Invasive Pulmonary Aspergillosis in Patients with SARS-CoV-2 Infection: A Systematic Review of the Literature

In this systematic review, we investigate the epidemiology, pathogenesis, risk factors, clinical manifestations, diagnosis and treatment of COVID-19-associated pulmonary aspergillosis (CAPA). We identified 85 cases from 22 studies. The frequency of CAPA is currently unknown but ranges between <5% to >30% in different case series; the possibility of colonization rather than invasive disease is the most important confounder. The vast majority of patients with CAPA did not have any of the classic host risk factors, such as immunosuppression from organ transplant or neutropenia, although a significant proportion (46%) had received corticosteroids. Age, pulmonary comorbidities and male sex were associated with higher mortality. Patients treated with voriconazole had numerically lower case-fatality rate. Clinical vigilance for CAPA is advisable in critically ill patients with COVID-19 who are not improving, even those who do not meet classic host criteria for invasive mycoses, especially if they are receiving corticosteroids. A thorough, multi-faceted diagnostic work-up and early initiation of a mold-active triazole may be lifesaving. Further research studies using standardized, uniform definitions of invasive disease and colonization are urgently needed.


Introduction
According to the World Health Organization (WHO), since December 2019 and as of 26 July 2020, there have been more than 15 million documented cases of coronavirus disease 2019 (COVID- 19) worldwide. Globally, more than 640,000 deaths have been attributed to the disease and its complications [1], including acute respiratory distress syndrome (ARDS) and secondary bacterial and fungal infections. According to a systematic review and meta-analysis of thirty studies (N = 3834 patients) from the earlier days of the SARS-CoV-2 pandemic (January-April 2020) [2], the rate of bacterial co-infections was 7% in hospitalized patients with COVID-19, but was as high as 14% in studies that solely included patients in the intensive care unit (ICU). The authors noted a relative paucity of data regarding COVID-19 associated fungal infections at the time.
Since then, a rising number of cases of invasive pulmonary aspergillosis (IPA) complicating COVID-19 infections have been reported in the literature. The aim of this systematic review is to

Cases Retrieved
A total of 85 patients with COVID-19-associated pulmonary aspergillosis (CAPA) from 22 articles were included in our analysis. Four case series were excluded, one where the authors reported isolation of Aspergillus from respiratory samples of patients with COVID-19, but did not specify whether these represented cases of IPA or colonization, and the data were inadequate to make that distinction [5]. Another study was excluded because of missing individual patient data [6]. The third one was an autopsy study where there was no evidence of IPA in any of the patients that met diagnostic criteria ante-mortem [7]. Lastly, we excluded the largest to-date case series of CAPA among intubated patients with COVID-19 since the authors did not provide patient-level information, with the exception of the number of patients treated with voriconazole and their mortality rates, compared to those who did not receive that treatment [8].

Epidemiology
The frequency of CAPA ranged from 35% [9] to 3.8% [10] of all ICU patients with COVID- 19. In comparison, the rate of influenza-associated (model post-viral) pulmonary aspergillosis (IAPA) is estimated between 16% and 23%. The majority of CAPA cases were documented in subjects from European countries (76/85, 89.4%), with only six cases described in in Asia, two in South America, and one in Australia. In 21 out of 22 articles (n = 65), the authors reported the patients' age and sex. The mean age at the time of presentation was 67 years, and most patients were male (75.4%, 49/65 with available information) (Table 1). With the exception of one study, the source of potential exposure to Aspergillus spp. was not investigated. In the study by Rutsaert et al. [9], an environmental source was ruled out by sampling room air and the oxygen and pressurized air supplies. In addition, high-efficiency particulate air filters were installed in the ICU. It is plausible that some of these cases had previous Aspergillus spp. airway colonization that progressed to clinical infection, since use of broad-spectrum antibiotics and especially corticosteroids, common in critically ill patients, promotes Aspergillus growth and tissue invasion [11][12][13][14][15][16][17][18].

