COVID-19 and Mucormycosis of Orofacial Region: A Scoping Review

During the second wave of coronavirus disease, or COVID-19, infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in the year 2021 around the globe, there is a surge in the number of cases of mucormycosis or “Black Fungus” that is directly/indirectly associated with COVID-19. In this review article, mucormycosis of the orofacial region has gained importance from the maximum published literature (45 articles) from various databases like PubMed, Google Scholar, Scopus, Web of Science, and Embase. Rhino-orbital cerebral mucormycosis (ROCM) is a fatal condition associated with COVID-19 among categories of mucormycosis such as pulmonary, oral, gastrointestinal, cutaneous, and disseminated. ROCM targets the maxillary sinus, also involving teeth of the maxilla, orbits, and ethmoidal sinus. These are of particular interest to dentists and oral pathologists for proper diagnosis and identification. Co-morbid conditions, especially diabetes mellitus type II, have to be monitored carefully in COVID-19 patients as they have a higher risk of developing mucormycosis. In this review article, various presentations of COVID-19-linked mucormycosis are mentioned having particular emphasis on pathogenesis, signs and symptoms, clinical presentation, various diagnostic modalities including histopathology, radiology like CT and MRI, serology, tissue culture, various laboratory investigations, treatment protocols, management with prognosis, and so on. Any suspected case of mucormycosis needs quick detection and treatment since it progresses quickly due to the destructive course of infection. Long-term follow-up along with proper care is a must to detect any kind of recurrence.


Introduction And Background
The coronavirus SARS-CoV2 (severe acute respiratory syndrome coronavirus 2) virus (enveloped RNA virus) resulted in a massive outbreak of COVID-19 in 2019, starting in China and then spreading all over India as well as other parts of the globe. It continued for two years with the first, second, and third waves until now. It was declared a pandemic by the World Health Organization. It resulted in acute respiratory distress syndrome (ARDS) in a maximum number of patients [1].
The launch and global distribution of vaccinations like Covishield and Covaxin at a rapid pace is a great relief for mankind and a ray of new hope to combat the disease [2].
COVID-19 pandemic's second wave gifted a superinfection named mucormycosis or "Black Fungus," an opportunistic fungal infection. COVID-19 is related to various devious co-infections both from bacterial and fungal origin. The trinity of typical co-infections arising secondarily of fungal origin of the oral cavity included candidosis, mucormycosis, and aspergillosis [1].
Mucormycosis became the second most popular interfering mold septicemia and is an epidemic within a pandemic. COVID-19-associated mucormycosis (CAM) is very challenging, as it was tough worldwide to sway the rapidly mutating COVID virus and also for less knowledge to treat mucormycosis as it is considered an infrequent infection [3]. Untreated mucormycosis is rapidly fatal, having high morbidity and mortality, and the term was first coined by Baker in 1957 [4].
Mucormycosis resulted from different fungi species, mainly Mucorales. Mucorales spores have their existence widely in nature in the mucosa of the nose of individuals as a normal commensal. It is uncommon, Pathophysiology COVID-19 enters the cell through receptors namely angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS-2). ACE2-R has higher expressivity in the respiratory, kidney, and digestive tract epithelium. Receptors of TMPRSS-2 are located particularly in the respiratory and gastrointestinal epithelium among many epithelia. It tends to attack lymphocytes when binding with these cellular ACE2 receptors, inducing lymphopenia, lowering counts of CD4+ and CD8+ T-cells, and therefore depressing the immune system [2,4]. This destruction happens due to increased interleukin measures (interleukin-2,6,7, interferon-gamma inducible factor, granulocyte colony-stimulating factor) inducing cytokine storm. This results in the following: 1. deterioration of lymphoid tissue; 2. compromised defense system; 3. decreasing future production; 4. increased protective lymphocytes [2].
Also, the effect of lactic acidosis knocks down an alveolar type 2 cell and affects the rebuilding of bronchial cells, consequently causing respiratory difficulties. Therefore, increasing acid-base levels finally results in hypoxemia and hypoperfusion. Since the cytokine storm needs to be immediately treated by immunosuppressive steroids, an optimal environment is generated for fungal growth. Finally, two other conditions give energy booster to the increase of Mucorales in the body of infected individuals: 1. Ferritin levels get shot as there is a higher breakdown of red blood cells (RBCs) (iron is deadly to phagocytes).
2. Increased body temperature (these organisms can tolerate high temperatures).
Mucorales get their nourishment as a result of destruction by ACE-2 receptors to beta cells of the pancreas causing higher glucose levels of plasma. So, mucormycosis is more prevalent in diabetic patients [2,3].
Mucormycetes enter blood vessels through damage to the endothelium, insulin opposition, and elevated levels of glucose leading to the growth of the Mucorales, and continuous destruction of the already damaged immunity of the patient. So, it leads to the eventual deterioration of the patient [2].
The fungi being bigger in comparison to others get easy access to the paranasal sinuses. But small-sized species may also be found in the lungs.
The most important process in the pathogenesis of mucormycosis is as follows: Angioinvasion by Mucorales fungi results in embolism of the affected vascular supply, therefore resulting in tissue ischemia and necrosis.
Less blood supply as a result of blockage of blood vessels by Mucorales protects it by resisting systemic antifungal drugs and the host's defense system from approaching the infection site [6].
For thrombosis to occur, the fungi require adhering to endothelial cells and destruction of cohesion entering the bloodstream [6][7][8].

