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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2023  |  Volume : 66  |  Issue : 1  |  Page : 106-110
The baneful alliance of COVID-19 and mucormycosis: Histopathological concerns


1 Department of Pathology, Lala Lajpat Rai Memorial Medical College, Meerut, Uttar Pradesh, India
2 Department of Neurosurgery, Bhagya Shree Hospital and Trauma Centre, Meerut, Uttar Pradesh, India

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Date of Submission23-Feb-2022
Date of Decision25-Apr-2022
Date of Acceptance28-Apr-2022
Date of Web Publication18-Jan-2023
 

   Abstract 


Background: Mucormycosis, once a rare fungal disease, has now shown a surge in cases, and its epidemiology in India is intriguing. Rising incidence confers the necessity of defining the demography of the population at risk, early diagnosis, and recommendations for the management of mucormycosis in patients with coronavirus disease (COVID-19) infection. Aims: To study the clinico-epidemological profile, risk factors, and histopathological features of COVID-19-associated Mucormycosis (CAM). Setting and Design: This is a retrospective observational study. Material and Methods: Fifty-six biopsy specimens taken from patients of COVID-19 with clinical suspicion of mucormycosis were included. Results: Diabetes mellitus, corticosteroids, and oxygen therapy were the most common risk factors associated with CAM. The mean duration between the onset of symptoms of COVID-19 and onset of CAM was 25.2 ± 15.3 days. Rhino-orbito-cerebral mucormycosis (ROCM) was present in 24.5% of the cases, with a survival rate of 58.3%. Histopathology showed tissue invasion by branching broad-based, pale, fungal hyphae consistent with mucor in 49 (87.5%) cases. Inflammation was acute neutrophilic type in 61.2% of the patients with a survival rate of 63.33%. The survival rate with chronic non-granulomatous and chronic granulomatous was 100% and 81.8%, respectively. Conclusion: Keeping a high index of suspicion for mucormycosis in COVID-19 by all medical fraternities of the health community in the existence of risk factors is the need of the hour for prompt diagnosis. Biopsy of antral necrotic tissue should be performed immediately after suspicion for histopathological study to confirm the diagnosis of mucormycosis and predict prognosis depending on the type of inflammation incited, fungal morphology, load, and necrosis.

Keywords: Black fungus, corticosteroids, COVID-19, diabetes, histopathology, mucormycosis

How to cite this article:
Singh A, Kumar V, Kumar V, Verma N. The baneful alliance of COVID-19 and mucormycosis: Histopathological concerns. Indian J Pathol Microbiol 2023;66:106-10

How to cite this URL:
Singh A, Kumar V, Kumar V, Verma N. The baneful alliance of COVID-19 and mucormycosis: Histopathological concerns. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Feb 7];66:106-10. Available from: https://www.ijpmonline.org/text.asp?2023/66/1/106/367945





   Introduction Top


The coronavirus disease (COVID-19), the most devastating disease of the era, has brought up several unmet challenges with itself. During the massive second wave of COVID-19 in India, around 45,432 cases and 4,252 deaths due to mucormycosis have been reported in COVID-19-infected and patients recovered from COVID-19.[1] This incidence was 70 times higher than global data, which is highly alarming for a deadly disease with a mortality of 46-96%.[2],[3] Morbidity and mortality in COVID-19-associated Mucormycosis (CAM) depend on the underlying patient health, pre-existing comorbidities, and treatment taken. The triad of COVID-19, uncontrolled diabetes mellitus, and rampant use of corticosteroids has created havoc. Henceforth, we need to understand the clinical presentation, temporal sequence, emerging risk factors, and outcomes of CAM to undertake measures for its prevention and treatment.

Objectives

  1. To study the clinico-epidemological profile and possible risk factors for COVID-19-associated Mucormycosis with the objective of highlighting the population at risk.
  2. To evaluate histopathological features as a prognostic aid to guide the physician at the stage of tissue diagnosis.



   Material and Methods Top


This is a retrospective observational study conducted in the Department of Pathology, Lala Lajpat Rai Memorial (LLRM) Medical College, Meerut, on 56 biopsy specimens sent from patients with coronavirus disease (COVID-19) clinically suspected of mucormycosis who were admitted in the Mucor ward between April 22, 2021 to June 28, 2021 (67 days). Ethical approval was taken from the Institutional Ethical Committee. Demographic and clinical data were collected from medical record files. Proven diagnosis of COVID-19 was based on a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) test on nasopharyngeal/oropharyngeal swabs.

