Surge of mucormycosis during the COVID-19 pandemic

Abstract

Patients with respiratory viral infections are more likely to develop co-infections leading to increased fatality. Mucormycosis is an epidemic amidst the COVID-19 pandemic that conveys a ‘double threat’ to the global health fraternity. Mucormycosis is caused by the Mucorales group of fungi and exhibits acute angioinvasion generally in immunocompromised patients. The most familiar foci of infections are sinuses (39%), lungs (24%), and skin tissues (19%) where the overall dissemination occurs in 23% of cases. The mortality rate in the case of disseminated mucormycosis is found to be 96%. Symptoms are mostly nonspecific and often resemble other common bacterial or fungal infections. Currently, COVID-19-associated mucormycosis (CAM) is being reported from a number of countries such as the USA, Turkey, France, Mexico, Iran, Austria, UK, Brazil, and Italy, while India is the hotspot for this deadly co-infection, accounting for approximately 28,252 cases up to June 8, 2021. It strikes patients within 12–18 days after COVID-19 recovery, and nearly 80% require surgery. Nevertheless, the mortality rate can reach 94% if the diagnosis is delayed or remains untreated. Sometimes COVID-19 is the sole predisposing factor for CAM. Therefore, this study may provide a comprehensive resource for clinicians and researchers dealing with fungal infections, intending to link the potential translational knowledge and prospective therapeutic challenges to counter this opportunistic pathogen.

1. Introduction

During deadly pandemics or epidemics, there are occurrences of co-infections, which are often understudied. Patients with respiratory viral infections are more likely to develop one or more co-infections, leading to disastrous diseases with increased morbidity and mortality [1]. Both in the 1918 influenza outbreak and in the 2009 H1N1 influenza pandemic, history witnessed the fatalities associated with co-infections [2]. The COVID-19 pandemic and mucormycosis co-infection are vivid examples currently faced by mankind. Mucormycosis is an unembellished angioinvasive fungal infection with acute incidence, rapid progression, and high fatality [3]. Moreover, it is reported as the second most prevalent mould infection that affects immunocompromised patients [4]. The fungal casual agents mainly belonging to the Mucorales group are ubiquitous in nature and are generally present in decaying organic matter [5,6]. Currently, COVID-19 patients are presenting with several bacterial or fungal co-infections [7,8]. While opportunistic fungal infections represent a matter of concern, mucormycosis co-infections are rare, but substantially complicate the health status of COVID-19 patients and overall outcomes [9]. As part of the ECMM's (European Confederation of Medical Mycology) ‘One World One Guideline’ project, authors recruited from 33 countries (UN regions) analyzed the existing studies on mucormycosis management and presented a consensus guideline that addressed regional disparities [10]. Notably, this co-infection is now increasing worldwide, and India is the worst hit. India is grappling with a sharp rise of COVID-19 cases in a more fatal second wave compared to the first one. Mucormycosis or ‘Black Fungus' infections are reported to co-affect COVID-19 patients, with about 70% of the global mucormycosis cases reported in March 2021 [2,11]. We have reviewed its causal agents, determinants, distributions, clinical manifestations, pathogenesis pattern, virulence factors, clinical case reports, diagnosis methods, and management procedures along with the futuristic drugs in the pipeline. We have also predicted the possible reasons for its sudden increase in several countries, as well as a plausible future trend of fungal co-infections during the next waves of the COVID-19 pandemic.

2. Taxonomy of mucormycosis causing fungi

In the preceding morphology-based classification system, fungi reproducing by ‘zygospore’ were assigned under the phylum Zygomycota [12], which was not validated and was discarded due to a lack of molecular phylogenetic support. Consequently, its subphylum Mucoromycotina was designated as ‘incertae sedis', which means a taxon not allocated to a higher taxon [13]. Walther and co-workers, in their review, presented the fact that the Mucoromycotina was improperly cited under the subphylum Glomeromycota in several articles [14]. Later, in the phylogenetic analysis, it was revealed that zygomycetes were described by two new clades sharing paraphyletic connection: phylum Mucoromycota and phylum Zoopagomycota [15]. Mucoromycota was placed as a sister clade to the Ascomycota and Basidiomycota. Mucoromycota predominantly involves three subphyla i.e., Glomeromycotina, Mortierellomycotina, and Mucoromycotina, where the latter one is comprised of the orders Mucorales (commonly decomposers and parasites of plants), Umbelopsidales (e.g., endophytes and saprobes), and Endogonales (e.g., ectomycorrhizas and saprobes) [15]. Distinctively, Zoopagomycota includes the order Entomophthorales [15]. Because of the phylogenetic distance of Mucorales and Entomophthorales, the term ‘zygomycosis’ (infection caused by zygomycetes) was discarded in favour of 'mucormycosis' (infection associated with the order Mucorales as the causal agent) and 'entomophthoromycosis' (infection associated with the order Entomophthorales as the causal agent) [14]. In immunocompetent individuals from subtropical and tropical areas and especially from developing countries, entomophthoromycosis results in long-term subcutaneous and mucocutaneous infections. In contrast, mucormycosis exhibits acute angioinvasion in people with compromised immune systems, in the case of developing or developed countries, and accounts for high fatality rates [16,17].

3. Salient features of mucorales

The order Mucorales includes 55 genera and 261 species, of which 38 species are known to cause mucormycosis infection in humans [14]. Mucorales, unlike other ascomycetes fungi, require a greater level of humidity for survival, growth, and spore germination [18]. Roden and colleagues reported a variable extent of pathogenicity caused by different genera of Mucorales. Based on the clinical context, the major genera include Rhizopus (47%), Mucor (18%), Cunninghamella (7%), Saksenaea (5%), Absidia (5%), Apophysomyces (5%), and Rhizomucor (4%), as detected in the tissue samples of mucormycosis-infected patients [19]. In a recent review, it was mentioned that Rhizopus is the predominant genus, accounting for about 48% of the cases, where Rhizopus arrhizus single-handedly contributed to 33% of the cases, followed by Mucor (14%) and Lichtheimia (13%) [20]. Infection by Cunninghamella resulted in a considerably greater fatality (77%) than that of the other Mucorales [19,20].

These genera exhibit significant variation in geographical regions. Apophysomyces infection is not found in Europe or Africa, whereas the genus Lichtheimia is prevalent in Europe [20]. This variation in infection prevalence might be due to biotic or abiotic factors like temperature, humidity or the presence of organic content associated with the optimal growth of the causal agents in different geographical locations. In India, Rhizopus sp. (79.7%) is the main cause of the disease, and Apophysomyces variabilis (7.9%) comes in second place [21]. Rhizopus arrhizus is the predominant fungus of the Rhizopus genus in India, but cases of Rhizopus microsporus and Rhizopus homothallicus are reported to be increasing [22]. In this country, Lichtheimia sp. are widely found in alkaline soils with low nitrogen content and cause 0.5%–13% of cases [22]. Other rare species that contribute to mucormycosis in India are reported to be Rhizomucor pusillus, Mucor irregulari, Saksenaea erythrospora and Thamnostylum lucknowense [22].

When Aspergillosis is compared with mucormycosis, a lot of parallels and some significant distinctions are observed [23]. Both infections are caused by inhaling spores followed by the formation of invasive hyphal structures, subsequent invasion and obstruction in blood vessels in support of the hematogenous spread [24]. The characterization of Mucorales is based on their rapid proliferation and formation of distinctive mycelia. The single-celled asexual spores/sporangiospores are produced endogenously within the sporangium, a large spore sac that is even visible to the naked eye [14,18]. Often, merosporangia (elongated with countable spores) or sporangiola (globular with countable spores) are seen in place of sporangia (globular with numerous spores). They are accordingly named depending on their shape and quantity of sporangiospores inside them [14]. Unique for the order Mucorales is columella, which is a noticeable sterile mid-vesicle within the sporangium and becomes prominent after the liberation of the spores [18]. They produce pigmentless, broad (having an irregular diameter of approximately 5–20 μm), slender-walled, ribbon-shaped hyphae along with a small number of occasional septae (also called pauciseptate) and right-angled branches [25]. Hence, they are typically characterized by these features and described as aseptate fungi. The hyphae might have different widths with pleated, ruffled or fragmented forms [26]. Thick-walled globular structures may develop in the hyphal tip that might convert to a spore-forming body and eventually, on maturation, might disperse to air [26].

