Rhizopus, as already mentioned, is ubiquitously present in soil, manure and as moulds on food ^[1][18]. Transmission of the infection can occur by airborne fungal spores entering via the nasal, oral, and conjunctival mucosa or by ingesting contaminated food. It may also be a commensal on the skin and mucosa, thus precipitating in opportunistic infections among the immunocompromised ^[2][19].

As discussed before, ROCM accounts for about a third of the cases of mucormycosis, with a steep rise noted in the COVID-19 pandemic ^[3][20]. The second most common type is pulmonary mucormycosis (PM) ^{[3][4][5][6][7]}[13,14,20,21,22]. ROCM and PM originate in the nose and paranasal sinuses. The hallmarks of the disease are vascular invasion, thrombosis, and tissue necrosis. It results in bony destruction in the walls of the sinuses, leading to orbital and cranial spread ^[3][8][20,23].

Smith and Kirchner’s diagnostic criteria for ROCM from 1958 still remain the gold standard for clinical diagnosis ^[5][14]. In an immunocompromised setting, a combination of the following features ought to raise suspicion of mucormycosis:

• Facial pain and blood-tinged nasal discharge on the same side;
• Soft periorbital or peri-nasal swelling with skin and mucosal discoloration progressing to induration;
• Conjunctival suffusion, eyelid ptosis, eyeball proptosis, and complete ophthalmoplegia;
• Necrotic, black turbinates appearing like clotted, crusted blood;
• Cranial nerve palsies.

The other non-specific features include fever, unilateral headache, sinus tenderness, puffiness of the face, loosening of teeth, and nasal congestion ^[9][24].

PM has non-specific symptoms such as fever, cough, chest pain, dyspnoea, and haemoptysis ^[9][24]. The symptoms appear innocuously similar to the underlying viral infection. Hence, the early signs are written off as COVID-19. Suspicion of PM should arise in COVID-19 patients when these symptoms persist, or new symptoms develop after the resolution of the viral disease. Strict monitoring for reappearance or prolonged persistence of symptoms in diabetics and those receiving steroids and immunosuppressive therapies should prompt evaluation for mucormycosis ^[3][4][5][13,14,20].

In the immunocompromised population, the invasive nature of the fungus quickly turns the disease lethal ^{[4][5][6][10]}[13,14,21,25]. A mortality rate of 30–50% is seen in CAM ^[3][4][5][6][13,14,20,21].

The first line of investigation performed is an imaging study ^[3][20]. The modalities recommended are magnetic resonance imaging of the paranasal sinuses with cerebral contrast for ROCM and simple computerized tomography (CT) thorax for pulmonary disease.

For pathologies of the paranasal sinuses, the imaging preferred is a CT scan ^[11][26]. IM may show as a unilateral hypodense opacification of the sinuses. It has a limited role as the soft tissue changes, and critical features such as perineural spread and cavernous sinus involvement can be missed. Its use is limited to picking up bony destruction only. Magnetic resonance imaging (MRI) has higher sensitivity than CT (86% vs. 57–69%) and similar specificity (83% vs. 81%) for acute invasive fungal sinusitis ^[11][12][26,27]. It produces hypo- or isointense lesions in all sequences with variable enhancement on contrast. The invasion of the fungus along the soft tissue and fatty planes is delineated with an increased clarity by T1-weighted images, aiding in the staging of the disease ^[13][28]. Detection of neurological complications such as cavernous sinus thrombosis, meningitis, necrosis, and subtle perineural invasion requires the utilization of cerebral contrast material such as gadolinium ^[12][14][27,29].

In pulmonary disease, with concomitant COVID-19 infection, it is exceedingly difficult to differentiate due to the similarities in the lesions and the acute respiratory distress syndrome picture. CT thorax shows ground-glass opacities and infiltration. Some cases may show features of consolidation and cavitation ^[15][30]. Angio-invasion in the lungs may cause thrombosis of the pulmonary vessels, appearing as wedge-shaped infarcts.

The suspicion of mucormycosis is based on direct microscopic examination of a wet mount. Staining with comparative fluorescent fungal stain, calcofluor white, and Giemsa is also performed on the clinical specimens at the outset ^[3][16][20,31].

The standard practice for confirmation of the diagnosis is a biopsy from the infected area demonstrating the presence of broad, wide ribbon-like aseptate hyphae branching at right angles, on a background of necrotic debris on histopathology ^{[2][3][17][18]}[19,20,32,33]. Stains commonly used are haematoxylin-eosin, periodic acid-Schiff, or Grocott–Gomori’s methenamine-silver stain (GMS) ^[3][16][20,31].