Inclusion Criteria: Adults, patients with a proven diagnosis of COVID-19 either prior to or at the time of development of clinical suspicion of mucormycosis, patients having co-infections of mucormycosis and other fungal infections, were included. Enrolled patients comprised both immunocompromised and immunocompetent individuals.

Exclusion criteria: Children, patients having fungal infections other than mucormycosis, patients without a confirmed diagnosis of COVID-19, and patients who failed to follow-up, were excluded.

The biopsy specimens were taken from sinus mucosa in all cases and orbital fat additionally, in 12 cases. The specimens were then put in normal saline and 10% formalin and were processed and studied. Routine Hematoxylin and Eosin (H&E) stain and, when necessary, special stains like Periodic Acid Schiff stain (PAS) and Silver stains were done. Diagnosis of mucormycosis was based on clinical features, culture, and histopathology from direct microscopic examination showing fungal hyphae with associated tissue damage. Patients were treated with intravenous liposomal amphotericin B with/without the addition of posaconazole and surgical debridement of necrotic tissue. The mean diameter of fungus was calculated by taking an average of diameters of five randomly selected hyphae in the area of maximum fungal load under the high power objective of the Olympus Penta Head Microscope. The degree of tissue necrosis was semi-quantified as a percentage (%) of the total area of the biopsy.


   Observation and Results Top


Within a span of 67 days, 56 biopsies were received from 38 males and 18 females. Male: Female ratio was 2:1. Out of these, 49 were histopathologically confirmed positive for mucormycosis. Five females (27.77%) and two males (5.26%) did not show the presence of fungal hyphae. In this study age range of patients was from 29-68 years, with the mean age 56.4 ± 4.[4] years The most common age group was 50-60 years with 18 patients (36.7%), followed by the 40-50 year age group comprising 12 patients (24.5%). As shown in [Table 1], 43 patients had diabetes mellitus with a mean blood glucose level of 263.2 ± 218.6 mg/dL. The most common presenting symptoms of CAM were headache, blurring of vision, nasal discharge, facial and periorbital edema, ptosis, orbital pain, and ophthalmoplegia. One patient had a history of recent tooth extraction for caries. All the patients had a history of previous hospitalization and use of oxygen therapy for more than five days at some point in time. The mean duration between the diagnosis of COVID-19 (first positive report) and the onset of mucormycosis was 19 ± 16.5 days (range 12-56 days). The mean duration between the diagnosis of COVID-19 (onset of symptoms) and the onset of mucormycosis was 25.2 ± 15.3 days. A total of 87.5% of the patients were detected by direct microscopy with 10% potassium hydroxide (KOH) wet mount and histopathology, whereas the culture was positive only in 58% of the cases.
Table 1: Clinico-epidemiological profile of COVID-19-Associated Mucormycosis

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Histopathological examination showed tissue invasion of hyphae consistent morphologically with Mucor in 49 (87.5%) patients, three being mixed infections with 1 Candida and 2 Aspergillus species. Seven (12.5%) patients did not show any presence of tissue necrosis or fungal hyphae. There was the presence of acute suppurative inflammation in 30 patients, no inflammation in five patients, and chronic inflammation in 14 patients. All three cases of mixed infection showed acute inflammation. Inflammation was not necessarily present around the fungal hyphae but in the sinonasal mucosa present adjacent to the area of necrosis. Other histological features and survival in different groups are described in [Table 2]. Fungal hyphae were exclusively present in areas of tissue necrosis. Cases negative for fungal hyphae showed chronic inflammation but no tissue necrosis. The combined medical and surgical intervention showed an improved survival rate (71.43%) over medical treatment alone (57.14%). Liposomal amphotericin in the initial dose of 5 mg/kg body weight, and 10 mg/kg body weight (in cerebral involvement) was given diluted in 5% dextrose till favorable response and stepped down to oral posaconazole 300 mg twice a day for one day followed by 300 mg daily till clinical resolution as per the World Health Organization (WHO) guidelines. Endoscopic Surgical debridement (ESS) of sinuses was done with or without orbital clearance. In a few cases, extensive debridement of the sinonasal areas was done, leading to cosmetic disfigurement, and subsequent surgeries were needed to rectify them.
Table 2: Histopathological findings in COVID-19 Associated Mucormycosis patients

