Clinical Characteristics And Outcomes of 16 Cases With COVID19 and Mucormycosis: Experience From A Tertiary Care Center In India and Review of Literature

Background: - The sharp uptick in the cases of mucormycosis in the background of the COVID19 pandemic is a cause of concern and the reasons and it’s impact remains to be seen. We studied the clinical characteristics in patients with mucormycosis and COVID19 co-infection and performed a literature review. Methods: - This retrospective study was conducted at tertiary centre in India. All patients admitted with COVID19 and mucormycosis were included, clinical details were obtained from hospital records. We did review of literatures using the terms “SARS-CoV2” OR “COVID19” AND “Mucormycosis” AND “co-infection” on Pubmed published before February 20, 2021. Results: - Sixteen cases (M:F–13:3), mean age 46·5 years (24-75years), were included. Fourteen had known risk factors for mucormycosis, the most common being diabetes mellitus. Most patients (n=14) were symptomatic with mucormycosis before diagnosis of COVID19. There was delay in surgery by 22.5 days (IQR–>17.75–29.5), pending SARS-CoV-2 RT-PCR negativity. There were six deaths in this cohort, unrelated to the COVID19 severity. The literature review revealed eleven case reports on co-infection. Patients who had developed mucormycosis were found to have history of mechanical ventilation. Conclusion: - The apparent increase in the incidence of mucormycosis may be due to decompensation of underlying comorbidities (decreased access to healthcare), and increased use of immunosuppressants in COVID19. Patients with co-infection were noted to have poorer outcomes.


Introduction
Even after more than one year following the origin of the pandemic the pathogenesis of COVID19 remains partially understood and understanding of the same continues to evolve with time. As the highly infectious virus continues to give rise to new cases globally, one of the biggest concerns is co-existing infections. Co-infections or superinfections are deemed to occur as the pandemic tends to overlap with geographical realm and seasonality for few infections or due to the encounter with other community or nosocomial pathogens (during hospitalisation for COVID -19). However, information on co-infections in COVID19, especially with relation to invasive fungal infections, is lacking.
Multiple cases of invasive pulmonary aspergillosis (CAPA -COVID19 associated pulmonary aspergillosis) and invasive candidiasis (CAC -COVID19 associated candidiasis) have been reported in patients with severe COVID19 (1). This has been linked to the use of steroids, long-term antimicrobials, prolonged mechanical ventilation, and the extended duration of hospital stay in these patients. There is growing concern over the increase in cases of mucormycosis in COVID19 as well. Mucormycosis is an angio-invasive disease caused by opportunistic fungi of the order Mucorales in immunocompromised patients.(2) Untreated mucormycosis is almost always fatal. It remains to be seen if this increasing incidence of mucormycosis in COVID19 is related to the illness itself, the steroids and immunomodulators administered for treatment, or the worsening of underlying predisposing factors in the socio-economic upheaval caused by the pandemic. Further, it is unknown how the co-existence of COVID19 would affect the natural history and treatment of patients with mucormycosis.
To answer some of these questions, we performed a retrospective clinico-epidemiological study on patients with COVID19 and mucormycosis co-infection.

Methods
This was a retrospective, observational study, conducted at All India Institute of Medical Sciences, New Delhi, India. Patients admitted between March 20, 2020 and January 31, 2021 with current, recent, or remote history of COVID19 AND diagnosed mucormycosis were included in the study. The diagnosis of mucormycosis was based on either the histopathological demonstration of hyphae consistent with mucormycetes, i.e., hyaline, broad, aseptate, or pauciseptate, ribbon-like hyphae with right/obtuse-angled branching in the hematoxylin and eosin (H & E)/ Periodic Acid Schiff's (PAS)/ Gomori's methenamine silver (GMS) staining or on culture isolation of the mucormycetes, in the tissue specimens obtained from suspected sites, as per the ECMM/MSG-ERC-2019 (European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium) diagnostic criteria.
