Mucormycosis: Issues, Pathophysiology, Diagnosis and Treatment

Mucormycosis (Black fungus) is an infection caused by opportunistic fungi of order Mucorales affecting the immune-compromised individuals. Cases of Aspergillus and Candida (White fungus) were also reported. The cases of fungal co-infection in COVID-19 patients are increasing globally affecting mainly diabetic patients and individuals that have undergone corticosteroid therapy. Iron metabolism and hypoxia plays an important role in pathophysiology of this disease. Mucormycosis causes thrombosis of blood vessels hindering the blood circulation causing black discoloration of affected site. Histopathological examination of specimen is the most effective way of its diagnosis. Surgical removal of affected site is the best treatment practice in rhino-orbital and cutaneous cases whereas lipid formulation of Amphotericin B is effective in other forms of fungal infection.


INTRODUCTION
COVID-19 is a viral disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 belonging to coronaviridae family.Airway epithelial cells, alveolar epithelial cells, vascular endothelial cells, type II pneumocytes and macrophages are the primary target sites of SARS-CoV-2 virus. 1 High glucose level in diabetic patients, ketoacidosis, and low oxygen environment inside host body provides favorable condition for the virus to replicate in COVID-19 infected patient using host metabolism.
Phycomycosis or zygomycosis was first described in 1885 by Paltauf and was later coined by Baker, an American pathologist, in 1957 as Mucormycosis. 2ucormycosis is a rare type of life threatening invasive fungal infection in immune-compromised hosts caused by species of order Mucorales, phylum Zygomycota and subphylum Mucoromycotina and are generally found in soil containing decaying organic matter, such as leaves compost piles or rotten wood. 3Rhizopus oryzae is responsible for almost 70% of all cases of mucormycosis.

Overall Epidemiology of Mucormycosis and other fungal co-infection
Mucorales are ubiquitous in nature, so is the cases of mucormycosis.Globally, the incidence rate of mucormycosis fluctuates from 0.005 to 1.7 per million population 9 whereas, in Indian population its prevalence is 0.14 per 1000 i.e., about 80 times higher than developed countries. 10According to the estimate of Leading International Fungal Education (LIFE), the annual prevalence of mucormycosis might be around 10,000 cases in the world excluding India and reaches 910,000 cases globally including the cases in India. 11he cases of mucormycosis is waxing rapidly globally but the growth rate is higher in Asian countries like India and China.However, W. Jeong et al., revealed that the prevalence of disease to be 34% in Europe, followed by Asia (31%) and North or South America (28%), Africa (3%), Australia and New Zealand (3%) based on a recent review of 851 cases over the period January 2000 through January 2017. 12The most common risk factor associated with the disease was found to be Diabetes Mellitus in Asian continent and hematological malignancies and transplantation European countries and US. 13,14The cases of nosocomial mucormycosis are also increasingly being reported after the use of contaminated wooden tongue depressors, umbilical catheter, bandages etc. 15 Chen et al. performed fungal culture on all 99 COVID-19 patients and discovered five cases of fungal infection out of which Aspergillus flavus, Candida glabrata and C. albicans causes one, one and three cases respectively. 16Five cases of COVID-19 associated invasive pulmonary aspergillosis (IPA) was found among 19 consecutive critically ill patients with moderate to severe ARDS in study performed by Germans. 17Among 31 ICU patients 5 out of 6 presumed IPA patients were found to have fungal infection associated with A. fumigatus in Netherlands. 18Pathophysiology This disease is not contagious and no cases of transmission through contact between humans or animals have been reported yet.However, the fungal spores i.e., sporangiospores plays pivotal role in its occurrence. 19Lower respiratory infection by SARS-CoV-2 causes diffuse alveolar damage with severe inflammatory exudation and patients always have immunosuppression with a decrease in CD4 + T and CD8 + T cells. 20o corticosteroids like Methylprednisolone and Dexamethasone were used to ameliorate inflammation mediated lung injury and reduce chances of respiratory failure in COVID-19 patients. 21Major risk factors that possess higher susceptibility to fungal co-infection includes use of corticosteroids as immunosuppressant during organ transplantation, uncontrolled diabetes (diabetic ketoacidosis) and other forms of metabolic acidosis, cancer, neutropenia, trauma, etc. 22 The mortality rate of mucormycosis varies between 40-80% based on the underlying condition and site of infection. 23Suspected fungal infection requires immediate medical attention as it may affect the entire body through hematogenous route and leads to progressive and destructive nature of infection.After the entry of spores through cutaneous route or entry into the lungs after angio-invasion, the spores must pass through the mononuclear and poly-nuclear phagocytes and are terminated by the oxidative factors and defensins manufactured by the host cell in immunecompetent organisms.The angio-invasion results in thrombosis inside blood vessel and subsequent tissue necrosis.But in case of immune-compromised organism, the movement of factors produced as defense against fungal infection is impeded. 24