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   Discussion Top


Mucormycosis was first reported as a cause of human disease in 1885.[5] Mucormycosis is extremely rare in healthy individuals, but several immunocompromized co-morbid conditions predispose. The last few decades have seen a dramatic rise in the occurrence of invasive fungal infections like mucormycosis worldwide largely due to an increase in the size of the population at risk in developing and tropical countries.[6]

Earlier studies implicated uncontrolled type-2 diabetes (44–88%) cases as one of the main risk factors for mucormycosis.[7],[8],[9] In a review of 179 cases of paranasal sinus mucormycosis, 70% of patients had diabetic ketoacidosis.[10] The present study also found diabetes mellitus to be most commonly present, seen in 87.7% of the cases, with Type 2 diabetes constituting 79.6% of the cases and Type 1 diabetes in 8.16% of the cases. Out of these, 16.3% had recent-onset/diagnosed diabetes after COVID-19 infection. Patients with diabetes mellitus had a mean blood glucose level of 263.2 ± 218.6 mg/dL. In a study by Bala et al.,[6] the cut-off for uncontrolled diabetes mellitus was a fasting blood glucose level >140 mg/dl, and diabetes mellitus (56%) was the most common risk factor for mucormycosis followed by intramuscular injection (10%), incision and drainage, deferoxamine therapy, and trauma.[11] The establishment of mucormycosis in diabetic patients is explained by Morales-Franco et al.,[12] stating that the tissue microenvironment in a diabetic patient is altered; high levels of glucose, free iron, and ketone bodies generate endoplasmic reticulum (ER) stress in the adjacent epithelial/endothelial cells in response to which glucose-regulated protein 78 (GRP78) is overexpressed and relocated to the cell surface. Exposed GRP78 favors interaction with the fungal hyphae through the CotH3 proteins causing cellular damage, epithelial penetration, and association with laminin and type IV collagen in the basement membrane. This permits the entry of fungus into the blood vessel, causing induction of extrinsic coagulation pathway leading to thrombus formation, infarction, and necrosis.[11],[12]

Rampant corticosteroid use in COVID-19 infection has contributed to the upsurge in the cases of CAM, as seen in the present study also. Corticosteroid therapy was given to 91.8% of the patients. This indirectly establishes a causal effect relationship between COVID-19 and mucormycosis. But, mucormycosis also depends on climatic factors such as seasonal variation, humidity, and ambient temperature.[13] In a study by Pal et al.,[14] COVID-19 infection was present in 63%, and prior COVID-19 infection was seen in 37%.[15],[16] In the present study, concurrent COVID-19 infection was present in 26.5% of the patients, and a history of prior COVID-19 infection was noted in 73.4%. In the present study, 67.3% were not vaccinated, 26.5% had taken a single-dose vaccination, and only 6.1% of the patients had received double-dose vaccination. Similar findings were seen in a study conducted by Lav Selarka et al.;[17] most patients (66.0%) were not vaccinated, and only 18.2% had received just a single dose. In another study, Rhino-orbito-cerebral mucormycosis (ROCM) before 17 days was seen in patients who had not received even a single dose of COVID-19 vaccine. In another report of 47 cases of ROCM, 66% had not been vaccinated.[18] Thus, the protection offered by the COVID-19 vaccination against ROCM is worth exploring. Lav Selarka and coworkers showed that the mean time interval between the diagnosis of COVID-19 and the appearance of symptoms suggestive of mucormycosis was 12.1 ± 4.6 days. In their study, presenting symptoms of CAM were headache (74.5%), nasal congestion, followed by diplopia, visual disturbances, facial weakness, etc. The most common presenting symptoms in the present study were headache, blurring of vision, nasal discharge, facial, and periorbital edema. The mean duration between the diagnosis of COVID-19 (first positive report) and the onset of mucormycosis was 19 ± 16.5 days, and the mean duration between the onset of symptoms and onset of mucormycosis was 25.2 ± 15.3 days. Mucormycosis can involve the nose, sinuses, orbit, central nervous system, lung, gastrointestinal tract, skin, jawbones, joints, heart, kidney, and mediastinum, but ROCM is the commonest variety seen in clinical practice worldwide.[17],[19],[20] Similarly, in the present study, ROCM was present in 24.5% of the patients with a survival rate of 58.3%. Rhino-orbital (RO) involvement constituted 20.4%, with a 60% survival rate. A study by Priya et al.[21] showed a better survival rate of 66.5% among 18 patients with rhino-orbito-cerebral involvement, compared with survival rates of <3% among patients with gastrointestinal and disseminated disease. The higher survival rate in this group may be because, in the rhino-orbito-cerebral or cutaneous category, patients are diagnosed early and treated quickly.