4. Determinants and distribution of mucormycosis

For the onset of this fungal infection, diabetes mellitus (DM) and DM-associated ketoacidosis (DKA) (in the case of Asia), as well as haematological malignancy and solid organ/bone-marrow transplantations (in the case of Europe and USA) represent the major risk factors. Moreover, history of other surgeries, trauma, neutropenia, protein-calorie malnutrition, autoimmune disease, chronic kidney disease or renal failure, HIV infection, deferoxamine therapy, corticosteroid therapy, etc. are also reported to be on the list of risk factors across the globe [27,28]. Along with human health conditions, other factors like seasonal variation, survival from natural disasters, etc., contribute to the predisposing agents [29,30]. Jeong and team investigated the cases during the 2000–2017 time period and reported that DM was more frequently observed in 340 patients out of 851 cases (40%) suffering from mucormycosis, whereas steroid use, haematological malignancy, solid organ transplantation and neutropenia were seen in 33%, 32%, 14%, and 20% of the cases respectively. Patients from African or Asian countries were found to be prone to DM. Among the major trauma-related cases, 33% of cases were observed in case of accidents (motor vehicle-related and others) and 30% of cases were involved with health-related issues, including gynaecological, gastrointestinal, orthopaedic and cardiovascular procedures. About 5% of the cases were documented in survivors of natural disasters (tsunami or tornado) [20]. As per the US-based study (1992–1993) by Rees and colleagues, 17.3 cases/million population/year were recorded for the infection among 2.94 million populations in three cumulative countries (Alameda, San Francisco, and Contra Costa), where the predisposing factors included chronic lung disease (9.3%), DM (9.9%), non-haematological malignancy (14.7%) and, interestingly, HIV infection (47.4%) [31]. But mucormycosis cases gradually displayed a sharp increase worldwide. For instance, in Japan, cases climbed from 0.01% to 0.16% within twenty years (1969–1989) [32]. This upward trend of mucormycosis was also noted in European countries like Belgium, Spain, France, and Switzerland. In Belgium, as per a ten-year (2000–2009) study report, the cases increased to 0.148 cases/10,000 patients from 0.019 cases/10,000 patients, with an average increase of 0.058 cases/10,000 patients [33]. Belgian patients with haematological malignancies were found to be the individuals most vulnerable to mucormycosis. A ten-year (1997–2006) case analysis report from France showed that 0.7 cases/million population were documented in 1997, which amounted to 1.2 cases/million population in 2006. DM and the arbitrary use of antifungal drugs were thought to be the main reasons for this rise in France [34]. In Spain, 1.2 cases of mucormycosis were observed among 100,000 patients admitted before 2006, but in the 2007–2015 period, the cases increased to 3.3 per 100,000 patients [35]. According to the analysis, 52.6% of patients were suffering from both trauma or surgical wounds and haematological malignancies, and 68% of patients received antifungal drugs [35]. Nevertheless, in Switzerland, a sharp rise in clinical cases was documented after 2003 (6.3 cases/100,000 admissions/year) in comparison with the time before 2003 (0.57 cases/100,000 admissions/year) [36]. The risk factors were found to be variable, and the study revealed that several reasons might have triggered the invasive cases, including the use of antifungal drugs such as caspofungin and/or voriconazole, etc., the high number of allogeneic bone marrow transplants or immunosuppressive drugs [36]. However, the mucormycosis burden in different countries such as Algeria, Argentina, Australia, Belgium, Brazil, Cameroon, Canada, Chile, Colombia, Czech Republic, Denmark, Dominican Republic, France, Greece, India, Ireland, Japan, Jordan, Kazakhstan, Kenya, Korea, Malawi, Mexico, Nigeria, Norway, Pakistan, Philippines, Portugal, Qatar, Romania, Russia, Serbia, Spain, Thailand, Ukraine, United Kingdom (UK), USA and Republic of Uzbekistan were thoroughly reviewed by Prakash and team [27].

The mucormycosis burden in India was 0.14 cases per 1000 population, with 38.2% deaths per year during the pre-COVID-19 period [37]. The usual occurrence of mucormycosis in India is estimated to be 70-times higher than global data [22]. However, an individual health facility in India has recorded three successive case series of mucormycosis: 129 cases over the subsequent ten years (from 1990 to 1999), 178 cases in five years (from 2000 to 2004) and 75 cases in an eighteen-month timeline (from 2006 to 2007) [[38], [39], [40]]. The total count increased from an average of 25 cases/year (1990–2007) to an average of 89 cases/year (2013–2015) [41]. Several researchers from various regions of the nation also published numerous reports regarding this infection in diverse risk categories [[42], [43], [44], [45], [46], [47], [48]]. However, it is clear that mucormycosis cases exhibit an increasing trend. In a recent study of 388 mucormycosis cases, the mortality rate was found to be 46.7%, which was significantly associated with poorly managed DM (56.8%) and trauma (10.2%) [41]. Most cases and mortality incidents were reported in North India (82.7%), and cases of post-tubercular mucormycosis were notably observed [27,41].

5. Clinical manifestations

According to the clinical spectrum or the epidemiological features of mucormycosis, the infection includes chromoblastomycosis, mycetomas, sinusitis, and superficial (Tinea nigra), subcutaneous, cutaneous, and systemic phaeohyphomycosis [49,50]. Based on the clinical demonstration and foci of infection, mucormycosis is classified into five main clinical patterns: (A) rhinocerebral; (B) cutaneous; (C) pulmonary; (D) gastrointestinal; (E) disseminated; and also other rare patterns, including peritonitis, endocarditis, osteomyelitis, renal infection etc. [[51], [52], [53], [54], [55], [56], [57], [58], [59]]. The most familiar foci of infections were reported to be in the sinuses (39%), lungs (24%), and skin tissues (19%) [60] where dissemination occurred in 23% of these cases [30]. The mortality rate was found to be 96% for disseminated mucormycosis, 85% for gastrointestinal-infected patients, and 76% for pulmonary infection [30]. Infant skin and gut are more vulnerable in comparison with adults [61]. DM was found to be associated with rhinocerebral mucormycosis cases, whereas haematological malignancies were linked with disseminated infection. Trauma was associated with cutaneous mucormycosis, and a history of solid organ transplantation was involved in the cases of pulmonary, gastrointestinal or disseminated mucormycosis [20]. Clinical manifestation patterns in India are parallel with the worldwide trend. Rhino-orbital-cerebral mucormycosis (with/without brain involvement) is the most common example in India, followed by cutaneous and pulmonary infections [21]. (Fig. 1 ).

Fig. 1.

Different types of mucormycosis clinical manifestation. Based on the clinical manifestation and foci of infection, mucormycosis is classified into four main clinical patterns: rhino-orbital-cerebral infection affects the sinuses, orbital area and can be transmitted to the brain; pulmonary mucormycosis infects the respiratory system; gastrointestinal mucormycosis commonly attacks the large intestine, stomach and small intestine; cutaneous mucormycosis is limited to superficial or deep skin tissues, and occasionally extends to the bones or muscles. Pathologic symptoms are manifested as the result of infection caused by the fungi, which belong to the order Mucorales. Tissue biopsy during laboratory analysis enables visualization of typical Mucorales characteristics from tissue samples collected from infected areas of mucormycosis patients.

Rhino-Orbital-Cerebral mucormycosis Rhino-orbital-cerebral mucormycosis is the most common type of mucormycosis. After inhaling the sporangiospores into nasal mucosa, the infection proliferates from the sinuses to the brain via the orbital area [62]. General non-ocular signs and symptoms are fever, headaches, facial numbness and pain, often accompanied by nerve palsy, nasal discharge, nasal ulceration, sinusitis, hemiplegia, eschars (black necrotic tissues), and mental deterioration [27]. Initial symptoms resemble bacterial sinusitis [63]. The ocular symptoms are reported to be eye pain, visual changes, proptosis, chemosis, ptosis, ophthalmoplegia, orbital cellulitis, and periorbital discolouration along with necrosis [27]. Imaging studies reveal thick mucosal lining, sinusitis with mild to severe lesions, and erosion of nasal septal bones, orbital, and jawbones. However, extended infection exhibits soft tissue infiltration, orbital cellulitis, optic neuritis, bone rarefaction, infarcts, intracranial abscess, and skull bone erosion. The infection route follows the sino-nasal or retro-orbital region, ethmoid-sphenoid sinus and/or superior orbital fissure. It reaches the brain by the perineural pathway or the way through the cribriform plate [27]. The pterygopalatine fossa was found to be the reservoir of fungi [62]. The mortality rate harshly increases soon after cranial invasion [63]. Cerebral infection can also be acquired through dissemination from a distant organ following a hematogenous path [27].

Pulmonary mucormycosis Pulmonary mucormycosis clinical patterns are often misinterpreted as pulmonary Aspergillosis. Patients frequently have a high fever (>38 °C) that does not respond to broad-spectrum antibiotics. Symptoms include nonspecific cough and rarely dyspnea, hemoptysis, and pleuritic chest pain. Diabetic patients are often accompanied by endobronchial or tracheal lesions, especially in diabetics, resulting in airway obstruction and lung collapse [30,64,65]. Chest imaging of a patient with pulmonary mucormycosis exhibits lung infiltration and consolidation, thick wall cavity, multiple nodules, pleural effusion, hilar/mediastinal lymphadenopathies, pneumothorax or air crescent signs [27]. The reversed halo sign is the classic feature of pulmonary mucormycosis, which is generally found to be unilateral (usually the upper lobe is associated, followed by the lower lobe and middle lobe, respectively), seldom bilateral, hilar/mediastinal [27].

