Schizophyllum spp. in Covid-19-A Series of Ten Cases from a Tertiary Care Hospital of Central India.

Background: Schizophyllum species can cause diverse clinical infections like sinusitis, pulmonary disease, ulcerative lesions of the palate, atypical meningitis, cerebral abscess which can occur in both immunocompetent and immunocompromised individuals. Although it is one of the emerging fungi, data on clinical features and


Introduction
Severe Acute Respiratory Syndrome-Corona virus-2 (SARS-CoV-2) is responsible for lower respiratory tract infection which can lead to diffuse alveolar damage with severe inflammatory exudation and altered immune response [1].
Covid patients accompanied with comorbidities and immunocompromised state such as diabetes, prolonged neutropenia, Glucocorticoid use, transplantation, inherited or acquired immunodeficiencies, and tumour are more likely to develop different fungal co-infection.
Schizophyllum spp. is a mold of phylum Basidiomycota, Schizophyllaceae family distributed widely in nature and grows on rotting wood and other plants [2,3].This fungus is rarely reported in humans because it is difficult to diagnose in many clinical settings.It has been recognized as the etiological agent of a variety of hypersensitivity disorders, such as allergic sinusitis, bronchopulmonary mycoses, chronic eosinophilic pneumonia, and of invasive infections such as fatal disseminated infections involving the lungs, orbit, and brain [4,5].
Among them, bronchopulmonary infections and sinusitis account for more than 90% of the reported cases worldwide [6].The localisation or spreading of the infection from the original site to other tissues and organs depend on the factors such as the immune status of the host, deviation of the nasal septum, use of corticosteroid therapy and duration of exposure to the spores [4].This fungus is rarely reported till date as a causative agent of fungal co-infection in Covid-19 patients.To the best of our knowledge, this is the first study from this region of India reporting a series of ten cases of schizophyllum infection associated with Covid-19 patients during the 2 nd wave of the pandemic.

METHODOLOGY
This study was conducted in the Diagnostic Microbiology Laboratory of a tertiary care hospital in central India.We received nasal scrapings of suspected patients from various departments during April to September, 2021.Demographic details, detailed medical history, clinical features, COVID-19 infection and treatment history, final clinical diagnosis and treatment outcome were recorded for all the patients.Standard imaging was done for all patients which included Computed tomography (CT) and magnetic resonance imaging (MRI) of the paranasal sinus( PNS), Chest and orbit and the findings are mentioned in the table no-1.
Nasal scrapings were processed for potassium hydroxide (KOH) mount, Gram stain and Fungal culture according to the standard procedures.The specimens were inoculated on two Sabouraud's Dextrose Agar (SDA) slants, one incubated at 37•C and another at room temperature.From a fungal growth observed, slide culture was prepared on corn meal agar and incubated at room temperature in wet chamber and observed for sporulation on alternate days.Findings on Lactophenol Cotton Blue (LPCB) mount and microscopic characteristics of the slide culture were used for the presumptive diagnosis.The suspected cultures were kept in dark for better growth with enhanced pigment production.
Hyaline septate branched hyphae of variable thickness (figure no-1) were found on KOH mount.On SDA, spreading, woolly, whitish to pale colored growth was seen after 3-4 days with characteristic droplets of exudate.(Fig no -2) Later it became dark brown and spreading over the surface of the slant.It became rough and leathery with enhanced reddish pigment when grown in the dark at room temperature (figure no-3).On microscopy, branched septate hyphae was found with clamp connection morphologically suggestive of Schiziphyllum spp.(figure no-4)

Results
The patients included in this case series were from the second wave of the covid pandemic and all of them were post covid.Most of the patients were from the age group of 30 to 70 years with male preponderance.Paranasal symptoms alone were present in 50% patients followed by ophthalmological symptoms in 40% patients.Both these symptoms were found in 80% of the patients.
Majority of the patients had comorbidities like diabetes, hypertension and history of steroid therapy.Seven of them were treated surgically with endoscopic debridement followed by liposomal amphotericin-B and oral posaconazole for 14 days and the remaining three patients were treated with only antifungals like Amphotericin-B, posaconazole and itraconazole.After clinical improvement all the patients were discharged from the hospital.Detailed history of all the patients is mentioned in table no-1.

Discussion
In COVID-19 patients, overexpression of inflammatory cytokines, and impaired cellmediated immune response with decreased CD4 + T and CD8 + T cell counts make them susceptible to fungal co-infection [7].The combination of diabetes, immunosuppression and increased use of corticosteroids to combat infection with COVID-19 appears to be the additional factors increasing the risk of development and aggravating existing opportunistic fungal infections like Schizophyllum spp.[8].There is increase in the frequency of fungal infections caused by basidiomycetes particularly, Schizophyllum spp.and it has become a much more common human pathogen in both immunocompetent and immunocompromised hosts [9,10].
CT PNS of seven of the patients was suggestive of sinusitis.Studies from different countries have reported Schizophyllum as a causative organism of fungal sinusitis in both immunocompromised and immunocompetent patients [11][12][13][14][15].In this study, all the ten patients were post-covid with the history of corticosteroid therapy.Ophthalmological infection caused by Schizophyllum spp.also reported by Sa HS et al. in 2012, Kaur et al. in the year 2020 [11,13].Similarly, there is case report of pneumonia caused by S. commune in a immune compromised individual of acute myeloid leukemia [16].
It has clinical similarity with aspergillosis and mucormycosis and clinically it is very difficult to differentiate them.There was a sudden outbreak of mucormycosis during the second wave of covid-19 and these ten patients were clinically suspected to have mucormycosis.Hence, seven of them were treated with endoscopic debridement followed by liposomal amphotericin B and oral posaconazole therapy.Surgical debridement and antifungal therapy are the treatment of choice in invasive fungal sinusitis and the treatment outcome is found to be good.However, the type, dose and duration of antifungal agents to be administered remain uncertain as, currently no sufficient data available regarding epidemiological cutoff values or clinical breakpoints for Schizophyllum spp.[17].Drugs known to have anti-mold activity are considered as favorable antifungal agent and recommended as the initial choice in patients with respiratory tract invasive fungal disease caused by Schizophyllum [16].Cases of allergic bronchopulmonary mycosis caused by Schizophyllum were successfully treated with oral Itraconazole therapy and recrudescence of the disease was prevented for four years [18].

Conclusion
During the second wave of Covid-19, infections showing similar symptoms were misdiagnosed clinically as Mucormycosis and treated accordingly.But unlike mucor, it has good prognosis with proper oral antifungal therapy.We want to emphasize that accurate identification and proper treatment approach can prevent the disease burden caused by Schizophyllum spp.Hence, broad knowledge of different clinical presentations and high index of suspicion are required to ensure accurate diagnosis and correct treatment in immunocompromised patients.

Limitations of the study:
Elaborate species identification could not be done due to constrained resources in the laboratory.
Highlights: Schizophyllum spp.Can be a causative fungal agent in post-covid patients showing similar symptoms as mucormycosis.