A Narrative Review of the Pathophysiology of Mucormycosis Infection Among COVID-19 Patients in India: Epidemiology and Clinical Implications

Mucor Infection Among COVID-19 Patients

Bhatt et al.² report the high risk of opportunistic infections such as mucormycosis among COVID-19 patients, as the COVID-19 infection induces significant lymphopenia (as seen in >85% of patients). Such high incidence rates of opportunistic infections among COVID-19 patients justify the early detection and treatment of mucor infection and other fungal infections such as candidiasis, pneumocystis pneumonia, pulmonary aspergillosis, and cryptococcal disease. Laboratory investigations of severe COVID-19 patients show an increase in pro-inflammatory markers such as IL-1, IL-6, and tumor necrosis alpha, reduced expression of CD4 interferon-gamma and lowered count of CD4+ and CD8+ T cells.² Each of these markers increases the susceptibility to bacterial and fungal infections. Such airborne fungal organisms can invade the alveolo- interstitial lesions of the damaged pulmonary organ system of a severe COVID-19 patient.

Singh et al.³ opine on the facilitating environment for germination of Mucorales spores in COVID-19 patients, which include: hypoxia, hyperglycemia (either pre-diabetes, steroid-induced, or new-onset), acidic media (metabolic acidosis, diabetic ketoacidosis), increased ferritin causing high iron levels and immunosuppression (due to co-morbidities, SARS-CoV-2 infection or steroid-induced), along with other risk factors such as prolonged hospitalization with or without mechanical ventilation.

As depicted in Figure 2, severe COVID-19 patients treated with a high dose of steroids, patients with uncontrolled diabetes, and those with a long duration of stay in the ICU are susceptible to mucor infection. The acidic nature of the patient's blood during diabetic ketoacidosis creates a fertile environment for the thriving of Mucorales fungi.⁶ COVID-19 induced high ferritin levels (due to hemolysis), and lymphopenia might contribute to the steep rise in infections.

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Figure 2:

Factors Associated with Mucormycosis Occurrence Among COVID-19 Patients

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The study by Lv et al.¹¹ shows that the fungal load of the gut among patients with COVID-19 or H1N1 infection was significantly higher than that among healthy controls. Such fungal overgrowth in the gut is attributed to factors such as dysbiosis of bacterial microbiota, the presence of an inflammatory environment, and extensive tissue damage. The gut-lung axis plays a crucial role in the association between lung infection with SARS-CoV-2 and H1N1 and alterations of the gut mycobiota. Thus, pneumonia could be the result of the depletion of important fungi in the gut, as well as the growth of opportunistic pathogenic fungi. Pneumonia due to SARS-CoV-2 or H1N1 infection could also spawn the gastrointestinal symptoms and crucially alter the mycobiota of the gut, in addition to direct viral infection of the gastrointestinal tract.

The high mortality among mucormycosis patients is due to complications such as cavernous sinus thrombosis, osteomyelitis, and disseminated infection. The vascular invasion by fungal hyphae leads to thrombosis and tissue necrosis. Laboratory investigation involves blood analysis, biopsy, and radiological imaging. The management protocol includes the reversal of risk factors, surgical debridement, and intravenous antifungal medication such as amphotericin B.²

Table 1 lists the salient features of 5 studies (out of the 16 research articles reviewed), which comprehensively address the issues associated with the cause-effect relationship of interest.

Steroid Usage During the COVID-19 Pandemic: Its Implications for Mucormycosis

The immunosuppressive activity of corticosteroids along with its ability to induce hyperglycemia can aggravate the clinical course of COVID-19 patients.² Balsari et al.¹³ attributes the inappropriate usage of steroids to the rise in mucormycosis among patients recovering from COVID-19 in India. The authors¹³ mention the ingrained culture of polypharmacy among healthcare practices in India. Such gestalt practices have resulted in the indiscriminate prescription of remdesivir, azithromycin, doxycycline, and plasma therapy, with the presumption that the organized whole (improving COVID-19 outcomes in this case) is perceived as more than the sum of its parts (indiscriminate use of drugs). The authors¹³ opine that regardless of the drug efficacy or severity of the disease, more drugs such as favipiravir, baricitanib, and bevacizumab have been added to this list.

