European Journal of Cardiovascular Medicine

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with a wide range of opportunistic bacterial and fungal infections [1]. Both Aspergill us and Candida have been reported as the main fungal pathogens for co-infection in people with COVID-19 [2]. Recently, several cases of mucormycosis in people with COVID-19 have been increasingly reported world-wide, in particular from India. The primary reason that appears to be facilitating Mucorales spores to germinate in people with COVID-19 is an ideal environment of low oxygen (hypoxia), high glucose (diabetes, new-onset hyperglycemia, steroid-induced hyperglycemia), acidic medium (metabolic acidosis, diabetic ketoacidosis [DKA]), high iron levels (increased ferritins) and decreased phagocytic activity of white blood cells (WBC) due to immunosuppression (SARS-CoV-2 mediated, steroid-mediated or background comorbidities) coupled with several other shared risk factors including prolonged hospitalization with or without mechanical ventilators. [3]

Globally, the prevalence of mucormycosis varied from 0.005 to 1.7 per million population, while its prevalence is nearly 80 times higher (0.14 per 1000) in India compared to developed countries, in a recent estimate of year 2019–2020. [4] In other words, India has highest cases of the mucormycosis in the world. Notwithstanding, India is already having second largest population with diabetes mellitus (DM) and was the diabetes capital of the world, until recently. [5] Importantly, DM has been the most common risk factor linked with mucormycosis in India, although hematological malignancies and organ transplant takes the lead in Europe and the USA. [6] Nevertheless, DM remains the leading risk factor associated with mucormycosis globally, with an overall mortality of 46% [7].

Indeed, presence of DM was an independent risk factor (Odds ratio [OR] 2.69; 95% Confidence Interval 1.77–3.54; P < 0.001) in a large 2018 meta-analysis of 851 cases of rarely occurring mucormycosis, and the most common species isolated was Rhizopus (48%) [8]. While long term use of corticosteroids have often been associated with several opportunistic fungal infection including aspergillosis and mucormycosis, even a short course of corticosteroids has recently been reported to link with mucormycosis especially in people with DM. A cumulative prednisone dose of greater than 600 mg or a total methyl prednisone dose of 2–7 g given during the month before, predisposes immunocompromised people to mucormycosis [9]. There are few case reports of mucormycosis resulting from even a short course (5–14 days) of steroid therapy, especially in people with DM [10]. Surprisingly, 46% of the patients had received corticosteroids within the month before the diagnosis of mucormycosis in the European Confederation of Medical Mycology study [11].

This prompted us to conduct a study and to published research work of mucormycosis in people with COVID-19, to know its temporal associations in relation to comorbidities, association with drugs being used in COVID-19 and overall characteristics of patients with its outcome. We additionally postulated a mechanistic explanation as to why mucormycosis could be increasingly linked to COVID-19 and is being reported increasingly from India.

The study was carried out in the Department of Medicine, Shadan Institute of Medical Sciences, Teaching Hospital and Research Center. All consecutive samples that were received during April–July 2021 from private facilities or from the Department of ENT of the hospital were included in the study. The demographic and clinical details of the patients such as signs, symptoms, site, imaging findings, laboratory parameters, such as complete blood count, blood sugar levels, C-reactive protein (CRP), serum ferritin, d-dimer, and KOH wet mount preparation, were noted in predesigned forms from the patient’s clinical sheets and the histopathology request form. The patient’s follow-up details as they survived or died were also noted. Patients who had antifungal treatment (oral or intravenous) within 72 h prior to surgical debridement were also included in the study.

Microdebrider and forceps were used for good debridement of the tissue. Adequate representative samples (volume of tissue ranging from 2 to 16 g) were sent for histopathology in 10% neutral-buffered formalin. The tissue was examined for gross characteristics. At least 10 g of tissue was submitted in five blocks (2 g of tissue per cassette) for processing and if more tissue bits were available, an additional sample was processed when required (no invasion was seen on histology). The specimen for the KOH mount was sent separately in normal saline. Cases with non-invasive lesion on histology, insufficient tissue (less than 2 g) for grading and additional scoring, or specimen with only nasal crust removal were excluded from the final analysis. Patients whose survival follow-up data were missing were also excluded from the final analysis.