On culture, the presence of cotton white or grey-black colonies at 28–30 °C and 35–37 °C is characteristic. The media commonly employed are Sabouraud Dextrose Agar and Brain and Heart Infusion Agar. Morphological identification from the fungal culture can be carried out by microscopic examination or by DNA sequencing on the basis of barcodes 18s, ITS, and MALDI-TOF ^[19][20][21][5,34,35]. Isolation from a fungal culture may be performed for differentiation of genus and species, and for antimicrobial susceptibility testing ^[17][19][5,32]. The treatment, however, is independent of the genus and species and is of epidemiological importance ^[22][36].

Serological investigations such as galactomannan assays and 1,3-β-D glucan assays support a diagnosis of fungal infection ^[3][21][20,35]. These tests are, however, typically negative in a pure mucor infection ^[3][20]. The utility of using the negative test to rule out the disease is questionable, as the tests are positive in mixed fungal infections ^[20][34]. While, presently, there is no serological test available for the rapid diagnosis of mucormycosis, ELISA and lateral flow immunoassay (LFIA) are in the process of development ^[23][37].

The clinical features, though characteristic of a fungal infection, are unfortunately non-specific, and may be mistaken for aspergillosis. Further, there exists a degree of misidentification of the Mucorales for the more ubiquitous Aspergillus spp. on microscopy ^[3][10][24][20,25,38]. There is thus a challenge in obtaining a confirmation of the diagnosis without immunohistochemistry staining with monoclonal antibodies or DNA-PCR of the tissue section. Both of these modalities are not only expensive but also not readily available at the point of contact ^[3][25][20,39].

The utility of complete blood counts is not established, especially in the present cohort of COVID-19 patients, where the white cell picture is deranged as a result of the primary infection ^{[15][17][22][26]}[30,32,36,40]. For any analysis into the association of the derangement of these parameters due to the secondary fungal infection, confounding variables of the primary disease will have to be considered. The data on this are insufficient to comment upon and require further study.