The incidence of steroid-induced hyperglycemia (SIH) is estimated in the range of 40 to 50%.¹⁴ The known risk factors for steroid-induced hyperglycemia include increased body mass index (BMI), pre-existing DM, and older age. The 3 types include transient hyperglycemia among nondiabetics, newly discovered DM, and exacerbation of existing type 2 DM. Cortisol and other glucocorticoids tend to inhibit insulin secretion from the β cells of the pancreas and increase the glucagon secretion from α cells. This reduces the insulin sensitivity of fat tissue and skeletal muscles and stimulates the synthesis of glucose in the liver.¹⁴ The usage of steroids also results in lowering of immunity (reduced phagocytic activity of white blood cells), and impairment of bronchoalveolar macrophage functioning (migration, ingestion, phagolysosome fusion).³

When the morning dose of Glucocorticoid is administered, steroid-induced hyperglycemia tends to occur during the afternoon and evening hours, followed by a gradual decline in the overnight blood glucose levels. This mandates the monitoring of diurnal variation in blood glucose levels. Higher doses of glucocorticoid and longer duration of treatment tend to increase the risk of steroid- induced hyperglycemia, irrespective of the type of glucocorticoid (considering equivalent doses). The Joint British Diabetes Societies Guidelines recommend clinical vigilance of patients during the initial phase of administration of any dose of glucocorticoid.¹⁴

Pathology of COVID-19 Infection – Enigmatic Association with Mucormycosis

COVID-19 infection by itself increases the levels of glucose in the blood, as the virus has the potential to infect the beta cells of the pancreas which produce insulin. Further research is indicated to determine the pathogenicity of the delta variant (which precipitated the second wave) is damaging the beta cells. The low production of insulin increases blood glucose levels. COVID-19 infection also increases the level of iron in the blood, which is a good feed for the molds of Mucor. A few microbiologists in India have opined that the samples from humidifiers (for oxygen administration) or the oxygen supply pipelines have not isolated the Mucor fungi.⁴

As depicted in Table 2, the second COVID-19 wave in India (April – May 2021) was different from the first wave (August – October 2020), in terms of symptoms, severity, and secondary infections. The characteristics of viral behavior during each wave could determine the incidence of comorbid mucor infection. Apart from black fungus (Mucor), white fungus (Candidiasis) and green fungus (Aspergillosis) were more common during the second wave. An increase in the number of deaths among the young population was also reported during the second wave.¹⁵ Scientists used the wild-type virus strain (the Wuhan, China variant) across the globe to develop testing kits, plans of treatment, and the COVID-19 vaccines.¹⁶ The delta variant which has been implicated for the second wave is known to possess immune escape mechanisms. Further research will determine whether the delta variant is more virulent or circulating to a greater extent among the vulnerable population and the response of health systems to the pandemic.

The activation of humoral immunity following two to three weeks of COVID-19 infection leads to the formation of antigen-antibody complexes which results in the extrapulmonary manifestations. COVID-19 induced organ damage is largely immune-mediated, quite similar to other autoimmune diseases. The debilitating symptoms for many months following COVID-19 infection could be due to the occlusive and propagative injuries, caused by the prothrombotic state which has resulted from the immune dysregulation. The etiology of long COVID-19 disease could be due to the aberrant immune response, antigen-antibody reactions, endotheliopathy, and the hypoxemic injuries.¹⁷

According to data from Public Health England,¹⁶ the effectiveness of two doses of COVID-19 vaccination against related hospitalization was 96% for Pfizer's mRNA vaccine and 92% for AstraZeneca vaccine, respectively. These rates are comparable to the effectiveness against the alpha variant. Although it is imperative to receive both doses of a particular COVID-19 vaccine, the efficacy against the delta variant warrants further investigation with regard to breakthrough infections and the requirement of booster doses.¹⁸ Such vaccine- induced immunity against COVID-19 might ward off the disease and, in turn, the possibility of comorbid mucor infection.