Pathogenesis
The complex pathophysiology behind the development of CAPA is not well understood, but several hypotheses have been proposed ( Figure 1). As with all infectious disease syndromes, the development of IPA in association with COVID-19 infection is a result of the interplay between host, pathogen, and environmental factors. Aspergillus spp. are abundant in the environment in the form of airborne conidia that can easily reach the alveoli. Alveolar macrophage-driven neutrophil recruitment and transient flux of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) are key factors in eliminating these conidia from the airways. Structural damage to the lungs by SARS-CoV-2 infection and an impaired immune system may provide adequate conditions for the conidia to germinate and produce hyphae that then invade the tissues and vessels.
Diagnostics 2020, 10, x FOR PEER REVIEW 4 of 12 With the exception of one study, the source of potential exposure to Aspergillus spp. was not investigated. In the study by Rutsaert et al. [9], an environmental source was ruled out by sampling room air and the oxygen and pressurized air supplies. In addition, high-efficiency particulate air filters were installed in the ICU. It is plausible that some of these cases had previous Aspergillus spp. airway colonization that progressed to clinical infection, since use of broad-spectrum antibiotics and especially corticosteroids, common in critically ill patients, promotes Aspergillus growth and tissue invasion [11][12][13][14][15][16][17][18].

Pathogenesis
The complex pathophysiology behind the development of CAPA is not well understood, but several hypotheses have been proposed ( Figure 1). As with all infectious disease syndromes, the development of IPA in association with COVID-19 infection is a result of the interplay between host, pathogen, and environmental factors. Aspergillus spp. are abundant in the environment in the form of airborne conidia that can easily reach the alveoli. Alveolar macrophage-driven neutrophil recruitment and transient flux of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) are key factors in eliminating these conidia from the airways. Structural damage to the lungs by SARS-CoV-2 infection and an impaired immune system may provide adequate conditions for the conidia to germinate and produce hyphae that then invade the tissues and vessels. Dexamethasone use has emerged as an important supportive treatment for COVID-19 [19]. Steroid use hampers the activity of alveolar macrophages and thereby facilitates the germination of conidia. Corticosteroids exert an anti-inflammatory effect by repression of the Nuclear Factor-kB (NF-kB)-dependent production of pro-inflammatory cytokines [20]. This was demonstrated in a mouse model of Aspergillus infection where mice treated with corticosteroids showed an absent TNF-α response [21]. The study also reported an elevated level of interleukin-10 (IL-10) with corticosteroid use. Elevated anti-inflammatory cytokines, such as IL-10, may favor IPA by inhibiting phagocytosis of Aspergillus spp. [22]. In addition, pathology analysis of neutropenic and glucocorticoid-treated mice models infected with intranasal A. fumigatus conidia, showed that, compared to neutropenia, corticosteroid-associated aspergillosis demonstrates extensive necrosis, less angioinvasion, and a lower fungal burden [22,23].
Notably, structural damage to the respiratory tissue caused by viral infections, such as influenza, may favor the development of concurrent fungal infections [24][25][26][27]. In moderate to severe COVID-19 Dexamethasone use has emerged as an important supportive treatment for COVID-19 [19]. Steroid use hampers the activity of alveolar macrophages and thereby facilitates the germination of conidia. Corticosteroids exert an anti-inflammatory effect by repression of the Nuclear Factor-kB (NF-kB)-dependent production of pro-inflammatory cytokines [20]. This was demonstrated in a mouse model of Aspergillus infection where mice treated with corticosteroids showed an absent TNF-α response [21]. The study also reported an elevated level of interleukin-10 (IL-10) with corticosteroid use. Elevated anti-inflammatory cytokines, such as IL-10, may favor IPA by inhibiting phagocytosis of Aspergillus spp. [22]. In addition, pathology analysis of neutropenic and glucocorticoid-treated mice models infected with intranasal A. fumigatus conidia, showed that, compared to neutropenia, corticosteroid-associated aspergillosis demonstrates extensive necrosis, less angioinvasion, and a lower fungal burden [22,23].
Notably, structural damage to the respiratory tissue caused by viral infections, such as influenza, may favor the development of concurrent fungal infections [24][25][26][27]. In moderate to severe COVID-19 infection, extensive damage to the lung parenchyma and vascular tissue can develop during the pro-inflammatory cytokine storm phase [28,29].