Role of iron
Rhizopus oryzae requires ferrous/ferric for main cellular activities for cell advancement with evolution. In a normal way, the serum of the mammalian body contains iron in bounded forms like transferrin, ferritin, and lactoferrin. These protect from toxicity resulting from free iron. The human body's free iron introduction is very much needed to uplift R. oryzae by distinct mechanisms. They naturally discharge iron chelators of low molecular weight and are termed "siderophores," which are iron lovers. R. oryzae produces polycarboxylate siderophore "rhizoferrin" and gets iron from the body of the host by an energy-dependent receptormoderated process. But, rhizoferrin is not able to draw out iron from the serum of the host. Endogenous siderophores have a limited role in its pathogenicity [5,6].
In renal dialysis patients, due to excess iron production, deferoxamine is included during treatment, since they have a chance of developing toxicity. They chelate directly bound iron in transferrin, forming an ironferrioxamine complex. The ferrous iron gets free by depletion at the surface of the cell of Mucorales by Fob receptors [6,7].
Rhizopus spp. is 8-40 times the quantity of iron by deferoxamine. Free iron obtainability depends upon the seriousness of the disease caused by Rhizopus spp. Conditions like acidosis decreasing the iron-adhering capability of transferrin through proton-modulated substitution of ferric ions create beneficial environments for Mucorales growth. In patients having continued blood transfusions as a result of diseases like myelodysplastic syndrome, iron overload is caused ultimately causing mucormycosis [6].
Risk factors could be local or systemic in nature.

Local Factors
Inhaler usage, sinusitis of acute origin, injection, trauma, or burns can be considered.

Systemic Factors
Diabetes mellitus (uncontrolled) is one of the major causative factors.
Decreased immunity, drug use, hematological disorders or diseases, malignancy-related disorders, hematopoietic stem cell transplantation, and solid organ transplantation [5,6] are other important factors. Uncontrolled diabetes is likely to be one of the most important causative factors to produce mucormycosis in developing countries like India.

Environmental Factors and Causes of COVID-Associated Mucorrmycosis (CAM)
Humans acquire mucormycosis disease (universal distribution) through inhalation, consumption, or traumatic infusion of the sporangiospores of Mucorales fungi from the atmosphere. But the spore load is higher in tropical countries. Mucorales spores are found in the air in India's indoor and outdoor environments. Rhizopus arrhizus, the major pathogenic species, is also the predominant species isolated from the environment [7]. Diabetes mellitus is a significant consideration for the spread of mucormycosis infection In COVID-19 patients ( Figure 2).

FIGURE 2: Causes and pathogenesis
Graphical representation of causes and pathogenetic mechanisms of mucormycosis.

