key: cord-0886725-1zv510du
authors: Benhadid-Brahmi, Yasmine; Hamane, Samia; Soyer, Benjamin; Mebazaa, Alexandre; Alanio, Alexandre; Chousterman, Benjamin; Bretagne, Stéphane; Dellière, Sarah
title: COVID-19-associated mixed mold infection: a case report of aspergillosis and mucormycosis and a literature review
date: 2021-11-26
journal: J Mycol Med
DOI: 10.1016/j.mycmed.2021.101231
sha: 5aa265ea07f69642835181471c741d6201d83464
doc_id: 886725
cord_uid: 1zv510du

COVID-19-associated mold infections have been increasingly reported, and the main entity is COVID-19-associated aspergillosis (CAPA). Similarly, COVID-19-associated mucormycosis has been reported in hematology, and its prevalence is high and has been increasing in the diabetic population in India during the third COVID-19 pandemic wave. Simultaneous infection with Mucorales and Aspergillus is rare and even rarer during COVID-19. Here, we report the case of a previously immunocompetent patient with severe SARS-CoV-2 infection complicated with probable CAPA and mucormycosis co-infection. Specific diagnostic tools for mucormycosis are lacking, and this case highlights the advantages of analyzing blood and respiratory samples using the quantitative polymerase chain reaction to detect these fungi. We further reviewed the literature on mixed Aspergillus/Mucorales invasive fungal diseases to provide an overview of patients presenting with both fungi and to identify characteristics of this rare infection.

There have been few studies on mixed mold diseases, and they are rarely reported in immunocompromised patients with neutropenia, malignant hemopathy [1, 2] , solid organ transplantation [3] , or poorly controlled diabetes mellitus [4, 5] . Viral infections, especially severe influenza and COVID-19, which cause acute respiratory distress syndrome (ARDS), increase the susceptibility to mold infection in previously immunocompetent patients [6, 7] .

COVID-19-associated pulmonary aspergillosis (CAPA) and COVID-19-associated mucormycosis (CAM) are associated with a higher mortality rate in this patient population [8, 9] . CAPA and CAM prevalence rates vary widely between studies, which may be explained by different awareness and diagnostic strategies [8] . Our center screened every mechanically ventilated COVID-19 patient who clinically worsened despite adequate standard of care for CAPA. This case is a patient with Rhizopus/Aspergillus coinfection in a previously immunocompetent COVID-19 patient and a discussion about mixed mold infections.

A 74-year-old retired man with a history of hypertension that was being treated with calcium channel blockers presented in the emergency ward on October 21, 2020 with a cough and a fever that lasted 4 days. He had evocative chest computed tomography (CT) scan findings, and the COVID-19 diagnosis was confirmed using real-time reverse-transcriptase polymerase chain reaction (PCR) on a nasopharyngeal swab (RealStar® SARS-CoV-2 Kit, Altona Diagnostics). The patient had a history of gastric lymphoma that was treated surgically in 1975, and he has subsequently been in remission.

Upon admission in the pneumology ward, the patient received ceftriaxone, azithromycin, and preventive anticoagulation by enoxaparin sodium (0.8 mL × two per 24 hours subcutaneously). On October 23, 2020, the patient received dexamethasone 6 mg/day because of a persistent SpO 2 of 85% on room air and a fever (38.6°C). After multiple desaturation episodes despite increased oxygen support (up to 6 L/min), he received a bolus of 120 mg methylprednisolone and was transferred to the intensive care unit (ICU) on October 26, 2020 (Day 1). The patient presented with polypnea (respiratory rate, 34 breaths/minute) with signs of respiratory distress such as abdominal breathing and SpO 2 91% under 15 L/min of oxygen.

His white blood cell count was 10,000 cells per mm 3 (normal range, 4000 to 10000) with 90% neutrophils (9040, normal range 1700 to 7000 cells per mm 3 ) and 3.8% lymphocytes (400, normal range 1500 to 4500 cells per mm 3 ) , and his C-reactive protein level was 194 mg/dL (normal value <5 mg/dL), procalcitonin was 1.49 µg/L (normal value <0.05 µg/L), fibrinogen was 7.77 g/L (normal range 2 to 4 g/L), and D-dimer was 2830 ng/mL (normal value <500 ng/mL). He had metabolic alkalosis with profound hypoxemia at 55 mmHg and lactatemia at 1.4 mmol/L. The simplified acute physiology score II (SAPSII) and Sepsis-related Organ Failure Assessment (SOFA) at 24 h were 32 and 4, respectively. Ceftriaxone and azithromycin were discontinued on Day 2 after negative microbiological results from respiratory samples.

On Day 6, the patient's condition deteriorated due to acute heart failure. Mechanical ventilation was initiated. Dexamethasone was increased to 10 mg twice daily and piperacillin/tazobactam was empirically prescribed. The patient subsequently presented with multiple organ failure.