Clinical Presentation
COVID-19 initially presents with fever, dyspnea, cough, myalgia, and diarrhea, occasionally progressing to respiratory and multi-organ failure requiring mechanical ventilation and hemodynamic support [30][31][32]. In the majority of cases, a diagnosis of CAPA was established when respiratory status continued to worsen despite the management of ARDS. In addition to worsening respiratory failure, new, persistent, or rising fever can prompt further workup in some cases, leading to the diagnosis of CAPA.
A total of 77 patients with CAPA (all with available data) required admission to the ICU, whereas this information was not available in one study [35]. Acute Respiratory Distress Syndrome (ARDS), which is the main complication associated with multi-organ failure in COVID-19, was reported in 21/85 cases (25%). Hemodynamic instability requiring vasopressor administration developed in 13/21 patients with ARDS (62%). Acute kidney injury developed in 20/21 patients with ARDS (95%), and nine (43%) required renal replacement therapy. No patients were receiving antifungal prophylaxis.
Exceptions included (i) one patient who was a renal transplant recipient on maintenance immunosuppressive therapy (mycophenolate mofetil 1 g twice daily and prednisone 20 mg daily) [39], (ii) one patient who had a remote history of lung transplant on maintenance immunosuppressive therapy with tacrolimus and prednisone (unspecified doses) [40], (iii) one patient who was on chronic steroids for pemphigus foliaceus [9], (iv) one patient who had history of multiple myeloma and was on steroids [41], (v) one patient with history of rheumatoid arthritis on long-term leflunomide [33], (vi) one patient with a history of chronic myelomonocytic leukemia [40] and (vii) one patient with a history of unspecified hematologic malignancy [42]. For the last two patients, it was not specified in the studies whether their disease was active or in remission, and whether they were neutropenic at baseline or not. For the purposes of our review, we considered these five patients as immunocompromised hosts.
There were three additional "immunomodulated" patients with history of hematologic malignancy for which they were not being treated. Two of these patients had been treated for acute myeloid leukemia (AML) remotely and were in remission [9,43]. The third was a patient with myelodysplastic syndrome (MDS) who was neither neutropenic nor on chemotherapy [44]. One other patient had a history of Human Immunodeficiency Virus (HIV) infection on lamivudine/tenofovir/nevirapine with a viral load of less than 20 copies and CD4 count > 250 cells [9]. We considered these patients as "immunomodulated", non-immunocompromised hosts.
A total of 19 out of 21 studies reported information on corticosteroid use prior to the diagnosis of IPA. For one additional study, information regarding prior corticosteroid use was obtained after communication with the corresponding author, for a total of 72 patients [39]. Of these 72 patients, 38 patients (53%) had recently received systemic corticosteroids and one patient was on inhaled fluticasone for asthma prior to the diagnosis of IPA. Only four patients who received systemic corticosteroids had been on chronic steroids prior to diagnosis of COVID-19 (10%, patients (i)-(iv) above). The exact steroid regimens for patients (ii) and (iv) were not explicitly mentioned in the studies. All other patients in this subgroup (35/39, 90%) received corticosteroids at variable doses in the setting of their acute illness, either as an anti-inflammatory modality against severe COVID-19 or for refractory shock. A small number of patients received immunomodulators for the treatment of COVID-19 and prior to the diagnosis of IPA. Specifically, eight patients received tocilizumab [10,33,45,46] and one patient eculizumab [37].