Patients without microbiological or histopathological evidence of mucormycosis were excluded from the study. Written informed consent was obtained in all cases. Hospital records were scanned, and missing details were lled in through telephonic or direct interviews with patients. Information on demography, clinical features, investigations, treatment, and outcomes was collected. Mucormycosis was classi ed based on the site of involvement into sino-nasal disease, rhino-orbital, rhino-orbito-cerebral, or pulmonary disease. Sino-nasal disease was de ned as infection limited to sinus and nasal cavities only without orbital or intracranial extension. Rhino-orbital or rhino-orbital-cerebral disease included fungal invasion into orbit or intracranial cavity to involve orbital contents or intracranial structures, respectively. Orbital involvement was de ned as vision diminution or ophthalmoplegia and de nitive radiological evidence of the same. Brain parenchymal involvement was de ned as focal neurological de cits or altered mentation with radiological evidence of intracranial extension. Pulmonary disease was de ned as radiological, histopathological, or microbiological evidence of fungal infection in the lung parenchyma. The protocol for the study was approved by the Institutional Ethics Committee.

Results
Between March 20, 2020 and January 31, 2020, 6250 cases of COVID19 were admitted at our centre. Of these, sixteen patients were (M: F 13:3) with a mean age of 46·5 years (SD, 14·5) were diagnosed with mucormycosis and were included in our study (Table 1). Fifteen patients had one or more comorbidities (Table 3), most commonly uncontrolled diabetes mellitus (n = 12), chronic kidney disease (n = 2), solid-organ transplantation (n = 1), hematological malignancy (n = 1), decompensated chronic liver disease (n = 1) and chronic granulomatous disease (n = 1). Three out of twelve diabetic patients had presented with diabetic ketoacidosis.  Pulmonary Tuberculosis (MDR-TB) 1 (6.25%) All patients had con rmed COVID19 illness using SARS CoV-2 RT-PCR on nasopharyngeal or oropharyngeal swabs. Two patients were diagnosed with mucormycosis during convalescence from COVID19, whereas the rest had features suggestive of mucormycosis even before the diagnosis of COVID19. The median duration of symptoms was 15 days (range, 6-60 days). Laboratory abnormalities have been summarised in Table 2. The severity of COVID19 ranged from asymptomatic infection (n = 5, 31·25%), mild (n = 7, patients, 43·75%), moderate (one patient, 6·25%), and severe COVID19 requiring steroids and anticoagulation (n = 2, 12·5%). Most of our patients had sino-nasal mucormycosis (n = 15) with or without orbital or intracranial involvement, with only one patient with pulmonary disease. Out of the fteen patients with sino-nasal disease, ve patients had localised disease, three had orbital involvement, and seven patients had evidence of intracranial spread. The diagnosis was established by demonstrating the fungal hyphae, in KOH mount and calco uor uorescent staining, with the characteristic feature of mucormycetes, while the fungus could be cultivated and identi ed as Rhizopus arrhizius in two cases and Rhizopus spp. in one case. While six cases had histopathological features of acute disease process i.e., coagulative necrosis or angioinvasion or perineural invasion, seven cases had granulomatous in ammation with giant cells, features suggestive of chronic mucormycosis. Radiological (CECT or CE-MRI) ndings have been summarised in Table 1. All patients were managed with Liposomal Amphotericin B (Fungisome) with or without surgical debridement. It was started at a dose of 3 mg/kg and escalated to 5 mg/kg if tolerated well. Adverse events necessitating alteration or discontinuation of therapy were nephrotoxicity (n = 9, 56·25%), hypokalaemia (n = 8, 50%), hypomagnesemia (n = 2, 12·5%) and bone marrow suppression (n = 2, 12·5%). Surgical intervention was done following two consecutive negative SARS CoV-2 RT-PCR results (at least 24 hours apart). Twelve patients (75%) underwent surgery, two patients had fulminant disease and succumbed before medical stabilisation, and surgical intervention could be undertaken, one patient was planned for surgery, and one patient was lost to follow up. Surgical approaches included maxillectomy, orbital exenteration, and functional endoscopic sinus surgery, alone or in combination. The median duration of SARS CoV-2 RT-PCR positivity was 16 days (IQR 10-24 days). The median duration between diagnosis of mucormycosis and surgical debridement was 22·5 days (IQR 17.75-29.5 days).
Out of the sixteen patients, ve patients required admission to the intensive care unit (ICU), two due to severe COVID19, two patients due to diabetic ketoacidosis with severe metabolic acidosis, and one due to sudden drop in sensorium (due to progressive mucormycosis) ( Table 1). The median duration of hospitalization was 40·5 days (IQR 27-49 days). There were six (37·5%) mortalities in this cohort, all of them attributable to mucormycosis. Seven patients (43·75%) patients have been discharged with step-down long term maintenance therapy with oral Posaconazole tablets (at a dosage of 300 mg OD). Two patients (6·13%) were still admitted at the time of writing.