Passages of Access
Although they are universally present, these fungi are rarely found as normal commensal microflora of the dermis/abdominal tract.
Mucorales are not capable of permeating by undamaged epidermis/dermis or mucosa. Break in the mucocutaneous cohesion due to injury/tear and laceration/mechanical procedures/extraction sockets of teeth creates passages for fungal entry. Also, the Mucorales spores get access through the nose or get direct entry into the oral cavity by using uncleaned objects and hospital materials, etc., in dental setups like forceps, burs, or airotor handpiece [6].
The most common mode of entry is the ventilators (mechanical) giving oxygen to hypoxemic patients. The possibility of entry can be during drug delivery in the treatment of COVID-19/easy entry into the blood circulation by catheters used intravenously/subcutaneous injections [5,6,8].

Mucormycosis diagnosis [8]
Diagnosis It can be done by diagnosing Mucorales from the affected tissue by performing a biopsy and thereafter histopathology. A validatory fungal culture in microbiology is also important. Diagnostic imaging techniques, Molecular Assays, and Direct identification of fungus from the body's secretions like blood, plasma/serum, pleural fluid, and urine are equally significant.

Diagnosis Based on Clinical Approach
Identification of host-microbial interaction along with its varied clinical presentation is important for prompt response.
Double vision, exophthalmos, discomfort in the sinus, periorbital edema, palsy of the cranial nerve, palatal ulceration, and apex syndrome of the orbit are the "RED FLAGS" [8].
Based on Smith and Kirchner criteria, the gold standard to confirm mucormycosis, the diagnosis clinically comprises the following: a. If dried and crusted blood is present, black and necrotic turbinates may be misdiagnosed.
b. Nasal blood-colored secretions are linked to facial pain in the affected region.
c. Soft swelling with darkening around the peri-orbital or peri-nasal area that is related to the development of induration. d. Abnormally low-positioned upper eyelid with protrusion of the eyeball from the orbit, total oculomotor paralysis.

Direct Microscopy
It is done with biopsied samples with the help of optical brighteners like Blankophor and Calcofluor White, which aids in fast diagnosis. Fungal hyphae, whether non-septate/pauci-septate, differ in dimensions and have ribbon-like features and they are uneven. They are distinctly seen on periodic acid-Schiff (PAS stain), H&E stained sections, Grocott-methenamine, and Gomori's silver stains.

Histopathology
Inflammation (neutrophilic/granulomatous) and invasive lesions are differentiated since they have angioinvasion and large closure. Perineural invasion is visible in cases where nerve structures are involved.
Necrotic tissue with inflammatory constituents is found, namely neutrophils, lymphocytes, macrophages, and giant cells. Inflamed granulation tissue, hemorrhagic elements, and extravasated RBCs are also noticed. The necrotic tissue shows the appearance of broad, non-septate hyphal forms [9].

Tissue Culture
Sabouraud agar and potato dextrose agar were used. They are cultured at (25°C to 30°C). In them, every fungus, especially Mucorales, develops fast (3-7 days). In half of the cases, results are positive for Mucorales.

Immunohistochemistry
A certain monoclonal anti-Rhizomucor antibody of the mouse is used [8].

Antifungal Susceptibility Testing and Identification of Species
Mucorales fungi are easily differentiated from Aspergillus during culture. On inspection by specialists in the fungal field, morphological characteristics might help in prompt diagnosis. ID32C kit (bio Merieux, Marcy lÉtoile, France) is used for identifying Rhizomucor pusillus and Lichtheimia corymbifera and API 50CH (bioMerieux, Marcy-l'Étoile, France) for Mucor species [8].

Serological Examination
ELISA (enzyme-linked immunosorbent assay) technique, Western blot tests, and Ouchterlony tests (allows antigen detection) are among the most important ones.

Tests Using Molecules
Polymerase chain reaction (PCR) performed routinely, chaining of DNA of particular genes, melt curve investigation of products of PCR, and restriction fragment length polymorphism analyses are very significant.
All of the assays mentioned above can be used to detect or identify Mucorales for internal transcribed spacer or the 18S rRNA genes [8].