Cutaneous mucormycosis Cutaneous mucormycosis is classified into primary cutaneous mucormycosis involving the direct inoculation onto the skin by the degeneration of cutaneous barrier-breach and secondary cutaneous mucormycosis where dissemination from other infected areas (generally rhinocerebral infection) seeds the disease. Primary can be classified as (i) localized infection (limited to superficial or deep skin tissues in 32–56% cases) and (ii) deep infection (extends to bones or muscles in 24–52% cases) [27]; whereas 16–20% of the cases exhibit hematogenous dissemination and secondary mucormycosis [27,66]. Clinically, the infection is formed as dry, but rapidly growing ulcers associated with an erythematous halo. Secondary cutaneous mucormycosis often involves necrotic lesions, including erythematous edges and eschar as well as necrosis on orbital, nasal or palatal tissues, i.e. the rhinocerebral route [67].

Gastrointestinal mucormycosis Gastrointestinal infections are an uncommon pattern of clinical manifestation that primarily target the large intestine (∼43.2%) followed by the stomach (∼33%) and small intestine (∼28.4%). The stomach is commonly targeted in adults, whereas the intestine is considerably involved in children [68]. Symptoms are nonspecific and the diagnosis is often missed or impeded. Symptoms include fever, abdominal pain, gastrointestinal bleeding, bowel change, etc. [69]. It is significantly found in premature neonatal cases and patients with malnutrition, reaching a mortality rate of 85% [68].

Disseminated mucormycosis All of the above-mentioned infections might evolve into the disseminated type of mucormycosis, which involves two or more widely distant organ systems. These are highly rare and usually occur in critically immunocompromised people or patients who received deferoxamine therapy [70]. Dissemination commonly arises from the lungs, gastrointestinal tract, trauma, or deep cutaneous lesions. However, the brain is a usual target area after angioinvasion, and metastatic lesions are also discovered in the heart, liver, spleen, and other organs [30].

Phylogenetic analysis In the present review, we provide a phylogenetic analysis of the causative Mucorales organism [20] based on its ITS regions using MEGA7 [71] and the neighbor-joining method [72] on the Jukes-Cantor model [73] by applying 1000 bootstrap replications (Fig. 2 ).

Fig. 2.

The Neighbor-joining tree is based on ITS sequences of the causative agents of mucorales. The bootstrap value is shown at the branch node from 1000 replicants. The phylogenetic tree represents three varied clades: Clade 1 is composed of predominant species Apophysomyces spp., Lichtheimia spp. and Saksenaea spp.that cause cutaneous mucormycosis; Clade 2 is composed of Rhizopus spp, the most prominent causal agent for rhino-orbital-cerebral mucormycosis; and Clade 3 includes Mucor spp, which shows almost all clinical manifestations.

We have tried to focus the phylogenetic relationship on the varied clinical manifestation of mucormycosis. It has been noted in past studies that several predisposing conditions are associated with various clinical manifestations [20]. The role of the causative agents in the varied clinical manifestations may have a specific influence on the type of mucormycosis. The phylogenetic study has been carried out upon such a backdrop to find the segregation of the different Mucorales genera that is reflected in clinical manifestations. Compared with other genera, Apophysomyces sp., Lichtheimia sp. and Saksenaea sp. were more commonly found in patients with cutaneous mucormycosis. In the phylogenetic tree, Apophysomyces sp. and Saksenaea sp. appeared in the same cluster, whereas Lictheimia sp. appeared in a different cluster. Rhizopus sp. is often isolated from patients with rhino-orbital-cerebral mucormycosis and forms a prominent single cluster within the phylogenetic tree. Cunninghamella sp. and Rhizomucor sp. are more common in patients with pulmonary disease. Such clustering can be observed between Rhizomucor pusillus and Cunninghamella bertholletiae. Rhizomucor variabilis is found in a separate clade with Mucor sp. A closure look at the phylogenetic tree revealed three various clades: Clade 1, which consists of cutaneous mucormycosis predominant species Apophysomyces sp., Lichtheimia sp. and Saksenaea sp.; Clade 2 includes Rhizopus sp., the most prominent rhino-orbital-cerebral mucormycosis causative organism; and Clade 3 includes Mucor sp., which is present in almost all clinical manifestations, even though Mucor sp. elicits the predominance of cutaneous mucormycosis. The prevalence of causative organisms for pulmonary mucormycosis is found within Clade 1 only. It can be hypothesized that the Mucorales causative organism may show any type of clinical manifestations based on the predominant condition; however, the type of mucormycosis is based on the species dominance, well determined from the phylogenetic tree.

6. Pathogens and their virulence factors

The virulence factors associated with various types of mucormycosis are an interesting parameter to understand the epidemiology of the disease, particularly when the disease has many clinical manifestations, and is caused by several fungal causal agents of a given Mucorales order. Moreover, Rhizopus and Mucor species are found to be the most prevalent agents for this disease. Additionally, the reported virulence genes of Rhizopus arrhizus have been retrieved from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/), and each gene was subjected to protein-protein Blast analysis to obtain its occurrence in species of other Mucorales groups. Frt1, Fet3, Mpp, rfs, bycA, cotH, chi3 are the major virulence genes reported associated with Rhizopus arrhizus pathogenesis [[74], [75], [76]]. It has been found that many of these genes are distributed in species of Rhizopus, Mucor, Lichtheimia, Apophysomyces, Phycomyces, Absidia, Parasitella, and Thamnidium, many of which are not reported as pathogenic strains. Based on the abundance of these virulence genes among various closely associated and so far identified pathogenic and non-pathogenic genera, a significant concerns still arise, as the non-pathogenic species might become pathogenic due to virulence genes. This possibility makes them an additional source for the development of a new type of mucormycosis.

7. Pathogenesis and host defense

The genome sequence in Rhizopus arrhizus is extremely repetitive compared to other fungi, with several transposable genetic elements, accounting for nearly 20% of the genome [77]. An ancestral whole-genome duplication, along with the recent gene duplication events, have provided shreds of evidence in support of the amplification of several gene families related to energy production, cell growth, signal transduction, as well as fungal virulence proteins (e.g., aspartic protease, etc.) leading to substrate-degradation and angioinvasion in the host [77]. Mucormycosis has been characterized by extensive angioinvasion (Fig. 3 ) with limited inflammatory response, which leads to thrombosis in the blood vessels and necrosis of neighbouring tissues [78]. Tissue necrosis inhibits the accumulation of the immune cells into the foci of infection and promotes angioinvasion that, subsequently, may lead the pathogens to spread to several target organs. Hence, pathogen interaction with the endothelium of blood vessels is thought to be the crucial stage in its pathogenesis. Rhizopus, dead or alive, damages the endothelial cells, followed by adhesion and phagocytosis [79].

Fig. 3.

Molecular mechanism of mucormycosis pathogenesis: interaction of Mucorales with host endothelial cells and host factors impacting the immune response. Acidifying factors present in the host's blood vessels induce the release of iron from host carrier proteins (e.g., transferrin, lactoferrin, ferritin, among others). Consequently, the concentration of free irons increases in the blood serum. Additionally, a higher concentration of glucose, free iron and ketone bodies, including β-hydroxybutyrate (BHB) (as found in DKA patients), increases GRP78 expression in the host endothelium. Fungal CotH interacts with GRP78 and promotes angioinvasion. Fungal ferric iron reductase and siderophores help to absorb the maximum iron available and facilitate fungal growth and proliferation in Mucorales. Higher concentrations of glucose, free iron, and BHB also act as the positive regulators of fungal growth. Moreover, ketone bodies and free iron present in the serum inhibit the host immune response cascades. In contrast, sodium bicarbonate (NaHCO₃) can reverse this phenomenon.

Researchers have recognized glucose-regulated protein 78 (GRP78) as a unique receptor present in the host endothelium that selectively interacts with Rhizopus [80]. Moreover, higher glucose and iron concentrations, usually found in DKA patients (Fig. 3), were reported to increase GRP78 expression and facilitate angioinvasion in a receptor-dependent mode [80]. These findings shed light on the reason that DKA patients have been vulnerable to mucormycosis. Another fungal ligand, CotH (spore coat protein homologs), was found in Mucorales (Fig. 3). Therefore, CotH-GRP78 interaction was considered to be the key strategy in specific host-pathogen interaction during mucormycosis pathogenesis [81,82]. In order to install an infection, fungal spores have to penetrate and evade the host immune system for further spore germination and hyphal growth. The study confirms that the immunocompetent hosts do not necessarily develop mucormycosis [83]. As sporangiospores are readily aerosolized and released into the atmosphere, the respiratory tract is the most common entry point for the pathogen [84]. An immunocompetent host exhibits several defence strategies against fungal invasion, including innate immunity, mechanical barriers of skin/mucosa, and nutritional immunity (i.e., generation of the iron-poor environment), etc. [85]. For successful pathogenesis, metal nutrients (e.g., iron) are required to be absorbed by the microbes for their growth. In turn, the host organism regulates the concentration of available metals to defend itself from microbial invaders [86]. The strategy of narrowing iron availability by binding it to the sequestering proteins like transferrin, lactoferrin, and ferritin present in the host, is considered the universal host defence mechanism against fungal invasion [87,88]. During lower iron availability, fungi produce high-affinity ferric iron reductase and synthesize iron-chelators or 'siderophores' to absorb the maximum iron available [87] (Fig. 3). Rhizopus secretes rhizoferrin, a polycarboxylate-type siderophore [89]. Notably, DKA patients are reported to have high levels of free iron in serum, which aids the growth of Rhizopus arrhizus at a range of acidic pH (7.3–6.88) [88].