A literature search on pubmed for articles, published before February 20, 2021, using the terms "SARS CoV2" OR "COVID19" AND "Mucormycosis" AND "coinfection", yielded 11 published cases of COVID19 and mucormycosis. We analysed these twenty-seven cases (eleven case reports, sixteen cases from our study and divided them into categories based on if they developed mucormycosis before or after the COVID19 infection (Table 5). In patients with pre-COVID mucormycosis, sino-nasal disease (with or without orbital or cerebral involvement) was the most involved site, whereas in patients with post-COVID mucormycosis, the most common site was the lungs. Moreover, the need for mechanical ventilation was more common in post-COVID patients (p < 0.05).

Discussion
This study summarizes the experience of co-existing mucormycosis and COVID19 from our center and those reported in the literature so far. The incidence for the speci c at-risk population for mucormycosis varies between 0·005 to 1·7 per million population (2). A recent multicentric study on the epidemiology of mucormycosis from India reported an increase in incidence rates from 24·7 to 89 cases per year (3); while another single-center based survey on people with diabetes reports an incidence of 0.15 % (4). Only a few cases of COVID19-mucormycosis co-infection have been reported in literature to date (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In our centre, the occurrence rate of co-existing COVID19-mucormycosis was 2·56 per 1000 COVID19 patients; the incidence rate of mucormycosis following COVID19 was 3·2 per 10,000 COVID19 patients.
Comparing with the data prior to year 2020, current data is suggestive of increasing case of mucormycosis. There may be many causes for the increase in the incidence of COVID19. As part of the pandemic control strategy, active tracing of all contacts of COVID19 cases was being carried out along with screening of all cases, presenting to out-patient or emergency services, for SARS CoV-2 infection. As per national recommendations, all patients who tested positive were admitted regardless of symptomatology (17). This possibly explains the clustering of cases with mucormycosis in hospitals that were otherwise asymptomatic or previously undiagnosed. In our study, fourteen patients had symptoms suggestive of mucormycosis at admission, while the remaining two patients developed rhino-orbital mucormycosis nearly one month after the resolution of COVID19. The duration of diagnosis was variable, ranging from one week to three weeks.
Common mucormycosis (CM) occurs following inhalation of spores or inoculation of spores in wounds in icted during trauma e.g., bomb blast injuries or road tra c accidents (18, 19). However, hospital acquired mucormycosis (HAM) secondary to surgical intervention, adhesive bandages, wooden tongue depressors, osteomy bags or contaminated linen etc. also has been reported (20,21). Most common sites involved in HAM were skin followed by intrabdominal disease. The increased incidence of mucormycosis in the COVID19 may have many causes. The pandemic has caused widespread sociological and economic disturbances, severely restricting adequate access to healthcare. This has caused decompensation of previously well-controlled comorbidities in patients with chronic illnesses, like diabetes and chronic kidney disease. Furthermore, the incidence of hospital-acquired mucormycosis (HAM) has increased due to more exposure of at-risk individuals to Mucorales, in the setting of an altered host-immune response. Pre-existing conditions such as uncontrolled diabetes mellitus, solid organ transplants, haematological malignancy, neutropenia, or use of immunosuppressants predispose to the development of mucormycosis. It is noteworthy that exposure to molds is common in ICUs or wards close to construction sites, which was common in our case, where several newly built or previously unused buildings were repurposed as COVID19 facilities. Moreover, contaminated air, equipment, or linen may serve as sources of spores. Interventions such as swab tests (along with use of wooden tongue depressors), Ryle's tube insertion, insulin injections, etc. serve as a mode of inoculation into human tissues. The situation is further complicated using steroids or immunomodulators for severe COVID19, worsening glycaemic status, rampant use of antibiotics and voriconazole (empirically for CAPA), prolonged mechanical ventilation as well as prolonged hospitalisation.
In our cohort, sites of involvement were sino-nasal (with or without orbital or cerebral involvement) or lung parenchyma. Of the two patients (at our center) who developed mucormycosis following COVID19, one on immunosuppressants for renal transplant (and had received steroids for severe COVID19), and the other had end-stage renal disease. Both patients were diagnosed with rhino-orbital mucormycosis.