3. COVID and Mucormycosis

As explained above, high-risk factors for mucormycosis include DM and metabolic acidosis, steroid usage in COVID-19, neutropenia, elevated serum iron levels, immunomodulatory therapies, and concomitant chronic illness. An increased likelihood of fungal infection has also been noted with haematological malignancies, organ and bone marrow transplant recipients, steroid therapy, patients on maintenance haemodialysis and iron chelation, trauma and burn victims ^[7][22]. SARS-CoV-2-mediated immune dysregulation of the cytokine storm in the background of a compromised immune system predisposes to invasive fungal infections ^[27][56]. This hyper-inflammatory state is also noted as a consequence of some malignancies, autoimmune diseases, and immunosuppressive medications ^[28][57]. High inflammatory markers, interleukins, interferon-gamma, tumour necrosis factors and hyperferritinaemia produce a picture similar to secondary haemophagocytic lymphohistiocytosis ^[29][30][3,58]. This hypercytokinaemia of severe COVID-19 produces diffuse alveolar lung damage, microvascular thrombosis, hyaline membrane formation and fibrosis, leading to an acute respiratory distress picture ^[31][32][33][59,60,61]. This unregulated increase in inflammatory markers and acute phase reactants downregulates the CD4+ T lymphocyte count, causing lymphopenia, propagating a decrease in viral clearance ^[34][35][62,63]. Additionally, there is a diffuse systemic vasculitic endotheliitis and microvascular damage, causing multiorgan dysfunction ^[36][37][64,65]. The vascular endothelial and alveolar damage is due to angiotensinogen converting enzyme- 2 (ACE-2) receptor-mediated viral entry into these cells. Other tissues with higher expression of these receptors such as gastrointestinal mucosa and proximal tubular cells of the kidney show a greater localised inflammatory response ^[38][39][66,67]. Patients with already compromised systems, i.e., with cardiovascular comorbidities such as hypertension, severe dyslipidaemia, obesity, DM, and those with chronic kidney disease thus have poorer outcomes in the event of disease progression to a cytokine storm ^[40][41][68,69]. Cardiac infection-producing myocarditis and infarctions are similarly hypothesized ^[42][70]. Immunosuppressive therapies such as steroids are among the few interventions proven to blunt this unchecked inflammation and improve survival. The high rate of usage of steroids in the period of cytokine storm in these patients exponentially increases the risk of them acquiring the IFIs in the recovery period ^[4][11][13,26]. The association of steroid therapy with mucormycosis has been the most constant in the recent upstroke of cases. Steroids are one of the only therapies proven to consistently reduce mortality in COVID-19 patients ^[4][11][13,26]. They are considered an essential therapy for COVID-19 patients on supplemental oxygen therapy ^[43][71]. Their use, however, has a multitude of complications attached. Worsening hyperglycaemia or new-onset DM and immunosuppression are among the common mechanisms that predispose to angio-invasive mucormycosis. Corticosteroids are implicated in immune system destruction by impairing leukocyte chemotaxis and prevention of phagolysosome fusion ^[44][72]. This steroid-induced immune dysregulation is an added burden on the virus-induced lymphopenia and hypercytokinaemia, leaving the patient wide open to a host of super-added co-infections. This may be compounded by the fact that steroids are given as over-the-counter pills in India leading to rampant misuse of the drugs ^[5][14]. DM remains the maximally associated factor predisposing to mucormycosis, with a mortality of 46% ^[45][48]. SARS-CoV-2 has been seen to worsen the glycaemic profile in patients with DM through multiple mechanisms, though they are all not completely understood. Notably, the virus can infect the pancreatic islet cells causing damage to the β-cells, causing hypoinsulinaemia and worsening hyperglycaemia ^[7][46][47][22,73,74]. DM, by nature, causes a degree of endothelial dysfunction due to chronic inflammation, leading to microvascular complications. Direct viral invasion of the vascular lining, causing endothelial damage, is mediated by ACE-2 receptors. This results in apoptosis and pyroptosis of the endothelium predisposing to angio-invasive secondary infections ^[33][61]. Among diabetics, the risk multiplies in patients with diabetic ketoacidosis (DKA). The virus and the hyperimmune stress state themselves predispose to dysglycaemia and DKA, even in the absence of poorly controlled sugars ^[7][47][22,74]. Hyperglycaemia produces a state of secondary immune deficiency. The dysglycaemia and acidic pH in ketoacidosis results in phagocyte dysfunction and defective intracellular killing of the fungi. The low-grade chronic inflammation causes an impaired immune response—both innate and adaptive response to infections. This also paves the way for the cytokine storm. The acidotic serum pH of 6.88–7.3 in DKA is also conducive to florid fungal proliferation ^[48][75]. The endothelial dysfunction and cytokine storm are procoagulant states leading to thrombosis and tissue ischemia ^[31][33][59,61]. The ischemic necrosis prevents leukocyte chemotaxis and effective delivery of antifungal agents to the foci of infection. The concomitant endotheliitis that occurs as a result of oxidative damage causes adherence of the fungal elements to the vessel walls. These factors combined with the angio-invasive nature of the fungus are responsible for the haematogenous dissemination of the disease ^[49][76]. Iron is required for fungal hyphal growth and development. One of the main host defence mechanisms against mucormycosis is limiting the availability of free iron to the fungus by having it bind to forms such as transferrin, ferritin, and lactoferrin. The acidic pH in DKA displaces the iron from its bound forms in the human body, increasing its availability to the fungal elements. The presence of increased unbound iron plays a vital role in predisposing patients with DKA to developing the disease. Some Mucorales secrete high-affinity iron chelators or siderophores such as rhizoferrin to acquire the iron from the host cells. Those species without rhizoferrin utilize exogenous xenosiderophores such as deferoxamine that are administered in DKA patients to treat iron overload, in order to fulfil their iron requirements. This explains why patients who are on long-term iron chelation therapy are at elevated risk of mucormycosis. Severe COVID-19 disease is also a hyperferritinaemic state, due to interleukin-six-mediated ferritin synthesis and downregulation of iron transport. There is an intracellular accumulation of iron in the hepatocytes, eventually causing necrosis and release of the accumulated iron. The high serum iron forms a fertile ground for florid fungal proliferation ^[50][51][77,78]. Tocilizumab, an immunomodulatory monoclonal antibody that targets the deregulated interleukin-six pathway implicated in the COVID-19 cytokine storm, is associated with an increased risk of infections. It modulates and suppresses the immune system, leaving the body vulnerable to other pathogens such as bacteria and fungi ^[52][53][79,80]. Neutrophil and phagocyte activity are the main defences against fungal infection. Mononuclear and polymorphonuclear cells generate reactive oxygen species and defensin peptides that inhibit fungal growth. Patients with impaired neutrophil function (primary immune deficiencies) are thus at higher risk. These are unaffected in COVID-19 infection; thus, a primary cause–association relationship is unlikely ^[44][72].