Mononuclear and polymorphonuclear phagocytic cells are responsible for host defence and pathogen killing by oxidative and non-oxidative mechanisms [83,90,91]. Macrophages phagocytose the invaded spores. Spores that escape from the macrophage defence and germinate into newly synthesized hyphae stimulate chemotaxis of neutrophils, which phagocyte these fungal pathogens [85,92]. Neutrophils produce reactive oxygen species (ROS), cationic peptides, perforin, and other enzymes for pathogen degradation. These cells also express pro-inflammatory cytokines, including TNF-α, IL-1, INF-γ, etc. that induce other inflammatory cells to trigger the host's immune response [85]. In addition, Rhizopus hyphae activate TLRs (toll-like receptors) present on the phagocytes (e.g., polymorphonuclear neutrophils) and bind to PAMPs (pathogen-associated molecular patterns) of the pathogen surface. TLRs, along with the other receptors, perform a critical role in fungal recognition and activate the signalling cascade in host cells [84,93]. Platelets with their granule-dependent mechanism adhere to fungal hyphae and conidia [94]. They facilitate hyphal damage by inhibiting hyphal elongation and conidia germination in a time-dependent manner [94]. Platelets secrete pro-inflammatory and anti-inflammatory cytokines and chemokines, leading to the activation of monocytes and dendritic cells. However, natural killer (NK) cells also produce chemokines and cytokines [85]. NK cells exhibit cytotoxic effects against Rhizopus arrhizus hyphae and damage the structure when assisted by perforin and other antifungal factors [95].

8. Mucormycosis: amidst COVID-19 period

The actual prevalence of mucormycosis is hard to determine, due to the lack of a population-based investigation. Kanwar and colleagues hypothesized that the actual number of COVID-19-associated mucormycosis (CAM) cases is likely to be much higher than those reported due to challenges faced during diagnosis or detection, aided by emerging case reports [96]. During the COVID-19 period, many studies were reported globally on CAM complicating COVID-19 or vice-versa. The cases have been documented in the USA, Turkey, France, Mexico, Iran, Austria, UK, Brazil, and Italy, etc. Still, the number of cases reported was far less than in India, which emerged as a global hotspot of CAM infection (Table 1 ).

Table 1.

Worldwide clinical case reports on COVID-19 associated mucormycosis (CAM).