Amongst the cases reported in the literature, eight patients had risk factors for mucormycosis, i.e., diabetes mellitus (n = 6) and hematological malignancy (n = 2) (Table 4). Moreover, eight cases received steroids for moderate to severe COVID19. Six had pulmonary mucormycosis, four had rhino-orbital-cerebral disease, and one patient had gastro-intestinal mucormycosis. Ten patientswere on mechanical ventilation before the diagnosis, while no such intervention was noted in the one with gastro-intestinal mucormycosis. Noteworthy here is the case reported by Pasero et. al. who developed pulmonary mucormycosis without any known risk factors; severe COVID19 and mechanical ventilation are potential risk factors in this case. Mechanical ventilation was a signi cant risk factor for developing post-COVID mucormycosis (Table 5). We did not nd any patient developing mucormycosis, following COVID19, in the absence of obvious risk factors or severe COVID19 (possible use of steroids, mechanical ventilation). Lymphopenia is a common nding in COVID19, than neutropenia, whether COVID19 illness itself predisposes to mucormycosis is unclear yet (22,23). According to one study, a protective role of Mucorales speci c CD4 and CD8 T cells have been hypothesized during active mucormycosis (24). However, it will be interesting to know, whether COVID-19 related lymphocytopenia, results in impaired T cell Mucorales speci c response and predispose to mucormycosis (25). It is worth noting that thirteen patients (81·25%, n = 16) had an absolute lymphocyte count less than 1500 cells/cumm (Table 2), however, the difference was not signi cant between pre-COVID and post-COVID mucormycosis cases (Table 5). The starkest aspect of co-existing mucormycosis and COVID19 was the impact on the management of mucormycosis. Mucormycosis is a fulminant disease and a surgical emergency. Its management requires a combination of extensive surgical debridement to remove infected tissue as well as medical therapy (2, 18, 26). Despite both surgery and antifungal, it is associated with high mortality and morbidity. It has been observed that surgery performed during active COVID19 disease is associated with poor outcomes (27,28). Furthermore, there is a risk to operating room personnel during surgery due to aerosol-generating procedures like intubation and endotracheal tube suction. A general consensus during COVID-19 pandemic is to postpone all elective surgeries, while all emergency surgery requires utmost consideration to indication, urgency of surgery, surgical approach, risk of aerosol spread, and availability of staff and resources (29,30). All our patients were started on medical management with antifungal agents at diagnosis. We found that in most cases, surgery was delayed till medical stabilisation and RT-PCR negativity. The median duration between diagnosis of mucormycosis and surgical debridement was 22·5 days (IQR 17.75-29.5 days). Surgery was performed in twelve cases. Two patients deteriorated rapidly and could not be taken up for surgery; one was lost to follow up, and one patient was awaiting surgery following medical stabilisation. Our cohort had six mortalities, including four patients who had undergone surgical debridement. Of these, ve were of sino-nasal mucormycosis, while one patient had pulmonary involvement. In reported literature, two out of eleven cases, had rapidly worsening severe COVID19 and succumbed to severe illness before surgery or medical therapy. Other patients were managed medically with one or more antifungals, i.e., Liposomal Amphotericin B or isavuconazole. Surgery was undertaken in only three patients, while the rest were deemed medically un t for surgical intervention. Ten out of eleven patients died despite medical therapy and surgery.
This study is the largest account of COVID19 and mucormycosis co-infection available in the literature. Having been done at a tertiary care centre equipped to diagnose and treat both COVID19 and mucormycosis with alacrity, our study is uniquely equipped to identify disease-related, patient-related, and treatmentrelated factors leading to poor outcomes. Limitations of our study include a descriptive design. The lack of a non-COVID19 infected control group limits our ability to distinguish COVID19 speci c considerations in the broader background of the socio-economic upheaval caused by the pandemic. Further research is required to establish if COVID19 itself predisposes to mucormycosis.

Conclusion
There has been an apparent increase in the incidence of mucormycosis in patients of COVID19. The causes for this are multi-factorial, including decreased access to healthcare leading to decompensation of underlying comorbidities, increased exposure to healthcare facilities leading to more Hospital Acquired Mucormycosis (HAM), and increased use of immunosuppressants in the setting of COVID19. Patients with mucormycosis with COVID19 co-infection have poorer outcomes which may be related to immunosuppression administered for the COVID19 infection and delays in access to adequate surgical management. Declarations