Bronchoalveolar lavage (BAL) was performed on Day 7 and identified 5×10 3 colony forming units (CFU)/mL of Pseudomonas aeruginosa that was resistant to piperacillin/tazobactam, and a chest CT angiography showed a bilateral segmental pulmonary embolism with D-dimer >3000 ng/mL. Ceftazidim and intravenous unfractionated heparin were introduced on Day10.

On Day 15, a second BAL identified ventilation-associated pneumonia with 10 3 CFU/mL P. aeruginosa, 10 4 CFU/mL Stenotrophomonas maltophila, and herpes simplex virus (HSV)-1 reactivation at 8 log copies/mL. He was administered cefepime, amikacin, and sulfamethoxazole-trimethoprim (800 mg twice daily). Acyclovir was introduced on Day 16 before HSV-1 and cytomegalovirus viremia (4.37 log copies/mL and 2.2 log IU/mL).

The evolution was initially favorable, but a new degradation with hemodynamic and acute renal failure on Day 19 required initiation of renal replacement therapy. A new CT-scan highlighted a right apical excavation with peripheral condensation, which was associated with overall stability in the areas with a ground glass appearance and an increase in bilateral subpleural condensation in the postero-inferior segments. BAL performed on Day 19 was positive for Aspergillus section Nigri and was associated with a positive BAL galactomannan index >4.63 (Platelia Aspergillus enzyme immunoassay, Biorad). The concomitant serum galactomannan index was also >4.63 (Table 1) We retrospectively studied the two strains that were identified. Aspergillus section Nigri was identified as Aspergillus welwitschiae by sequencing using calmodulin as a target with CL1/CL2a primers [11] , and Rhizopus sp. was identified as Rhizopus delemar by sequencing with an internal transcribed spacer (ITS) as a target. A. welwischiae and R. delemar underwent antifungal susceptibility testing using the EUCAST method and showed minimal inhibitory In the ICU that cared for the patient, 153 patients were admitted for severe COVID-19

between March 2020 and February 2021, of whom 84 were mechanically ventilated. Among these latter patients, five (6.0%) developed CAPA and one (1%) developed a mixed mold infection, highlighting the rarity of this triple association. No other mucormycosis cases were reported.

References for this review were identified by searching the PubMed database using a combination of title keywords that referred to mixed mold infection (mucormycosis AND (aspergillosis OR aspergillus), mixed fungal infection, mixed mold infection). Thirty five cases reporting Aspergillus and Mucorales were analyzed ( Table 2 ). These cases were Sinuses, brain, and skin were involved in 11 (31%), 4 (11%), and 2 (6%) cases, respectively.

When identified to the Aspergillus species level (n=22; 69%), A. fumigatus was isolated in 15 (69%) patients followed by A. flavus (n=5; 23%), and A. niger (n=3; 14%). In two cases, multiple Aspergillus species were isolated. Mucorales were rarely identified to the species.

The most frequently identified genera were Mucor (n=11; 31%) and Rhizopus (n=8; 23%) followed by Lichteimia (n=4; 11%), Cunninghamella (n=3; 9%) and Rhizomucor (n=2; 9.1%).

In fifteen cases, Mucorales order members were only identified upon histopathological biopsy examination. In twelve (34%) cases, aspergillosis was diagnosed before mucormycosis and voriconazole was prescribed, and the patient was subsequently switched to amphotericin B. Overall, no specificities emerged from these cases compared to other patients with mixed Aspergillus/Mucorales infection.

Here, we report the case of a patient with severe SARS-CoV-2 infection with the probable complications of CAPA and CAM.

One important aspect of our case is the co-infection with both A. welwischiae and R. delemar.

Aspergillus and Rhizopus are two fungal genera with an angioinvasive ability. This association has been rarely described in the immunocompromised subject with an often fatal outcome in pulmonary and brain localizations ( Table 2 ). More cases are described in case series of mucormycosis with up to 44.4% of Aspergillus co-infection [12] . Furthermore, the VITAL trial that assessed the efficacy of isavuconazole for treatment of mucormycosis also reported six cases among 37 patients, suggesting that this co-infection may be underdiagnosed [13, 14] . The triple association COVID-19/Aspergillus/Mucorales has been reported in severely immunocompromised patient after stem cell transplantation and in patients with diabetes mellitus [15, 16] . In one other case, a mixed mold infection was reported in a patient receiving high-dose corticosteroids for dermatomyositis [17] .

There have been several publications on the subject of COVID-19 and mold co-infection since the beginning of the first COVID-19 pandemic wave. The main co-infection that was studied was aspergillosis [7, 18, 19] , and these studies highlighted several potential risk factors including intubation and mechanical ventilation, high-dose corticosteroids [20, 21] , azithromycin for ≥3 days [22] , tocilizumab [23, 24] , and immunological storm including high inflammatory cytokine levels [18] . CAM was reported less frequently especially in the first two COVID-19 pandemic waves, but it appears to be an increasing problem in India [25] .

