caused by different species, particularly from the genera Candida, Aspergillus, and the order Mucorales. It is worth mentioning that new species have been identified, and many species that had not previously been associated with infections in humans are now recognized as pathogenic within these fungal groups. Further, many of these new species may have different susceptibility to the antifungals of choice for mycosis treatment, for example, we can cite C. auris and Aspergillus section Fumigati. While it is true that the limitations of conventional diagnostic tests have been gradually overcome with the development and implementation of different tests, particularly molecular ones, not all tests are available in intrahospital laboratories due to the lack of adequate infrastructure [91].
Diagnosing mycoses with molecular techniques is often complicated because they require trained staff. It is also impractical to routinely analyze multiple samples. The use of PCR and other molecular methods provides limited detection and identification due to the presence of inhibitors in clinical samples. Such inhibitors can lead to underestimating fungus at different levels, from the extraction process to the amplification of nucleic acids. As for antigen detection, it is not always specific as there is potential antigenic crossreactivity, which would prevent fungal identification [109].
The third challenge related to diagnostic tests is sample collection. For example, for COVID-19-associated pulmonary aspergillosis diagnosis, the sample must be collected from a bronchial washing that can generate aerosols representing a source of infection for healthcare personnel [62,75,110,111]. Another critical complication in diagnosis is the lack of algorithms and standardized diagnostic methods since the European Organization for Research and Treatment of Cancer (EORTC)/Mycoses Study Group (MSG) criteria are not specific for aspergillosis. For this reason, in an effort to establish a timely diagnosis, the AspICU algorithm for non-immunocompromised patients has been modified.
Overcoming these challenges will allow identification of cases of co-infections caused by SARS-CoV-2 and fungi, and enable definition of risk factors, affected populations, species distribution, and its antifungal profile.
Treatments reported in the revised literature fail to mention antifungal effectiveness as it is unknown whether the reported mortality was due to the stage of the disease per se or the fungal infection. In addition, treatment depends on the adequate diagnosis, which is complicated, as already mentioned. Thus, by not diagnosing the fungi properly, the correct treatment may not be given, and therefore it may not be effective.
It is important to mention that this work has limitations. This study did not include mycoses that were either reported just once, like fusariosis or caused by fungi that have rarely been associated with human infections, like Saccharomyces cerevisiae. Another limitation is that, unfortunately, not all fungal infections developed in COVID-19 patients have been reported in the literature. It should be noted that most references included in this study come from non-American countries, even though America has been the continent most affected by the pandemic. It is highly likely that fungal co-infections have occurred but have not been detected. It is our hope that the information presented in this study may serve as an alert for health workers to be aware of possible fungal co-infections in COVID-19 patients and the challenges posed by diagnosis. We also hope to remind them of the relevance of establishing acceptable infection control measures, prophylaxis, and adequate antifungal therapies.