Pre-Existing Diabetes as a Predisposing Factor for Mucormycosis in this Conundrum

Afroze et al.¹⁹ study on mucor infections among diabetic patients discusses the various ways by which the body's immunological response changes among diabetics. The proliferation of fungus is stimulated by hyperglycemia, and this status also decreases the efficiency of phagocytes and the process of chemotaxis. Bhogireddy et al.²⁰ explain the low incidence of mucor infection among HIV/AIDS patients due to neutrophils and non-T lymphocytes playing a vital role in inhibiting the proliferation of fungal spores. Future research should determine the mechanisms by which diabetes and corticosteroid intake render the phagocytes dysfunctional. The innocuous mucor organism thrives in the acid-rich environment. In diabetic ketoacidosis, the ketone bodies in biological tissue enable the growth of Rhizopus oryzae organisms which produce the enzyme ketoreductase. This results in an increased risk of mucormycosis, as the host defense mechanism among such patients is further compromised by the inability of transferrin in binding iron.

Garg et al.¹⁰ report that diabetes status increases the risk of death among patients with severe COVID-19. For some of these patients, their diabetic condition may be poorly controlled resulting in renal dysfunction. The dysregulation of immunity caused by COVID-19 can aggravate the mucor infection among diabetic patients. Such co-morbid illness along with the administration of glucocorticoids and monoclonal antibodies for severe COVID-19 increases the net state of immune suppression. Mucorales have the ketone reductase enzyme and thrive in states of hyperglycemia and diabetic ketoacidosis.⁸

Hyperglycemia and High Ferritin Levels in Blood – An Unholy Mixture Predisposing the Mucor Infection

Figure 3 depicts the pathological mechanism by which hyperglycemia increases free iron in the blood through glycosylation of transferrin and ferritin, thus reducing the binding of iron. During the course of COVID-19 infection, there is an increase in the level of cytokines, especially interleukin-6. This also contributes to the increase in free iron in the blood by increasing the synthesis of ferritin and decreasing the transport of iron. Concomitant acidosis compliments these mechanisms and reduces the ability of transferrin to chelate iron.³ These processes increase the expression of glucose-regulator protein 78 (GRP-78) of endothelium cells and fungal ligand spore coating homolog (CotH) protein. COVID-19 uses processes to increase serum iron levels, which promotes the growth of Mucorales. Mucor infection is facilitated by the described pathological change which further enables channels such as angio- invasion, hematogenous dissemination, and necrosis of tissue.³

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Figure 3:

COVID-19 Induced Hyperglycemia and the Resulting Increase in Ferritin Levels

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Mucor Due to Treatment of COVID-19 Patients with Il-6 Inhibitors

TCZ is administered to reduce the levels of interleukin-6 (IL-6) during the cytokine release syndrome (CRS) among COVID-19 patients. It is a recombinant humanized monoclonal antibody that is used against the interleukin-6 receptor. The inflammatory effects of interleukin-6 can otherwise damage the lung tissue, and subsequent COVID-19 induced multiorgan failure. The weakened immune system due to large doses of TCZ may pave the way for opportunistic infections such as mucor infection. Suppressing interleukin-6 with TCZ will reduce the clearance of viruses and bacteria, which in turn can cause secondary infections. There are concerns regarding other toxicities associated with the use of TCZ, such as liver dysfunction, hypertension, diverticulitis, gastrointestinal perforation, neutropenia, and reactions related to the infusion.¹²

Kimmig et al.²¹ studied the independent association between TCZ administration for severe adult COVID-19 patients and the presence of bacterial infections (OR: 3.960, 95% CI: 1.351-11.607, p=0.033). The logistic regression model analyzed in the study includes bacterial infections as the outcome and age, sex, and Charlson Comorbidity index as the independent variables.

Pettit et al.¹² report a 61% possible post-TCZ complication among COVID-19 patients with a hyper-inflammatory response. The study¹² reports a significantly higher proportion of late-onset (≥48 hours) infections among COVID-19 patients who received TCZ (cases) when compared with controls who did not receive it (23% vs. 8%, p=0.013). However, the overall infection rate was similar between the groups. This variation could be explained by the long half-life of TCZ: 11 days with a dosage of 4 mg/kg.¹² Additional research should explore the infectious complications and drug- related toxicities associated with the use of TCZ. A stringent selection criterion for the administration of TCZ needs to be followed, with the dosage not exceeding a single dose of 400 mg.²²