Origin	No. of cases	Age, Sex	Comorbidity/Past medical history	Symptoms associated with COVID-19	COVID-19 Confirmed/Suspected And Active/Recovered	Treatment received for COVID-19	Mucormycosis Confirmed/Suspected	Mucormycosis type	Clinical presentations	Fungi isolated	Antifungal treatment	Surgery	Outcome	References
USA	2	36/M	Type-II DM, DKA	–	Confirmed	Remdesivir	Confirmed	Rhino-cerebral	Proptosis, periorbital edema and conjunctival chemosis, optic disc pallor, retinal whitening	–	Amphotericin B, isovuconazole, micafungin	No	Died	[164]
		48/M	Type-II DM, DKA	–	Confirmed	Remdesivir, convalescent plasma and intravenous dexamethasone	Confirmed	Rhino-cerebral	Right eye proptosis, right upper eyelid eschar, and conjunctival chemosis	Rhizopus sp.	Amphotericin B, isovuconazole	No	Ongoing
USA	1	60/M	Type-I DM, asthma, hypertension, hyperlipidemia	Dyspnea, hypoxia, ARDS	Confirmed/Active	Remdesivir, Dexamethasone, Convalescent plasma	Confirmed	Rhino-Orbital	Proptosis, opacification of sinuses, erythema and edema of the eyelids, conjunctival chemosis.	Rhizopus sp.	Amphotericin B, Caspofungin, Posaconazole	Yes	Died	[165]
USA	1	41/M	Type-I DM	Loss of taste, cough	Confirmed/Active	Steroids and hydroxychloroquine,	Confirmed	Rhino-cerebral	Deep radiating pain in nose, black eschar in palate, necrotic mucosa in nasal sinuses, intracranial abscess, sinus thrombophlebitis, vein thrombosis, extensive granulation in sinus.	–	IV abelcet	Yes	Discharged	[166]
USA	1	79/M	DM, hypertension	Fever, rigor, dry cough, severe shortness of breath, tachycardia	Confirmed/Active	Ceftriaxone, Azithromycin,	Suspected	Pulmonary	Extensive bilateral pneumonia and new development of bilateral upper lobe cavitations	Rhizopus arrhizus, Aspergillus fumigatus	Amphotericin B	Yes	Discharged to a long-term acute care facility	[53]
						Remdesivir, Dexamethasone
USA	1	68/M	Type-II DM, hypertension, chronic kidney disease, obstructive sleep apnea, orthotopic heart transplantation	Nonproductive cough, non-bloody diarrhea, fever, respiratory symptoms	Confirmed/Active	Remdesivir, Hydroxychloroquine, Methylprednisolone Convalescent plasma	Confirmed	Cutaneous	Purplish skin discoloration, infection burrowing towards omentum, extending deep in the right chest cavity, musculature and sternal wound.	Rhizopus microsporus	Amphotericin B, Posaconazole	Yes	Died	[113]
USA	1	56/M	End-stage renal disease on hemodialysis	Fatigue, shortness of breath, hemoptysis	Confirmed/Discharged and readmitted after 5 days	Methylprednisone, tocilizumab, convalescent plasma	Confirmed	Pulmonary	Dense consolidation in right lower lobe with lung necrosis, necrotic tissue and purulent exudate in lungs	Rhizopus azygosporus	Amphotericin B, posaconazole, isavuconazole	Yes	Died	[96]
USA	1	33/F	Hypertension, asthma, untreated diabetic ketoacidosis	Vomiting, cough, shortness of breath, mild tachycardia, hypertension, and tachypnea	Confirmed/Active	Remdesivir, Convalescent plasma	Confirmed	Rhino-orbital-cerebral	Acutely altered mental status, left eye ptosis, proptosis, fixed dilated pupil, complete ophthalmoplegia, secretions in palate, cerebral edema	–	Amphotericin B	No	Died	[125]
USA	1	49/M	Insignificant	Fever, cough, shortness of breath, pneumonia	Confirmed/Active	Ceftriaxone, Azithromycin, Remdesivir, Dexamethasone, Convalescent plasma, Tocilizumab	Confirmed	Pulmonary	Tension pneumothorax with bronchopleural fistula, necrotic pleural empyema	Rhizopus sp.	Amphotericin B.	Yes	Died	[138]
Turkey	11	61-88/M: 9, F: 2	DM, hypertension, hyperthyroidism, COPD, coronary artery disease, renal failure, myelodysplastic syndrome,	ARDS	Confirmed/Active	Dexamethasone	Confirmed	Rhino-orbital	Eye proptosis, ophthalmoplegia, orbital pain, conjunctival hyperemia/chemosis, ptosis, fixed and dilated pupil, endophthalmitis, vision loss	Mucor sp.	Amphotericin B, Voriconazole	Yes	Discharged: 4 Died: 7	[167]
Turkey	1	56/F	DM	Hypoxia, hyperglycemia, electrolyte imbalance and metabolic acidosis	Confirmed/Recovered	Broad-spectrum antibiotics, corticosteroids (methylprednisolone)	Confirmed	Rhino-cerebral	Proptosis in the right eye, restricted eye movements, edema and color change in the nasal area	Mucor sp.	Amphotericin B	Yes	Died	[168]
France	1	55/M	Follicular lymphoma	Fever	Confirmed/Active	Systemic steroids	Confirmed	Pulmonary	–	Rhizopus microsporus, Aspergillus fumigatus	Amphotericin B	No	Died	[169]
Mexico	1	24/F	DM, DKA, obesity	Hypoxia, Respiratory failure	Confirmed/Active	–	Confirmed	Rhino-cerebral	Left facial pain, lid swelling, edema & proptosis, maxillary hypoesthesia, hyperemic conjunctiva, swelling and proptosis of sinuses	-	Amphotericin B	No	Died	[170]
Iran	2	40/F	None	Respiratory symptoms	Confirmed/Active	Remdesivir, levofloxacin, dexamethasone	Confirmed	Rhino-orbito cerebral	Bilateral visual loss, periorbital pain, complete blepharoptosis, ophthalmoplegia, mild proptosis, opacifications of sinuses and spaces, necrotic tissues in sinuses	–	Amphotericin B	Yes	Died	[171]
		54/M	Type-II DM	–	Confirmed/Active	Remdesivir, levofloxacin, dexamethasone	Confirmed	Rhino-orbital	Orbital pain, periorbital swelling, vision loss, blepharoptosis, proptosis, chemosis, unilateral opacifications, blackish necrotic tissues	–	Amphotericin B; Posaconazole	Yes	Discharged
Iran	1	61/F	–	–	Confirmed/Recovered and readmitted after 1 week	Remdesivir, interferon-alpha, and systemic corticosteroid.	Confirmed	Rhino-orbital	Hemifacial numbness, decreased visual acuity, chemosis, black eschar on the skin, proptosis, frozen eye, complete loss of vision, fixed mydriasis, complete opacification of sinus, mucosal necrosis	–	Systemic antifungal drugs	Yes	Outcome not mentioned	[108]
Iran	1	50/F	Type-II DM, hypertension, gastric bypass surgery	Dry cough, shortness of breath, myalgia, fatigue	Confirmed/Recovered and readmitted after 5 days	Remdesivir, Dexamethasone	Confirmed	Rhinosinusitis	Facial swelling, facial numbness, periorbital edema, erythema, necrotic eschars on the palate and nasal turbinates	Rhizopus oryzae	Amphotericin B	Yes	Discharged after 28 days	[172]
Austria	1	53/M	Myelodysplastic syndrome, depression.	Fever, ARDS	Confirmed/Active	Tocilizumab, Prednisolone	Confirmed during postmortem	Pulmonary	–	Rhizopus microsporus	Not administered	No	Died	[173]
UK	1	22/M	Necrotic hemorrhagic pancreatitis	N.A.	Confirmed/Active	N.A.	Confirmed during postmortem	Disseminated	Florid fibrinous pericarditis containing fungal hyphae, thrombotic endocarditis	Mucorales	Not administered	NA	Died	[174]
Brasil	1	86/M	N.A.	Acute diarrhea, cough, dyspnea, and fever	Confirmed/Active	Ceftriaxone, azithromycin, oseltamivir, hydrocortisone	Confirmed	Gastrointestinal	Two giant gastric ulcers with dirty debris and a deep hemorrhagic base	–	Not administered	No	Died	[105]
Italy	1	66/M	Respiratory symptoms	Arterial hypertension, urinary tract infection	Confirmed/Active	Hydroxychloroquine, lopinavir-ritonavir,piperacillin-tazobactam, levofloxacin	Confirmed	Pulmonary	Buried cavitary lesions in the lingula of the left lung upper lobe, rupture of the cavities previously observed in the pleural space and bilateral pleural effusion	Rhizopus sp	Amphotericin B, isavuconazole	No	Died	[109]
India	1	Middle-aged/F	Newly detected DM	Fever	Confirmed/Active	–	Confirmed	Rhino-orbito-cerebral	Left eye ptosis, facial pain, ophthalmoplegia, vision acuity loss, orbital apex syndrome, opacification of sinuses, polypoidal mass into sinuses with pus, acute infarct	Rhizopus sp.	Amphotericin B	Yes	Discharged.	[175]
India	1	32/F	DM	None	Confirmed during hospitalization/Active	–	Confirmed	Rhino-orbital/Paranasal	Left eye complete ptosis and left facial pain, nasal pus, opacification of sinuses, subperiosteal abscess, optic neuritis	–	Amphotericin B	Yes	Discharged due to financial constraints.	[176]
India	23; M: 15, F: 8	Age range not mentioned	DM: 21, Hypertension: 14, Renal failure: 1	–	Confirmed: 23/Recovered and readmitted: 19 & Active: 4	Systemic corticosteroids	Confirmed: 23	Paranasal: 23, Intraorbital: 10, Intracranial: 2	–	–	Amphotericin B	Yes	Ongoing	[177]
India	6	46.2–73.9/M:6, F:0	Type-II DM: 6	–	Confirmed:6/Recovered and readmitted: 5 & Active: 1	Prednisolone, dexamethasone, methylprednisolone (except one case)	Confirmed: 5, suspected: 1	Rhino-orbito-cerebral: 5, Rhino-orbital: 1	Periocular swelling, drooping of eyelids, limitation of ocular movements, and painful loss of vision.	–	Amphotericin B, posaconazole	Yes	Not known	[178]
India	1	55/M	DM, hypertension, ischemic cardiomyopathy, end-stage renal disease	Fever, dry cough, breathlessness	Confirmed/Active	Dexamethasone, remdesivir	Confirmed	Pulmonary	Pleural cavity, pleural effusion	Rhizopus microsporus	Amphotericin B	Yes (scheduled)	Discharged	[9]
India	1	50/M	–	–	Confirmed/Recovered	–	Confirmed	Rhino-orbito-cerebral	Sinus thrombosis, conjunctival congestion, chemosis, corneal edema, sinusitis, panophthalmitis, proptosis	Rhizopus sp.	Amphotericin B	No	Died	[179]
India	1	38/M	Insignificant	High grade fever, body ache, cough, shortness of breath	Confirmed/Active	Remdesivir, methylprednisolone, dexamethasone	Confirmed	Sino-orbital/Rhino-orbital	Eye swelling and pain, malaise, proptosis, chemosis, periorbital cellulitis, partial ophthalmoplegia, polypoidal mucosal thickening in sinuses	Rhizopus oryzae	Amphotericin B	Yes	Discharged	[180]
India	10	37-78/M:9, F:1	DM, hypertension, chronic kidney disease.	–	Confirmed/Active	Steroids: 6, Tocilizumab: 1, remdesivir: 6	Confirmed	Rhino-orbito-cerebral	Facial pain, nasal block, mucosal thickening of sinuses, bony erosion,	-	Amphotericin B	Yes	Died: 4 Discharged: 5 Lost follow-up: 1	[181]
India	25	30-74/M:22, F:3	DM, Hypertension, Leukemia	–	Confirmed: 11/Active	–	Confirmed	Rhino-orbito-cerebral: 6, Rhino-orbital: 19	Unilateral facial swelling, retro-orbital pain, ptosis, headache	-	Amphotericin B	Yes	Died: 2, Discharged or lost to follow up: 23	[182]
India	17	39-73/M:15 F:2	DM	–	Confirmed/Active or recovered and readmitted	Steroids: 16	Confirmed	Maxillofacial and rhino-cerebro-orbital	Facial swelling, headache, orbital cellulitis, proptosis, loss of vision, sinusitis, maxillary necrosis, edema, intracranial involvement,	-	Amphotericin B	Yes	Died: 6, Discharged: 11	[183]
India	10	27-67/M:8, F:2	DM, Diabetes ketoacedosis	–	Confirmed	Dexamethasone, remdesivir	Confirmed: 6, suspected: 4	Orbital	Sinusitis, pansinusitis, apex or extraconal involvement	Rhizopus sp., Mucor sp.	Amphotericin B	Yes	Died:4, discharged: 6	[184]
India	1	60/M	DM	Breathlessness, pyrexia, tachypnea, generalized malaise	Confirmed/Active	Meropenem, oseltamivir, methylprednisolone, dexamethasone, tocilizumab, sitagliptin/metformin	Confirmed	Rhino-Orbital	Eyelid edema, proptosis, sinusitis, right orbital cellulitis, soft tissue necrosis, conjunctival edema, exposure keratitis.	–	Vancomycin, Amphotericin B	No	Died	[185]

Mucormycosis, colloquially called the deadly ‘black fungus' in India, has been increasing dramatically throughout the past months since the ‘second wave’ of the pandemic hit India. As per the central government's recommendation, states declared it as a ‘Notifiable Epidemic’ under the Epidemic Diseases Act, 1897, and urged people to follow ICMR, India, for the screening, diagnosis, and management [97]. Moreover, every case has been documented by the Department of Health, Govt. of India and Integrated Disease Surveillance Project (IDSP) surveillance system [97]. The national capital of India, Delhi, issued ‘The Delhi Epidemic Diseases (Mucormycosis) Regulations, 2021’ to combat the fungal infection immediately after declaring it an epidemic [98]. India had accounted for more than 28,252 cases up to June 8, 2021, and Maharashtra (6339), and Gujarat (5486) reported approximately half of the cases [99]. Black fungus infection is striking patients within 12–18 days after COVID-19 recovery, and nearly 80% require surgery. Nevertheless, the mortality rate can reach 94% if the diagnosis is delayed or untreated [100].

From September 2020 to December 2020, Patel and colleagues supervised a multicenter epidemiological investigation across India to assess the consequence of CAM cases and found a prevalence of 65.2%, i.e. 187 of 287 mucormycosis patients were affected by COVID-19. Interestingly, it was a 2.1-fold hike in mucormycosis cases if compared to the mucormycosis cases of September 2019–December 2019 [101]. Not only DM but also COVID-19 was the sole predisposing factor in 32.6% of CAM patients, but COVID-19-related hypoxia and erroneous use of steroidal drugs were also found to be associated with CAM incidence [101]. In this particular timeline, the mortality rate was 45.7%, with no distinction between CAM and non-CAM cases [101]. Report 1 of the COSMIC study (January 1, 2020–May 26, 2021) conducted by Sen and team, which involved rhino-orbital-cerebral mucormycosis and managed by Indian ophthalmologists, was recently published. It showed that Gujarat (22%) and Maharashtra (21%) are the worst hit states [102]. The patients' mean age was found to be 51.9 years with male dominance (71%) and oxygen support (57%), steroid use (87%), as well as DM (78%) reported as the predisposing factors [102]. Rhino-orbital-cerebral mucormycosis tended to begin between the 10th to 15th days after COVID-19 detection. Herein, 56% developed the disease during COVID-19 and 44% exhibited delayed onset. Overall mortality was found to be 14% after the final follow-up [102].