Imaging
CT scanning of both sinus and lungs: Partial or complete cloudiness para-nasal sinuses whether single or multiple, division line appearance between healthy and necrotic bone, also associated teeth movability, and the emergence of sequestrum in maxillary/zygomatic bone are seen; the orbit is also not spared with prominent soft tissue and of adipose tissue dissolution in and surrounding pterygopalatine fossa.
Chest CT scans imaging: Cavities, nodules, pleural effusion, halo signals, and wedge-shaped shadows are the changes seen. Blood vessel capture is also imaged, which denotes a sort of fungal infection but mucormycosis is not specified.
Patients having reduced neutrophil counts and blood cancer show a reverse halo sign (RHS) indicating mucormycosis.
MRI and MRI with contrast agent gadolinium are used in ROCM (method of choice). f) Extension appears as an intracranial hypointense dural amplifying infection.

Dental and Oral Presentations
The manifestations are as follows: Toothache, teeth mobility, foul-or bad-breath-causing halitosis, blockage in the nose, epistaxis and nasal ejection, black pus release, necrosis of bone/sequestrum development in palatal bone and alveolar bone of maxilla, oro-antral or oro-nasal communications or fistulae, pain in and around sinus, trismus-affecting muscles of mastication, unhealed extraction sockets having characteristics same as alveolar osteitis/chronic osteomyelitis, ulceration in the palate, erythematous face, skin getting discolored (black), draining sinuses intra and extra orally, nasal mucosa getting erythematous, erythema around the orbit, swelling and inflammation of subcutaneous connective tissue, the pain of orbit, drooping upper eyelid, double vision, blindness, paralysis of motor nerves of the eye, and migraine [8].
Tables 1-3 of Appendices depict the summary of our review findings .

Management [39-42]
There are different surgery protocols, antifungal treatment and follow-up, monitoring, and other advisories from different associations or institutes as follows:

Complications
It results from the disease or can be the management or therapies of mucormycosis.
a. From the disease: Mucormycosis arising from maxillary sinuses in diabetes mellitus patients usually involves the rhino-cerebral region.
c. Coverage of mucormycosis includes orbit-causing ophthalmoplegia and lessening of visual perceptivity. Complete blindness results from thrombosis of the central artery of the retina. d. Mucormycosis may reach out in a posterior direction from the sinus of the maxilla and cavity of the nose even to the infratemporal fossa, pterygopalatine fossa, and masticatory muscles resulting in trismus and reduction of mouth opening [7][8][9].
e. Disturbances of the gastrointestinal tract, toxicity of the liver, kidney, and heart, infusion-related complications, swollen face, allergy, sensorineural deafness, achromatic vision, migraine, fever, anorexia, loss of calories, disturbances of electrolytes, and creation of advanced mold septicemia are few reported complications of different medicines utilized in the therapies of ROCM [41][42][43].
f. Complications after surgeries are problems in chewing, speaking, and nasal backward flow of fluids in case of a palatal defect. Facial, nasal, and oral deformities can occur during surgical treatment of ROCM [8,44]. Prevention a. Patients with blocked noses should be considered in cases of poor immunity and/or COVID-19 patients on immunomodulators.
b. Early characteristics of mucormycosis should be advised to patients during discharge.
Finally, proper control of infections by dental surgeons/oral pathologists/oral radiologists/technicians is a must and they should follow everything according to new guidelines in a COVID-19 scenario from handling a patient to various dental procedures/radiological procedures whether in a dental setup or academic institute [46].
All healthcare professionals including those >40 years (having greater experience and Master's degree) have good knowledge regarding protocols to be followed at the time of COVID-19 including the use of personal protective equipment (PPE), travel restriction protocols, and all other information regarding COVID in India and Saudi Arabia. Still, further, continuing education programs are necessary [47].
Allied healthcare professionals (AHPs) must also develop further knowledge and keep a positive attitude to increase their skills about everything in the COVID-19 scenario, and in the private sector, strategies must be enforced for better performance of AHPs. This must be done in all countries including India and Saudi Arabia [48].