Diabetes
Mucorales possess a virulence factor that possess ability to acquire iron from the host as it is an essential element for cell growth and development. 25An iron is bound to host carrier proteins i.e., transferrin, ferritin, and lactoferrin and avoids the availability of free iron in the circulation which possess toxic effect. 26The strategy of iron sequestration is a major host defense mechanism against microbes and against Mucorales in particular.In patients with Diabetic ketoacidosis, the level of free iron in their serum is elevated and possess higher susceptibility to mucormycosis. 27Use of deferoxamine in patients also increases the incidence of mucormycosis because this chelant is used as a siderophore by Mucorales to obtain more iron. 28This theory has been proved and well-known that administering iron or deferoxamine to animals infected with Mucorales reduces their survival. 29

Starvation
A study revealed that Mucorales adhere to the extracellular matrix laminin and type IV collagen ultimately leading to angio-invasion.Glucose-regulated protein (GRP78) was found to act as a receptor that mediates penetration through and damage of endothelial cells by Mucorales which was later identified to be induced by glucose starvation.Research performed on a mouse model explained that the enhanced expression of GRP78 leads to increased endocytosis of fungal species by human endothelial cells and their damage. 30So, starvation also acts as a predisposing factor leading to mucormycosis.

Organ transplantation
Mucormycosis constitutes a small proportion of invasive fungal infections in solid organ transplant (SOT) recipients and the estimated incidence ranges from 0.4% to 16.0% depending on the type of organ transplanted: 31 ➢ 0.2%-1.2% in renal transplant recipients ➢ 0%-1.6% in liver transplant recipients ➢ 0%-0.6% in heart transplant recipients ➢ 0%-1.5% in lung transplant recipients All recipients underwent chronic immunosuppression with high doses of systemic corticosteroids.Dissemination of infection was found to occur in cases where the transplanted organ was rejected by the host immune system. 32o, understanding of mechanism by which the fungus disseminates and damages the structures on its route is imperative to develop approaches to treat and prevent fungal infections.

Forms of fungal infection and symptoms
Fungal infections are of multiple types based on its route of exposure and signs of fungal infection also depends on the type of fungal infection and parts of the organ associated with it.
• Pulmonary or sinus form: This form mainly affects the lungs, sinuses, eyes, and face as exposure occurs by inhaling fungal spores or sporangiospores from the environment. 33In rare cases, the infection can spread to the central nervous system.Fever, headache, and sinus pain are the major symptoms of sinus form along with coughing in case of Lung infections. 34 Cutaneous form: It is the most common form of fungal infection in case of immuno-competent organisms and the fungus can enter the skin through the various forms of trauma on the skin like cuts, scrapes, puncture wounds, or other forms. 35In case of cutaneous form of infection, the tissue undergoes necrosis leading to the black discoloration of affected tissue i.e., "Black fungus".Fever, and tenderness, pain, heat, excessive redness, or swelling around a wound are the other symptoms associated with cutaneous form of infection. 36he above-mentioned forms are most seen in COVID-19 patients.Some rarely seen cases are: • Gastrointestinal form: It is the type of infection generally affecting neonates and can show symptoms like other gastro-intestinal diseases and possess higher mortality rate.