9. Prevalence of CAM: statistical perspective from clinical case reports

The odds ratio was performed to determine the association between the outcome status (died or discharged) of patients affected by mucormycosis and different prognostic factors of those patients. Data from various countries and India, reported by several authors as mentioned in Table 1, were selected to carry out statistical analyses. Here, the outcome status of the patients is considered as the response variable and the other variables, i.e., age (in years), gender (male or female), comorbidity factors (diabetes, renal related disease, hypertension, etc.), location of the mucormycosis (rhino-orbital-cerebral, pulmonary, etc.), antifungal treatment by amphotericin B (yes or no), and surgery (yes or no) are considered as prognostic factors. The odds ratio (OR) and corresponding 95% confidence interval (CI) are carried out for India and the rest of the world to highlight the significance of the outcome status over the several above-mentioned prognostic factors. This study revealed that OR of <1 indicates that the presence of prognostic factors is less harmful in relation to expected death for the patients. The reverse result of OR, i.e., OR of >1, specifies the presence of prognostic factors that are more harmful to the patients, in relation to an expected fatal outcome. From the data set, we found that patients from different age groups are affected by mucormycosis worldwide, including in India. Considering the world data (except India), we found that a 22-year-old young male patient was affected by and died from mucormycosis, and an 88-year-old man also died from mucormycosis. These data exemplify how a wide range of age groups are affected by this disease with a mean (standard deviation; SD) age of 53.39 (SD = 17.25) years. Similar findings are also observed for India, where the minimum age of the affected patient was a 23-year-old male, and the maximum was a 78-year-old male. The summary of the patient's age data is observed with a mean age of 52.65 (SD = 13.18) years. In both the cases, the mean age of the patients was found near to 50 years old, which is also reported by Sen and colleagues (2021). For the sake of statistical analysis of OR, we separated the age of the patients into two non-overlapping age groups: (i) patients having an age less than or equal to 50 years old; (ii) and patients who are more than 50 years old. For the global data, the OR of the patients > 50 years old compared to ≤ 50 years old is 1.5 (95% CI: 0.2184–10.3039), which indicates that patients aged > 50 years have 1.5 times high mortality chance than those ≤ 50 years old. However, the reverse trend has been noted for the Indian context, where the OR of the patients ≤ 50 years old compared to those > 50 years old is 2.333 (95% CI: 0.6172–8.8206). The result indicates that Indian patients aged ≤ 50 years old had a more than twofold higher mortality rate than patients >50 years old. Gender-wise infection occurring by the disease has also been reported in India and the remaining world. Globally, it has been found that among all mucormycosis-affected patients, almost 75% were male (74.19%), and the remaining 25.81% were females infected by mucormycosis, whereas in India, more than 80% male (80.41%) and 19.59% of females were diagnosed with mucormycosis. In the context of death by mucormycosis within the gender classification, it has been found that male patients have a 1.8-fold higher mortality rate than females in India (OR: 1.8056; 95% CI: 0.3020–10.7960). However, based on world data, females were found to present 1.5-fold higher mortality rate than males (OR: 1.5; 95% CI:0.2184–10.3039).

Amphotericin B drug has been widely used as an antifungal treatment for mucormycosis in India and worldwide. It has been observed that 100% of patients in India had been treated with Amphotericin B, while more than 90% of patients (92.86%) were treated with Amphotericin B in other countries. It has been found that almost 99% of cases in India and 71% of mucormycosis cases in the world (except India) were reported with Rhino-orbital-cerebral location. DM among mucormycosis patients was also diagnosed with a maximum number of occurrences. Approximately 95% of patients in India were diagnosed as having DM and 75% of patients in the remaining countries were detected as having DM with one of the comorbidity factors. A similar finding was also reported in a study by Sen and colleagues (2021), in which DM was reported with a maximum number of cases in India associated with mucormycosis. The most important analytical finding was observed for the patients who have undergone surgery to recover from mucormycosis. The OR of the patients who have undergone surgery compared to those without surgery was found to be 0.0406 (0.0021–0.8038) for India and 0.1123 (0.0054–2.3314) for all the other countries. In other words, it was found that patients who were not given surgery had almost 25-times higher mortality chances compared to those who underwent surgery in India, and more than 9-times mortality rate worldwide. This analysis reveals that surgery within the stipulated time saved more lives.

10. Probable Reasons Behind Covid-19 Associated Mucormycosis

COVID-19 and mucormycosis exhibit shared risk factors and high mortality rates. Zhou and colleagues (2020) reported that 50% of COVID-19 patients died due to underlying secondary bacterial co-infections [103]. In this regard, fungal co-infections are an add-on to COVID-19 fatalities [104]. We anticipate the following reasons for the surge in mucormycosis cases during COVID-19. People with risk factors are predisposed to the infection. However, presenting COVID-19 alone and without traditional risk factors of mucormycosis is sufficient to build a predisposing environment, as seen in the case of reports [105]. Patients infected by SARS-CoV-2 are supported with mechanical ventilation and acquire the risk of ventilator-associated infection or nosocomial infections due to prolonged hospital stays. Patients acquire the infection from electrocardiogram (ECG) leads, contaminated intramuscular injections, adhesive tapes, or hospital environment airways [40]. Researchers anticipated that people with poorly controlled DM were at higher risk of reinfection with COVID-19, which is attributable to an impaired adaptive immune response [106,107]. In addition, freshly harboured glucocorticoid-induced diabetes during COVID-19 management adds fuel to the flames [108]. Patel and researchers already proved that 78.7% of CAM patients were provided with steroids during COVID-19 management [101]. Steroid-induced immunosuppression and immune deregulation (during or just after COVID-19 recovery) predispose patients to opportunistic fungal infection, i.e. mucormycosis, leading to their readmission into hospitals [108]. COVID-19 victims produce significantly high levels of inflammatory cytokines (cytokine storm), including IL-2R, IL-6, IL-10, TNF-α etc., resulting in impaired cell-mediated immune response and affecting both CD4 + and CD8⁺ T cells [105]. The most severe COVID-19 cases exhibit a significant drop in the absolute lymphocyte number (lymphopenia), especially T cells. Mucorales-specific T cells secrete various cytokines and directly damage the fungal hyphae. Reduction of T lymphocytes in the case of COVID-19 may be linked with the poorest disease outcome and high risk of opportunistic fungal infections [109]. Disruption of iron homeostasis is a classic factor for COVID-19 [110]. SARS-CoV-2 interacts with haemoglobin via ACE2 and CD147 receptors and a cascade of events occurs along with the viral endocytosis. This results in haemoglobin malfunction, hemolysis, and release of heme [110]. Another reason might lie in the SARS-CoV-2 spike glycoprotein, which mimics ‘hepcidin’, the key regulator of iron metabolism in host cells [110]. Thus, a severe COVID-19 case leads to the presentation of hyperferritinemia [111]. As a clinical marker of the inflammatory response, Ferritin stimulates SARS-CoV-2 pathogenesis [112]. Ferritin binds and stores iron in the cell. Nevertheless, a high ferritin level leads to the accumulation of more intracellular iron, which results in tissue injury by ROS [111]. In addition, macrophage activation and the cytokine storm (e.g., elevated IL-6) trigger hepcidin upregulation, ferritin production and lower iron export, thus causing cellular iron overload [110,111]. The consequent tissue injury results in the export of free iron into the blood circulation and stimulates Mucorales fungal pathophysiology. COVID-19 is linked to widespread lung parenchymal illness, including diffuse alveolar damage, hyaline membrane development, interstitial lymphocyte infiltration, and the production of vascular microthrombi. These lung abnormalities can take weeks to heal, providing an appropriate environment for fungal growth [113]. COVID-associated pulmonary endothelialitis (ROS-mediated and ferritin-free iron-catalyzed) in blood vessels exposes the body to fungal adhesion and invasion [110]. The SARS-CoV-2 spike glycoprotein induces endoplasmic reticulum stress and further increases GRP78 expression [114], almost five times more than normal [111]. Endothelialitis, as a single stress factor, also upregulates GRP78 expression. Interestingly, SARS-CoV-2 uses the same GRP78 receptor for internalizing into host cells [114]. Thus, it clears the way for Mucorales Pathogenesis and virulence. Moreover, acidosis and hyperglycemic stress conditions induce endothelial GRP78 and fungal CotH, building the perfect storm for mucormycosis [111].

During the first wave of the COVID-19 pandemic, India did not witness any significant rise in fungal infection cases. During the second wave, India's SARS-CoV-2 infections broke pandemic records, with over 300,000 positive test results daily for a week. On April 26, 2021, India had the world's largest daily tally of new COVID-19 infections (360,960), raising the country's total number to 16 million cases, next only to the United States of America (USA) and more than 200,000 deaths [115]. India has the second largest diabetes population (65.1 million) globally, with 70% of uncontrolled diabetes causes [37], which is adeptly predisposing the Indian community to an upsurge in mucormycosis. Furthermore, it is well known that reinfections can occur [116,117] and, therefore, recovered patients from the first wave of infection were infected once again during the second wave, even when these individuals were in the ‘long-COVID’ period [118]. Reinfections, being higher during the second wave [115], make the patients vulnerable to co-infections. After recovery, most people drop their guards (e.g., masks), and fungal pathogens may take advantage when entering through the nasal path and invade the body with lower immunity.