Five-micrometer-thick sections were stained with routine hematoxylin and eosin (H&E) stain and periodic acid Schiff (PAS) stain for histopathology evaluation and classification. The final diagnosis of the lesion was established on the basis of the histopathology characteristics. Patients with clinical features suggestive/suspicious of mucormycosis and showing the histopathology features of broad aseptate ribbon-like fungal hyphae with 90° branching were further evaluated. The slides were examined independently and blindly by three different pathologists. If there were differences in the classification between two pathologists, the histopathology findings were reviewed and discussed with the third pathologist to achieve consensus.

The grading of severity classification of mucormycosis infection was assessed for the following four parameters: (1) neutrophilic inflammatory cell infiltrates were assessed in tissue bits showing the presence of fungal hyphae in ×400 microscopic field; (2) degree of tissue necrosis was defined as the presence of non-viable tissue with fungal hyphae and was quantified as the percentage of the whole tissue under ×100 showing necrosis; (3) the fungal load was quantified as the number of ×400 microscopic fields showing fungal hyphae; and (4) number of blood vessels involved under ten ×400 microscopic fields. A consensus score of 1 to 3 or 1 to 2 was given on the assessment of microscopic examination of each parameter and the lesion was classified by adding up the scores. The lesion that scored in the range of 4–5 was graded I, 6–8 was graded as II, and grade III was given for a score of 9–11.

Table 1. Demographic, clinical and investigation details of post-COVID-19 mucormycosis patients (N = 70).

A male predominance (86%) was observed with a male to female ratio of 2.8:1 (Table 1) at an age range of 26–75 years (mean age 46.8 ± 11 years). All patients had a history of COVID-19 disease in the last 2 months. Maximum cases (94.3%) presented within 20 days of COVID-19 treatment/ recovery. All patients presented with one or other local or constitutional symptoms or signs. The most common complaint at the time of presentation was local facial pain (92.9%), swelling of the cheek (67.1%), and eye pain with periorbital swelling (35.7%). Other complaints at the time of presentation were epistaxis (in one patient with ipsilateral eye involvement), fever, numbness on the cheek skin, and moderate-to-severe toothache with or without loosening of the tooth. Most (87.1%) of the patients had symptoms for 5 days.

Table 2. Histopathology score and grades of severity in post-COVID-19 mucormycosis patients (N = 70).

According to the histology criterion, the severity of the grade III infection was detected in 48.6% of the cases, grade II in 40% of the cases, and grade I in 5.7% of the cases (Table 2).

Mucor is a saprophytic fungus that resides in soil, dung and dust. Mucormycosis is an extremely rare infection in healthy individuals. Most researches on post covid mucormycosis revealed that elderly immunocompromised individuals are prone to develop this opportunistic infection. According to study by Maini et al., 2021, most common comorbidity associated with mucormycosis is diabetes mellitus which is comparable with the present study. [12] Most common site of involvement is rhino-orbital region. Rare sites include appendix. The evidence on relationship between covid vaccination status and severity of post covid mucor mycosis is minimal. In majority of covid related mucormycosis studies, vaccination status of the study population was not analysed. [13]

Rapid growth of fungus, ability to utilize the host iron for growth, ability to adhere to the endothelial surface and downregulation of host defense genes responsible for immune defense are considered to be responsible for the virulence and pathogenesis of mucor infection (Jose A et al., 2021). [14] According to the study by Choksi et al., on outcomes of rhinoorbital mucormycosis following COVID 19 infection, 36% of patients were died on follow up. It was comparable with the present study where 40 % patients died on follow up. [15]

Microscopic identification of the hyphae based on diameter, presence or absence of septae, branching angle and positive staining for fungal stains like GMS differentiate it from other fungal infections. Most common coexistent fungal infections are candida and aspergillus (Pakdel F et al., 2021). [16] The associated different tissue reactions like suppuration, chronic granulomatous inflammation and foreign body giant cell reactions were not analysed in other studies. Tissue destruction, angioinvasion and bone invasions are characteristic of mucor infection. In our literature search, it was found that all patients were managed with either intravenous amphotericin B injection or surgical debridement of affected site.

Despite of these aggressive clinical management, a significant proportion of patients succumbed to death. That may be due to complications associated with mucormycosis especially intracranial spread. However, histopathological examination of the tissue sections have a significant role in early identification which aids in appropriate clinical management.

Mucormycosis is a rare but fatal fungal infection that should be kept in mind in covid 19 recovered patients especially those who have uncontrolled diabetes and treated with corticosteroids. Timely diagnosis by histomorphological assessment supported with special stains is the cornerstone to prevent an adverse clinical outcome.

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