Diagnosis
Diagnostic modalities included histopathologic examination of lung tissue on autopsy, recovery of Aspergillus sp. from tracheal aspirate, bronchial brush or bronchoalveolar lavage (BAL) culture (CX), Aspergillus sp. polymerase chain reaction (PCR) from a BAL sample, and fungal markers including serum galactomannan (sGM), BAL galactomannan (bGM) and serum beta-D-glucan (bDG). The following cut-offs were used for fungal marker positivity: sGM > 0.5, bGM > 1.0 and bDG > 7.0. Two case reports reported use of an Aspergillus-specific lateral flow device (LFD) test as an additional test for point-of-care diagnosis [34,35] (Figure 2). Diagnostics 2020, 10, x FOR PEER REVIEW 6 of 12 communication with the corresponding author, for a total of 72 patients [39]. Of these 72 patients, 38 patients (53%) had recently received systemic corticosteroids and one patient was on inhaled fluticasone for asthma prior to the diagnosis of IPA. Only four patients who received systemic corticosteroids had been on chronic steroids prior to diagnosis of COVID-19 (10%, patients (i)-(iv) above). The exact steroid regimens for patients (ii) and (iv) were not explicitly mentioned in the studies. All other patients in this subgroup (35/39, 90%) received corticosteroids at variable doses in the setting of their acute illness, either as an anti-inflammatory modality against severe COVID-19 or for refractory shock. A small number of patients received immunomodulators for the treatment of COVID-19 and prior to the diagnosis of IPA. Specifically, eight patients received tocilizumab [10,33,45,46] and one patient eculizumab [37].

Diagnosis
Diagnostic modalities included histopathologic examination of lung tissue on autopsy, recovery of Aspergillus sp. from tracheal aspirate, bronchial brush or bronchoalveolar lavage (BAL) culture (CX), Aspergillus sp. polymerase chain reaction (PCR) from a BAL sample, and fungal markers including serum galactomannan (sGM), BAL galactomannan (bGM) and serum beta-D-glucan (bDG). The following cut-offs were used for fungal marker positivity: sGM > 0.5, bGM > 1.0 and bDG > 7.0. Two case reports reported use of an Aspergillus-specific lateral flow device (LFD) test as an additional test for point-of-care diagnosis [34,35] (Figure 2).   Figure 2.
The most frequent combination of tests was: 1. both positive culture and bPCR, and 2. bGM positive with neither sGM nor bDG performed (Figure 2).
There were three documented cases of multi-azole resistant strains, the first was a patient from a Dutch cohort [47] who had no known prior exposure to azoles but developed CAPA from A. fumigatus resistant to voriconazole, posaconazole and itraconazole. He was treated with intravenous (IV) liposomal amphotericin B (LAmB) and survived.
The second was a patient from Ireland [43] who also had A. fumigatus resistant to voriconazole, posaconazole and itraconazole but this patient had both occupational exposure to fungicides and a past medical history of IPA in the setting of remote history of AML for which he had undergone chemotherapy. This patient also received IV LAmB but died.
The last patient was treated in a tertiary center in the Netherlands [34] and had no known history of exposure to azoles. Her tracheal aspirate cultures upon ICU admission grew pan-sensitive A. fumigatus and the patient was initially treated with voriconazole. However, upon clinical deterioration two weeks later, cultures were repeated, and this time the isolated strain was resistant to itraconazole (minimal inhibitory concentration (MIC) 16 mg/L) and intermediate to voriconazole (MIC 2 mg/L) and posaconazole (MIC 0.5 mg/L), but sensitive to amphotericin B. Subsequently, the patient was switched from voriconazole to IV LAmB (LAmB); however, she did not survive. The authors reported detection of TR34/L98H mutation upon sequencing of the A. fumigatus cyp51A gene.

Outcomes
Case-fatality rate (CFR) was 54.1% (46/85), which is not substantially different from the high mortality reported in some studies with severe COVID-19 alone [30]. The only factor significantly associated with mortality was age. We also noted a trend for higher mortality in males, patients with diabetes, and those with history of asthma or chronic obstructive pulmonary disease (COPD) ( Table 1).
Finally, we investigated if the SARS-CoV2 inoculum as quantified by RT-PCR cycle thresholds (Ct) could be associated with the severity of CAPA. However, only five studies (total of eight patients) referenced such data, and there was both specimen and gene target heterogeneity preventing further analysis. These data can be found in the Appendix A Table A1.