Moreover, the UK variant (B.1.1.7) and Indian variant (B.1.617) of SARS-CoV-2 were predominant during this second wave of the pandemic, and they exhibited an advantage over the pre-existing strains [115] and might have caused greater disease severity. Eventually, co-infections increase with disease severity. Tropical and subtropical humid climates, as well as high ambient temperatures in most regions of India, create an ideal setting for the growth of these fungi and may contribute to the infection burden [37]. In this context, CAM has been rising while the healthcare settings in India are facing challenges due to the breakdown of infrastructure, lack of sufficient medication and surgical equipment and the low number of healthcare professionals compared to admitted patients. Mucormycosis might have been caused by the use of contaminated medical equipment [29] (Fig. 4 ). The second wave of COVID-19 in India was associated with a drastic drop of oxygen saturation in patients, thus resulting in the crisis of oxygen cylinders [119]. The use of industrial/non-medical grade oxygen with contaminated water to treat the maximum number of patients might be a predisposing factor for mucormycosis, as it is not efficiently purified like medical oxygen [120]. The demand for immunity-boosting supplements increased during the pandemic [121]. Iron overload (a traditional risk factor for mucormycosis) and zinc overload might be contributing to the appropriate environment for fungi growth. Zinc deficiency has long been known to hamper fungal proliferation by inducing stress [122]. The arbitrary use of nutraceuticals and medicines represents the major concern for mucormycosis co-infection in the Indian scenario. Symbiotic microbiota protects the body from pathogen invasion and proliferation [123]. Over-usage of various antibiotics in patients during the treatment of COVID-19 is attributable to the elimination of symbiotic microbial populations and predisposition to co-infections (Fig. 4).

Fig. 4.

Probable reasons for the mucormycosis surge as a COVID-19 co-infection in India. In both diabetic and non-diabetic COVID-19 patients, corticosteroidal drugs reduce immunity, cause immune deregulation and raise blood sugar levels. The new onset of steroid-induced diabetes and immune suppression may act as the predisposing factor for mucormycosis. With COVID-19, disease progression demands more oxygen cylinders and intubation in patients and prolonged hospital stay, increasing the risk of nosocomial fungal infections. Due to the massive second wave outbreak of SARS-CoV-2 in India, the health infrastructure collapsed, and in turn, unhygienic and contaminated instruments were used to attend the hospitalized cases.The abrupt use of nutraceuticals might have caused iron overload, i.e., a conventional risk factor for mucormycosis and/or zinc overload, contributing to the suitable settings for fungal growth. Moreover, the over-use of antimicrobial medications results in the eradication of symbiotic bacteria from the host body. It is hypothesized that such factors and the SARS-CoV-2 variants (that have caused disease severity) may have triggered the increased case numbers of co-infections, i.e., mucormycosis in current circumstances.

11. Mucormycosis diagnosis

Co-infections are challenging to diagnose. The patient might have harboured the organism before the viral infection. It could also be associated with a pre-existing infection or might have been acquired as a nosocomial infection [2]. Detection or diagnosis technologies to identify a wide variety of possible infections and antibiotic resistance are preferred. Nevertheless, early diagnosis of any co-infection is mandatory to restrict harmful consequences [2]. Physicians should check the medical history i.e., risk factors and symptoms, before further proceeding with imaging, laboratory diagnosis and other available techniques. If a mucormycosis infection is suspected, tissue biopsy involving microscopic examination or fungal culture analysis and other known methods should be opted for after collecting tissue samples from infected areas or body fluid. Algorithms have been recommended for prompt detection of mucormycosis where the ‘red flag or warning signs’ should be of primary concern [124]. For instance, diplopia, proptosis, cranial nerve palsy, sinus soreness, periorbital puffiness, palatine ulcer, or orbital apex syndrome indicate rhino-orbital-cerebral mucormycosis [124]. Angioinvasion and vascular thrombosis result in tissue necrosis, which is frequently a late indication of mucormycosis [125].

Medical imaging Computerized tomography (CT) scan, Magnetic resonance imaging (MRI) and/or endoscopy can further be used to determine the degree of infection [108]. In the case of patients with suspected pulmonary mucormycosis, CT is advised to detect the reversed halo sign (a region of the lungs with ground-glass opacity along with the ring of consolidations) which is a classic hallmark for pulmonary mucormycosis. In addition, CT pulmonary angiograms look for vessel occlusions in the lungs [10,126]. If rhino-orbital-cerebral mucormycosis is suspected, cranial CT or MRI is advised. In case of its invasion into the eyes or the brain, MRI with high sensitivity is recommended instead of CT. If mucormycosis is confirmed, regular body imaging involving the brain, thorax or abdomen should be done to determine the degree of the infection or dissemination [10]. PET/CT (positron emission tomography/computerized tomography) using [18F]-fluorodeoxyglucose (FDG) can also be employed in the near future for greater sensitivity [127].

Histopathology and Culture techniques Mucormycosis is generally suspected on the basis of direct microscopic analysis of the samples. For better visualization of the hyphae with more optical brightness, the blankophor and calcofluor white (fluorescent brighteners) are used along with potassium hydroxide (KOH) [128]. The infection is confirmed after visualizing typical hyphal characteristics using hematoxylin and eosin stain, periodic acid Schiff stain and/or Grocott-Gomori's methenamine silver stain [25,26,129]. Due to tissue processing, pseudo-septae can be formed or a 45° angle of branching can be deformed. Hence, the wide and asymmetrical ribbon-shaped hyphal structure is a more consistent feature for microscopic analysis [10]. In acute lesions, hemorrhagic infarcts, coagulative necrosis, angioinvasion, neutrophil infiltration (in non-neutropenic patients), and perineural invasion (PNI) are the distinctive characteristics; while, in chronic lesions, the pyogranulomatous inflammation (PI), and often hyphae enclosed by the Splendore-Hoeppli phenomenon are observed [10]. However, lesions are nonspecific. Diagnosis based on histological characteristics is difficult and often misidentified as Aspergillosis [10]. Therefore, to overcome such bottlenecks, immunohistochemistry techniques can be used along with monoclonal antibodies (mAbs). These mAbs act against specific Mucorales and permit the differentiation between mucormycosis and aspergillosis [126,130]. For the preliminary identification of fungi up to the genus and species level (and antifungal susceptibility testing), the solid media culture is useful, followed by macroscopic and microscopic identification [10,128].

Molecular methods The confirmed genus and species level identification demands sensitive molecular methods. Molecular analysis is robustly supported and favoured over histomorphology. For instance, internal transcribed spacer (ITS) sequencing is encouraged over Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) because of its limitations [10,131]. Varied types of modern, specific and sensitive tools such as nested PCR, RFLP coupled nested PCR, qPCR, PCR/high-resolution melting analysis (HRMA), and PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS), etc. can be used for rapid detection of the pathogen. The prime target of these tools is 18S rDNA, 28S rDNA, the cytochrome b (cyt b) gene, the mitochondrial gene rnl, CotH gene, and ITS region for R. arrhizus, Mucor sp., R. microsporus [10,126,132,133].

Serology Various techniques, including ELISA, immunoblots, β-D-glucan test, and immunodiffusion tests are usually used to detect mucormycosis infection [134]. Mucorales-specific T-cells can be detected by the enzyme-linked immunospot (ELISpot) assay [135]. During sandwich ELISA, a monoclonal antibody named 2DA6 appears to be extremely reactive with cell wall fucomannan of the Mucor sp [136]. Nevertheless, a lateral flow immunoassay detecting fucomannan is more convenient than ELISA and may potentially be used in point-of-care tests [136].

Metabolomics-Breath Test This approach can be used to detect the volatile organic compounds in the expired breath of mucormycosis patients. Koshy and colleagues performed a breath volatile metabolite profile of invasive mucormycosis-causing fungus (R. arrhizus var. delemar, Rhizopus arrhizus var. arrhizus, R. microsporus) by using GC–MS and concluded that there is a distinct breath profile for the three causative fungal agents; they suggested this as a non-invasive diagnostic method for invasive mucormycosis [137]. Such real-time, metabolomics-breath tests could allow large-scale screening and thus enable earlier treatment of mucormycosis patients before it develops into its severe forms.