Histopathological examination of biopsies revealed tissue lined by stratified squamous/pseudostratified epithelium in viable areas along with extensive areas of ulceration, necrosis, and bone erosion. Inflammation is comprised of neutrophils, macrophages, lymphocytes, and plasma cells. Some cases showed granuloma formation comprising epitheloid cells, multinucleated giant cells and chronic inflammatory cell infiltrate, areas of inflamed granulation tissue, hemorrhage, and congestion [Figure 1]. Necrotic areas showed colonies of mucormycosis consisting of branching broad-based, pale-staining, wide, flat aseptate hyphae [Figure 2]. Inflammation was predominantly acute suppurative (neutrophilic) type in 61.2% of the patients in the present study, with the survival rate of 63.3%. The survival rate with chronic non-granulomatous was 100% and 81.8% in patients with chronic granulomatous inflammation. In a study by Arora et al.,[18] the presence of acute suppurative cellular reaction (2), necrosis with no cellular reaction (16), and granulomatous reaction in four samples was seen. Neutrophils are critical for inhibiting proliferation of fungal spores. Besides, both mononuclear and polymorphonuclear phagocytes of a normal host kill Mucorales by generating reactive oxygen species and cationic peptides, namely defensins.[22],[23],[24] Microscopically, all the cases of CAM showed that the density of fungal organisms was higher in necrotic tissues. Therefore, it is recommended that necrotic tissue must be well-sampled to identify the fungal elements. Furthermore, newer tests like probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples can be used to complement histopathology, especially where hyphae are not seen or screen high-risk patients and for species identification.[25]
Figure 1: Histopathology of Mucormycosis [H&E stain 100×] [bony erosion-red arrow, mucor -yellow arrow] (a) Antral mucosa showing extensive necrosis (b) Granulomatous inflammation with giant cell (blue arrow) (c) Chronic inflammation showing lymphocytes, plasma cells, macrophages, some hemosiderin-laden (d) Acute neutrophilic inflammation with mucor

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Figure 2: Mucormycosis [H&E stain 200×] (a)(b)(d): Broad aseptate fungal hyphae with acute angle branching (c): Fungal hyphae with necrosis and neutrophilic inflammation [H&E stain 200×]

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A similar study done earlier in mucormycosis patients before the COVID-19 pandemic by Goel et al.[26] showed that the survival rate was 100% in cases with a moderate to marked granulomatous inflammation, and 67% in those who had mild granulomatous host response. Castillo et al.[27] found that multinucleated giant cell granulomas may be correlated with the disease with a better prognosis. It is still unclear whether the difference between the granulomatous and non-granulomatous forms is caused by immunologic dysfunction.[21] We observed a mortality rate of 30.6%, which is similar to that reported in the literature. Pal et al. reported a mortality of 34% in patients with mucormycosis. Sarkar et al. reported a mortality of 40% in cases of orbital mucormycosis. Garg et al. reported higher mortality of 87.5% among patients with severe COVID-19 infections.[14],[15],[17],[26],[28],[29]


   Conclusion Top


Prevention of COVID-associated mucormycosis rests on better glycemic control in diabetics, appropriate use of corticosteroids, and promotion of vaccination. Keeping a high index of suspicion for mucormycosis in COVID-19 by all medical fraternities of the health community in the existence of risk factors is the need of the hour for prompt diagnosis for improved survival. Biopsy of antral necrotic tissue should be performed immediately after suspicion for histopathological study to confirm the diagnosis of mucormycosis and predict prognosis depending on the type of inflammation incited, fungal morphology, load, and necrosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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DOI: 10.4103/ijpm.ijpm_192_22

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