Diagnosis of fungal infection
Rhino-orbital and mucocutaneous mucormycosis is relatively easy to diagnose than the condition when deeper tissues are invaded like in case of pulmonary mucormycosis. 43Aggressive processing of the specimen before plating, especially when the samples are biopsy specimens, leads to reduced sensitivity of tests. 44 Fungal culture Microbiology of Mucorales ➢ Mucorales grow rapidly forming 'cotton candylike' mycelium on both non-selective and selective media to cover the entire plate in only a few days.
➢ Hyaline appearance, vigorous growth, light coloration on the reverse side of the plate (tan to yellow for most species) and variable degrees of coloration on the sporulating surface of the colonies (from pure white to tan, brown, grey, or even black) are the major macroscopic features of Mucorales.Despite the use of this technique, many false negative results have been reported compared to histopathological examination.
• Histopathology ➢ Suspected: Based on results of direct microscopy of clinical specimens, preferably stained with fluorescent brighteners.➢ Confirmation: Non-pigmented hyphae showing tissue invasion must be present in tissue sectionsstained with Haematoxylin-Eosin (HE), or Periodic Acid Schiff stain (PAS) The main diagnostic approach for confirmation of mucormycosis involves the detection of the invasion of vessels by wide and non-septate hyphae branched at right angles during histologic examination. 45Note: Similarly other major fungal infections associated with COVID-19 patients must be determined and the Ascomycetes group of fungi causing "White fungus" typically appears as: o The diagnostic feature of Candida albicans is the formation of germ tubes along with creamy-grey colonies filamenting along their outer border. 46 Apergillus species shows slender septate hyphae that exhibit angular dichotomous branching 47 Results obtained from culture of clinical samples and various clinical data must be studied to reach final decision.48 • Radiographic examination The efficacy of radiographic diagnosis depends on the presence of imaging techniques, tools, and trained personnel as the diagnosis by imaging is difficult to perform. Siple radiology and CT-scans usually show invasion of the sinuses, displacement of the orbit and invasion of the surrounding bone structures in cases of rhino-orbital lesions.Similarly, magnetic resonance imaging is the technique of choice when intra-cranial structures are affected.5

Treatment
Various treatment approaches are considered during treatment based on the condition of patient and nature of infection.
➢ Surgical Rapid and complete surgery proves to be better than partial resection of necrotic tissue in cutaneous and rhino-orbital form mucormycosis.It is impossible to operate in pulmonary and disseminated cases of mucormycosis. 49Irrigation of surgical site with Amphotericin-B has also been done to mitigate the vaso-occlusive nature of infection which might impede the flow of antibiotics to the site of infection. 50 Anti-fungal therapy ▪ Polyenes: Amphotericin B deoxycholate (AMP) remains the only licensed antifungal agent for mucormycosis whereas the lipid formulations of amphotericin B are also considered a safe and efficient alternative.Starting doses recommended for the lipid formulation of amphotericin are 5-7.5 mg/kg/day and higher dosages up to 10 mg/kg/day for cases with CNS involvement. 51iposomal amphotericin B has lower toxicity and better tolerated than any other formulations. 52 Azoles: ➢ Iron chelation therapy Iron chelators other than Deferoxamine, like Deferasirox and deferiprone have been proved to be effective agents against mucormycosis in animal models and deferasirox has also been used successfully as salvage therapy in a case of rhinocerebral mucormycosis. 56 Others Granulocyte stimulating factor and hyperbaric oxygen were also found to be used as a supportive treatment against mucormycosis along with mainline drugs. 57ote: Amphotericin B is well known kidney toxicity, so BGS005 is being used against the fungus as a safe alternative in India. 58

Conclusion
Fungal infection in immune-compromised patients is a life-threatening condition producing thrombosis inside blood vessel leading to necrotized black discoloration of infected site mainly affecting sinuses, lungs, skin, gut and various other parts of the body.New diagnostic tools must be discovered to detect the fungus and treat the patient in time.In recent scenario, co-infection with fungus in COVID-19 patients had led to the deaths of multiple individuals.So, effective study must be done to tackle with this disease in coming days.

Table 1 :
5tiological agents responsible for mucormycosis5 6ww.jkahs.org.npJKAHS|Vol.6| No. 1 | Issue 16 | January -April 2023Aspegillus and Candida species causes infection known as "White fungus".Aspergillus fumigatus is the most notorious fungus endemic in Southeast Asia causing systemic infections and COVID-19 associated pulmonary aspergillosis patients particularly in immuno-compromised individuals such as HIV-positive patients or patients with impaired cell-mediated immunity.6Cases of white fungus is rarer than black fungus but possess higher mortality rate.Candida species (C albicans: most common) is a harmless commensal present as normal flora on skin and inside the body like mouth, throat, vagina gut etc.It remains harmless until any breaches are found in skin or internal organs or any natural defense mechanism in that site of infection.

Table 2 :
Classification of Aspergillus 7 and Candida 8

Table 3 :
38• Nervous form: This is the progressive form of rhino-orbital mucormycosis whereas some cases associated with intravenous drug addicts are also encountered.Meningitis was found to the nonspecific sign associated with it.38Various fos of fungal infection with their underlying factors

Table 4 :
Different anti-fungal drugs of azoles group