12. Mucormycosis management

Angioinvasive hyphae are expected to induce necrosis and thrombosis. Complex pathogenesis makes it hard for antifungal medicines to penetrate. Thus, vigorous surgical debridement is mandatory as a part of treatment [138]. Risk stratification based on disease severity, rapid and early detection, reversal of primary predisposing factors (if applicable), surgical debridement of damaged tissue, effective antifungal medication (monotherapy or combination therapy), reversal of immunosuppression (e.g., cessation of chemotherapy or enhancing neutrophils) and possible control of predisposing comorbidities are the fundamental strategies (Fig. 5 ) for the management of mucormycosis [17,139]. According to the universal instruction of EMCC, the first-line antifungal regimen includes high-dose liposomal amphotericin B (5 mg-10 mg kg⁻¹ per day). In contrast, moderate-strength posaconazole and isavuconazole are strongly recommended as a treatment [10]. Posaconazole delayed-release tablets or infusions, if available, are preferred over posaconazole oral suspensions. Most azoles are fungistatic drugs that act in the metabolic pathways to inhibit fungal growth [140,141]. Due to the high toxicity, amphotericin B deoxycholate is not advised (although it may be the only alternative in resource-constrained situations) [10]. Amphotericin B lipid complex improves the safety profiles of patients [142]. In a study on rhino-orbital-cerebral mucormycosis patients, whoever received the treatment of amphotericin B-lipid formulation along with caspofungin combination therapy exhibited better therapeutic success and longevity than people treated with amphotericin B lipid complex alone [143]. Nevertheless, polyenes (e.g., amphotericin B) and echinocandins (e.g., caspofungin) show synergistic effects, as echinocandins destroy some glucan on the fungal cell wall, which unmasks immune epitopes and accelerates phagocytosis by the host defence cells [139]. Early-stage diagnosis of mucormycosis and the treatment by using antifungal drugs such as amphotericin B increases the rate of survival by 40%–80% [144].

Fig. 5.

Diagnostic and therapeutic pathway for invasive mucormycosis infection. Computerized Tomography scan (CT scan), Magnetic Resonance Imaging (MRI), Positron Emission Tomography/Computed Tomography scan (PET/CT scan), Hematoxylin and Eosin stain (H&E stain), Grocott Methenamine-Silver and Periodic Acid-Schiff stain (GMS & PAS stain), Internal Transcribed Spacers (ITS), Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF-MS), Polymerase Chain Reaction (PCR), PCR-High Resolution Melting Analysis (PCR-HRMA), PCR-Electrospray Ionization/Mass Spectroscopy (PCR-ESI/MS), Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme-Linked Immunospot assay (ELISpot assay), Lateral Flow Immunoassay (LFI assay), and Gas Chromatography-Mass Spectrometry (GC-MS).

The cornerstone of mucormycosis management is the surgical excision of necrotic tissues. The surgical intervention combined with proper systemic antifungal drugs has been proven to considerably enhance survival in pulmonary mucormycosis compared to antifungal treatment alone [139]. Researchers revealed that surgical treatment was successful in 22 individuals with rhino-orbital-cerebral mucormycosis and local control enhanced the survival rate [145]. Therefore, adjunctive therapy to reverse the risk factors is essential. It is known that relapsed or refractory malignancy and chronic neutropenia are the primary agents for mucormycosis-associated mortality [146]. For example, adjunctive therapy to reverse neutropenia in haematological patients by hematopoietic growth factors or by white blood cell (WBC) transfusions should be encouraged. Immunosuppressed patients (as seen in autoimmune disease) should switch to alternative non-steroidal therapy to restore their immunity. Rigorous control of hyperglycemia of diabetic/DKA patients should be monitored. In this regard, reversing acidemia with sodium bicarbonate can partly limit the ability of Rhizopus arrhizus to infiltrate the vascular endothelium and restore host iron chelation along with neutrophil functioning [147]. Thus, iron chelators are potential options for adjunctive therapy. Hyperbaric oxygen is another option that improves neutrophil efficiency, reverses acidosis, and inhibits fungal proliferation [148]. Based on limited in vitro evidence, immune-augmentation approaches such as administering granulocytes (e.g., macrophages), colony-stimulating factors or interferon have been advocated as treatment options [149,150]. In a study by Grimaldi and colleagues, an immunodeficient trauma patient with chronic mucormycosis was effectively treated with a combination of interferon-gamma and a monoclonal antibody named nivolumab [151]. Therapy and management of mucormycosis do not involve any limited time duration. Decisions are taken on an individual level. As per the principle, the antifungal medications for mucormycosis are continued until all clinical manifestations are resolved; laboratory tests, as well as imaging signs and symptoms, are resolved using serial analysis, and continue until the immunosuppression has been reversed [10,139]. Finding a new drug design and target is of the utmost importance for efficient treatment. In this context, multicenter studies and meta-analytic research should be involved in developing more novel strategies.

13. Futuristic drugs in the pipeline

Mucorales infection, exhibiting a high level of antifungal resistance to most antifungal drugs found in the market, demands novel therapeutic agents for competent treatment. As monotherapy displays suboptimal efficacy, combination therapy might be a potential approach in the near future [152]. A clinical trial (Phase II) is ongoing to evaluate the efficacy of combined therapy for pulmonary mucormycosis involving amphotericin B deoxycholate inhalation with intravenous (IV) amphotericin B, compared to only IV amphotericin B [153]. Another study on the evaluation of Isavuconazonium Sulfate for the management of invasive mucormycosis in neonates is under Phase II clinical trial [154]. Various drugs are under in vivo experiments in model organisms (Fig. 6 ). Fosmanogepix (APX001) via manogepix targets Gwt1 protein, which interferes with the glycosylphosphatidylinositol (GPI) post-translational modification pathway and inhibits inositol acylation on the fungal cell surface, along with GPI-anchored proteins needed for fungal growth and virulence. In murine models, fosmanogepix was found to be effective when treating pulmonary mucormycosis caused by Rhizopus arrhizus [155]. Calcineurins can suppress bycA mRNA expression, promote hyphal growth and induce the virulent traits in Mucorales. Hence, as novel therapeutic drugs against mucormycosis infection, calcineurin inhibitors are considered the foremost agents in the pipeline of Mucorales management [156]. Statins inhibit HMG-CoA reductase acting in the mevalonate pathway, as well as ergosterol synthesis. The successive changes modulate the formation of siderophores, synthesis of hyphae, cellular growth and fungal virulence [157]. Another novel agent, fungal CYP51 inhibitor VT-1161, protects immunosuppressed rodents from the same pathogen [158]. Nevertheless, treatment with anti-CotH antibodies potentially shields DKA mice from mucormycosis by enhancing opsonophagocytosis and preventing invasion [159], which may offer insights that would be beneficial to the development of the therapeutic intervention in humans.

Fig. 6.

Mechanism of action of futuristic drugs/agents for mucormycosis treatment [1]. Fosmanogepix (APX001) targets GWT1 protein, which modulates glycosyl phosphatidylinositol (GPI) post-translational modification pathway and inhibits inositol acylation in the fungal cell membrane [2]. bycA mRNA is the negative regulator for hyphal growth that, in turn, is inhibited by calcineurin. Thus, calcineurin is the positive regulator for hyphal growth and the introduction of virulent traits [3]. Calcineurin inhibitors block calcineurin and appear likely to be a potential futuristic drug for mucormycosis infection. Additionally, ergosterol is the primary factor acting to induce the expression of virulent fungal traits [4]. Statins inhibit HMG-CoA reductase acting in the mevalonate pathway, as well as ergosterol synthesis [5]. VT-1161 inhibits fungal CYP51 and blocks ergosterol synthesis [6] Anti-CotH antibody targets CotH proteins which are widely present in the Mucorales surface and prevents invasion augmenting opsonophagocytosis.

14. Conclusion and future trends

The past months of 2021 witnessed a sharp exponential rise in global mucormycosis cases and infected individuals reached several thousand at a time. The exact reason behind this rise is still unclear. The population-based studies were not enough to conclude the exact count of the past years and the recent case numbers. Moreover, the much more fatal second wave of the COVID-19 pandemic, along with the double mutant or triple mutant variants of SARS-CoV-2, represents a mystery, and the world is grappling with two deadly pathogens. Amongst the fear of black fungus in India, ‘white fungus' and ‘yellow fungus' cases are also being reported. Health authorities reported that these three types of pathogens were found in a 45-year-old patient in Ghaziabad, India [160]. While some recognized white fungus as Aspergillus flavus, others argued that these names are colloquially given to the varied patterns of mucormycosis infections. However, it is comprehensible that fungal infections are showing a sharply increasing trend. According to the Centres for Disease Control and Prevention (CDC), Aspergillosis, Candidiasis, multidrug-resistant Candida auris infection, Pneumocystis pneumonia, etc. are all caused by fungi that attack immunocompromised individuals [161]. Hasty and improper use of antifungal drugs to manage the current mucormycosis surge in the present scenario may contribute to antifungal resistance over time [162] which, in turn, would further boost fungal infections. Indeed, we hypothesize that the world may witness the rise of many more fungal species while combating the present or during the next waves of the COVID-19 pandemic if prompt, effective countermeasures are not taken. The delta variant, i.e. B.1.617.2 of SARS-CoV-2, which is current in India, is now a matter of concern to the world [163]. Robust investigations to understand the basic pathology of co-infections during respiratory viral infections (previous influenza, SARS, or MERS epidemics can help), emergence and re-emergence of these pathogens, as well as the susceptibility of the hosts, are mandatory. Furthermore, an upgrade in the research criteria for genomic studies to identify the host factors that predispose people to a deadly infection such as mucormycosis will be highly valuable to the public health system in India and worldwide.