Although mucormycosis and mixed Aspergillus-Mucorales infection have been described less frequently, risk factors are expected to be similar [25, 26] . [27] were developed and used to identify invasive fungal diseases. These classifications are AspICU [28] , an influenza-associated pulmonary aspergillosis classification developed by Verweij et al. [29] , and most recently, a classification that was proposed by ECMM/ISHAM [7] . Both aspergillosis and mucormycosis are consistent with putative or probable CAPA or CAM because both BAL direct examination and culture results were positive. Additionally, a CT-scan showed patterns that were compatible with invasive fungal disease. Furthermore, a highly positive galactomannan index in both blood and BAL and a positive Mucorales PCR result in the blood strongly suggested an invasive disease. Therefore, the patient could have been classified as having a CAPA and CAM infection. However, careful investigation of our patient's corticosteroids doses indicated that he met the EORTC criteria [27] because he received a prednisone equivalent dose >0.3 mg/kg/day for 3 weeks (0.08 mg/kg/day of dexamethasone for 8 Days [0.6 mg/kg/day prednisone equivalent], 0.3 mg/kg/day of dexamethasone for 20 Days [1.9 mg/kg/day prednisone equivalent], and a methylprednisolone bolus of 120 mg [150 mg

prednisone equivalent]) [27, 30] . Therefore, a careful evaluation of the cumulative dose is required before concluding that the patient has no known EORTC/MSGERC risk factor for invasive mold infection.

Aspergillus section Nigri is also worth discussing because it is rarely responsible for infection compared to Aspergillus fumigatus due to its physiological characteristics such as its large conidia, which makes it more difficult to reach alveoli, and its optimal germination temperature, which is approximately 30°C [31] . Invasive aspergillosis due to A. section Nigri is mostly reported in severely immunocompromised patients [32] . One other case report describes a fatal CAPA infection due to A. section Nigri with a high galactomannan index at Day 10 after ICU admission [33] . The delay between hospitalization in the ICU and CAPA infection of 19 days was longer than the median time that was published in CAPA cohorts of approximately 6 days [29] . This could be explained because reaching a critical dose of corticosteroids was required for invasive disease to develop in a previously immunocompetent patient.

Molecular biology tools to diagnose invasive fungal infections (IFIs) have only recently been developed. Aspergillus qPCR have been included in international guidelines for invasive aspergillosis since 2020 [27] , and they are included when making a CAPA diagnosis [7] .

However, detection of circulating Mucorales DNA (cmDNA) is not recommended by default to diagnose mucormycosis [34] despite data showing a high sensitivity and its ability to predict a diagnosis and quantify the fungal burden [9] . The important angioinvasive ability of Mucorales makes it possible to detect the fungus in blood samples. Regular screening of atrisk patients such as severely burned patients suggests that cmDNA detection allows an earlier diagnosis of invasive mucormycosis in this population and earlier treatment initiation [35] .

Unlike galactomannan detection for the diagnosis of aspergillosis, there are no tools that target the Mucorales antigens, which emphasizes the need to include cmDNA detection in the mucormycosis diagnosis standards. This could be considered to be a screening tool for COVID-19 patients who are clinically worsening despite an appropriate standard of care and who have additional risk factors such as uncontrolled diabetes or high-dose corticosteroids.

Finally, there are no guidelines or standard practice for IFI management in COVID-19 patients, and clinical effectiveness of antifungal administration in these cases has not been demonstrated [7] . The treatment that is being promoted for patients with CAPA is intravenous voriconazole or isavuconazole, the latter of which covers the Mucorales species and Aspergillus species, and is showing promise in mixed mold infections [7, 14] . In 34.2% of coinfections that were analyzed in this literature review, Aspergillus was identified and treated first with voriconazole suggesting that the mucormycosis may be a breakthrough IFI that could be avoided using isavuconazole as a first-line treatment. Our patient was treated with voriconazole, against which Rhizopus delemar has high a MIC, which was confirmed by our data [36] . He was later switched to liposomal amphotericin B after Mucorales-type mycelium was identified in the direct BAL examination. However, this antifungal modification may have occurred too late in the infection's course considering that the serum PCR was retrospectively found to be positive 6 days before the antifungals were changed.

The SARS-CoV-2 virus has highlighted the existence of multiple fungal superinfections in patients who were not previously immunocompromised and who did not have common risk factors for invasive mold disease. However, the cumulative steroid dose for concomitant COVID-19 infection should be considered to be a risk factor for fungal infection in these patients. These superinfections, particularly CAPA and mucormycosis and the association between the two infections, as shown in the case of our patient, require adapting the management of these patients by screening respiratory and serum samples using biomarkers.

The authors have nothing to declare

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We thank Dea Garcia-Hermoso, Cécile Gautier and Agathe Bertho from the French national reference center for invasive fungal diseases and antifungals for their expertise. We thank all staff, nurses and lab technicians who were essential to patient care in intensive care and medical mycology from Saint-Louis Hospital and Lariboisière Hospital.