Prognosis
Prognosis depends on basically the extension of spread, aggressiveness, time of diagnosis and treatment, and immune status (especially the presence of any hematological diseases). The localized infection has a superior prognosis. Extension intracranially has a bad prognosis [40,45]. When there is no systemic ailment for rhino-cerebral disease, there is a 75% rate of survival.
ROCM (with systemic illness) has a 20% survival rate [6,8]. Diagnosis of ROCM can be done very advance than pulmonary mucormycosis. Therefore, ROCM with early detection and therapy has a better prognosis and survival rate. In total, reported mortality with all types of mucormycosis is 40%-80%. The chances of living are poorer in victims having malignancies of hematological origin and transplantation of any organ. The recommendation for follow-up is 36 months as there is a 13% recurrence in three-year postoperative patients. In a follow-up of five years as a whole, the rate of survival is 60% [8,45].
Social media played a good as well as bitter role during the COVID-19 outbreak. It was used for social awareness and it also created mass hysteria. Hence, social media platforms are a double-edged sword that has to be handled with government monitoring [49].
Several opportunistic infections were reported in COVID-19 patients, including Aspergillus spp., Candida spp., Cryptococcus neoformans, Pneumocystis jiroveci (carinii), mucormycosis, cytomegalovirus (CMV), herpes simplex virus (HSV), Strongyloides stercoralis, Mycobacterium tuberculosis, and Toxoplasma gondii [50]. The estimated prevalence of mucormycosis is approximately 70 times higher in India than in the rest of the world [51]. A total of 388 proven/probable mucormycosis cases were reported during the study period with overall mortality at 46.7%. The mortality rate was significantly higher in north Indian patients (50.5%) compared to 32.1% in south India (P = 0.016). The study highlights a higher number of mucormycosis cases in uncontrolled diabetics of north India and the emergence of R. microsporus and R. homothallicus across India causing the disease [52,53].

Clinical significance
Mucormycosis/"Black Fungus" is a fatal fungal infection that is associated with COVID-19 and came as an epidemic within the pandemic in the second wave of the infection. Mucorales fungi cause angioinvasion resulting in thrombosis of affected blood vessels and thereafter tissue ischemia and necrosis. Among various subtypes, rhino-orbital-cerebral mucormycosis is very dangerous and found in maximum cases of COVIDrelated lesions. This superinfection having a high mortality rate is seen especially in patients having comorbid conditions, particularly diabetes mellitus type II, immunosuppression states, and prolonged ICU stay during COVID-19 treatment. Black purulent discharge with palatal ulceration, bone necrosis, formation of sequestrum in the palatal region, and development of OAF (oro-antral fistula), oro-nasal or (OAC) oroantral communications or maybe fistulae, non-recovered extraction sockets, draining sinuses, difficulty in mouth opening, etc. are some of the clinical presentations. As an oral pathologist, the sole purpose is the earliest detection and diagnosis of risk factors so that they can be managed whether by surgery or an antifungal regimen. Patients having recovered from COVID-19 must be explained about mucormycosis and to seek a dentist's help immediately in any sort of such clinical manifestations. The treatment plan is to be very carefully decided, with long-term follow-up to avoid chances of recurrences and for a better prognosis.

Conclusions
COVID-19 and its association with mucormycosis can be a serious problem in the scenario of COVID-19 infection, especially in the second wave. There is a boost in mucormycosis cases in the COVID scenario. Oral and maxillofacial fungi, if exist, appeared jointly along with COVID symptoms or maybe in a straightaway healing phase. These are most commonly and frequently seen in patients having comorbid conditions, especially type II diabetes mellitus.
Patients infected with COVID-19 are prone to develop oral fungal opportunistic infections. Etiology can be many, under which decreased immunity as a result of latent viral infectivity, immunosuppression, and treatment of steroids for COVID, ventilator-supplemented Mucorales growth, xerostomia, and diabetes can be given importance. This was also found in patients who recovered from COVID-19 and prolonged ICU patients. They may have a certain degree of immunosuppression.
The research on the complex relationship between SARS-CoV-2 and mucormycosis is ongoing, as it is not properly established. ROCM is a serious condition and must be detected early and properly treated. It is a challenging fungal infection plus its mortality rate in COVID patients is towering, mainly in patients with pulmonary disease.
If attentive therapeutic planning is advocated, the rhino-orbital cerebral disease can be successfully managed. Faster recognition with control of CAM must be done properly; otherwise, they can be fatal. Dental surgeons and oral pathologists must detect and identify the symptoms, risk factors, and clinical manifestations at the earliest with proper investigations and treatment for a better prognosis.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.