Discussion
To our knowledge, this is the largest review and case series of patients with CAPA published in the English literature to-date. We highlight clinically relevant observations and uncharted areas that merit further investigation: The vast majority of patients with CAPA did not fulfill classic host criteria, such as neutropenia, solid organ or hematopoetic cell transplantation (HCT), but rather had critical illness as their main predisposing factor. Likewise, in the prospective cohort of CAPA by Bartoletti et al., only a minority of patients met classic host criteria [8]. Furthermore, we demonstrated significant heterogeneity in laboratory modalities to diagnose CAPA, frequently based on >1 positive tests, more often bPCR and bGM ( Figure 2).
Although IPA in the ICU is an increasingly recognized syndrome, its diagnosis is often confounded by the possibility of colonization. It should be noted that the specificity of fungal biomarkers, especially bGM in patients without hematologic malignancies or history of HCT remains suboptimal, with very high false positive rates [48,49]. Therefore, it is possible that some patients diagnosed with CAPA may not have IPA. However, the absence of more classic hosts at risk for IPA from our case series, especially those with hematologic malignancies (HM), could be due to more strict infection control measures and neutropenic precautions in this patient population, as well as the frequent use of mold-active antifungal prophylaxis [50].
An important observation was that almost half of all patients with CAPA had corticosteroid exposure (Table 1). Steroids are increasingly recognized as an important risk factor for invasive mold infections [11,12,51,52], and more specifically mixed invasive mold infections [51,52], even in patients with no other risk factors [11,52]. Bartoletti et al., also pointed out that the high incidence of CAPA in their cohort could be associated with high utilization of corticosteroids and tocilizumab for COVID-19. Corticosteroids were associated with a statistically significant increase in mortality rates in their population (p = 0.04) [8]. Clinicians should have a low threshold for aggressive diagnostic work-up, and potentially early initiation of empiric antifungals in patients with COVID-19 receiving corticosteroids, especially as they are sometimes standard-of-care in critically-ill patients, based on the results of the RECOVERY randomized-controlled trial [19]. One major limitation of our systematic review is that many studies included did not report the specific dose and duration of corticosteroids, which are important for risk stratification.
We did not find a solid benefit from antifungal treatment, although the diagnosis of some cases post-mortem introduces by definition significant "immortal" bias favoring the treatment groups. Moreover, the only factors significantly associated with higher mortality rates were the same as those reported in the general population of patients with COVID-19, mainly age [29,52], but also comorbidities (diabetes, asthma, COPD) and, potentially, male sex [52,53]. Therefore, it is still not entirely clear if patients with CAPA have higher mortality than other ICU patients with COVID-19. However, the prospective study from Italy reported a significant association between bGM index at ICU admission and 30 day mortality (p = 0.014), after adjustment for other confounders [8].
The above points further emphasize the need for better differentiation between colonization and IPA, with accurate non-invasive methods, such as "breath tests" [54,55], since colonized-only patients are less likely to benefit from treatment. It should be noted, though, that, in our review, we captured a signal for mortality benefit in patients with voriconazole treatment, which almost reached statistical significance. Larger, prospective studies evaluating the effect of early antifungal treatment are needed.
In conclusion, our findings indicate that the diagnosis of CAPA needs a strong correlation between clinical, radiographic and laboratory data [56][57][58][59][60], as the growth of Aspergillus or positive fungal biomarkers may not necessarily indicate invasive disease. High-risk patient groups need to be better defined, but administration of corticosteroids may be an important risk factor for CAPA. Clinical vigilance for CAPA is advisable in critically ill patients who are not improving, even those who do not meet classic host criteria. In such cases, a thorough, multi-faceted diagnostic work-up and early initiation of a MAT may be lifesaving.
Funding: D.F. has received consultant fees and research support by Viracor-Eurofins ® , as well as research support from Astellas.