Mucor Due to Industrial Oxygen, Primarily Used for COVID-19 Treatment

The usage of industrial oxygen (non-medical grade), was a possible route of contamination for mucor infection among COVID-19 patients during the second COVID-19 wave in India. Unlike medical oxygen, the hygiene of industrial oxygen cylinders is likely to be compromised. Also, oxygen is given to patients through humidifier bottles attached to the cylinder, and there is a possibility of the classified water being unsterile.²³ Patients are likely to inhale the fungal spores from such contaminated humidifiers. Due to the increased demand for oxygen during the second COVID-19 wave in India, hospitalized patients sometimes had to share the same cylinder or the non-rebreather mask. However, some of the microbiological samples taken from such items did not yield mucor fungus.¹⁵

The oxygen which fills the cylinder, whether medical or non-medical grade, is initially passed through water which traps the fungal spores.²³ If the premise regarding contaminated ICU pipes and other equipment is considered, then a plethora of viral/bacterial secondary infections are possible, not just the risk of mucor infection. There is a possibility of fungus colonization in the nose due to the continuous usage of the same mask (>2-3 weeks). This situation is complemented by the inhaled oxygen from the cylinders which is humid and by the frequency of steam inhalation multiple times a day.

Since mucor fungus is omnipresent (mucor resides in many indoor and outdoor environments, including the ducts of air conditioners, in soil, and in decaying organic matter), any compromise in the immune status of an individual makes it invasive. Some preventive interventions include a ventilated and hygienic environment for COVID-19 patients, using sterile water for humidification in the oxygen tube, and cleaning these tubes with betadine at a frequency of 2-3 times/day. The oxygen concentrators used at home should be filled with clean distilled water.

Diagnosis of Mucormycosis and Other Fungal Infections

Other fungal infections such as invasive aspergillosis could develop into a super-infection among severe COVID-19 patients requiring care at the ICU. Across the globe, the varied incidence rates of such fungal infections could be due to the challenges in diagnosing them. Clinical symptoms, as well as the abnormalities found on chest imaging, could be non-specific. The positive sputum samples or tracheal aspirates may not profoundly differentiate colonization and infection, and the COVID-19 status of the patient dissuades invasive diagnostic procedures such as bronchoscopy/lung biopsy/bronchoalveolar lavage. Given the difficulty in isolating mucor and the lack of biomarkers such as beta-d-glucan and galactomannan for invasive aspergillosis,¹⁰ under-diagnosis is a fallacy in the case of mucormycosis.

Treatment of Patients with Mucormycosis

The patient's current health condition precludes timely diagnosis and testing, which increases the risk of invasive mold infections. The overwhelming number of COVID-19 patients places a strain on the essential services of the hospital, including diagnostics and surgeries. Good glycemic control in the hospital and along with appropriate initiation of liposomal amphotericin B treatment will enable favorable outcomes among such patients. Systemic administration of liposomal amphotericin B is more effective and less toxic, thus facilitating prolonged administration without side effects.⁸ Due to its side- effect of nephrotoxicity, the renal function needs to be regularly monitored during admission. The second line of management includes the triazole antifungals: posaconazole, and isavuconazole. Since Isavuconazole has an extended spectrum of action, it is used in the treatment of invasive mucormycosis.²⁴

Due to the barrier created by vascular thrombosis and extensive ischemic necrosis, the entry of antifungal agents in adequate concentration to the infected tissue is affected. Hence, antifungal treatment alone may not be effective. To minimize the fungal load in the tissue, we need to perform a radical debridement of infected and necrotic tissue along with drainage of infected paranasal sinuses. For rhino-orbital mucor infections, the line of management includes retrobulbar antifungal therapy and intravitreal amphotericin B injection.⁸

The European Confederation of Medical Mycology and Mycoses Study Group Education and Research Consortium, provide the 2019 guidelines for the diagnosis and management of mucormycosis.²⁵ This includes immediate and complete surgical intervention followed by administration of systemic antifungals as the first line of management. The recommendation is to initiate high-dose liposomal amphotericin B in association with an adequate dosage of intravenous isavuconazole and posaconazole. Posaconazole can be given as prophylaxis for high-risk individuals such as neutropenic patients with graft versus host disease.

Limitations

1. Given the difficulty in microbiological/histo- pathological diagnosis of mucormycosis during the course of an ongoing pandemic, the morbidity of this infection could be under-represented.

2. Many studies did not associate the duration of diabetes and baseline glucose (and/or HbA1c) data for all the cases.

3. As we cannot define the at-risk individuals infected with COVID-19, the value of the denominator cannot be estimated and thus the incidence rate cannot be computed.

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