Study on a Deadly Uninvited Guest During the Historical Second Wave at a Tertiary Health Care Center in South India

Original Research Article

Study on a deadly uninvited guest during the historical second wave at a tertiary health care center in south India

Sunil Kumar Katari1, Suneetha Sunkari2, Bhargavi3*, T Khadeeja4, C Aparna5, G Baleswari6

1Assistant Professor, 2Assistant Professor, 3,4PG, 5Professor, 6Professor and HOD, Department of Pathology, Kurnool Medical College, Kurnool 518002. Andhra Pradesh, INDIA. Email: [email protected]

Abstract Background: Mucormycotina are widely distributed fungal elements in nature and cause no harm to immunocompetent individuals, but they infect immunosuppressed people causing mucormycosis. Mucormycosis in Severe coronavirus disease (COVID-19) during the first wave escaped unnoticed in many healthcare centers while only a few cases were reported during 2020 in India. As mucormycosis is an invasive fungal infection, accurate identification and diagnosis of Mucormycotina on histopathological examination assists the clinician in further treatment. Aim of the study: To know the incidence of Mucormycosis in the histological specimens received. To know the Age, Sex and Site wise distribution of the same. To compare the present study with other global studies regarding incidence, age and gender wise distribution and suspect: histopatholocally confirmed ratio. Material and Methods: This is a prospective study done in the department of Pathology, Kurnool Medical College, Kurnool, on 162 biopsy specimens sent from patients of Severe coronavirus disease (COVID-19) suspected with mucormycosis of the Rhino-orbito-cerebral regions who underwent surgery in Government General Hospital, Kurnool, from 25st April 2021 to 20th June 2021 (56 days). The biopsy specimens were processed and studied. Results: Within a span of 56 days, out of 162 specimens received, 116 were histopathologically confirmed positive for mucormycosis. Conclusion: Severe coronavirus disease (COVID-19) is changing its course through unexpected life-threatening manifestations of which mucormycosis is one such entity in this second wave. As per the literature mucormycotina is a frequent contaminant in culture media, so histopathological examination always stands as a gold standard. Being invasive fungus with high mortality rate, any hint of mucormycosis in patients of Severe coronavirus disease (COVID-19) should be promptly treated medically and also surgically wherever required in order to contain the morbidity and mortality by this deadly black fungus. Keywords: Mucormycosis, Covid 19, Black fungus, diabetes, Histopathology, angioinvasion, immunosuppressed.

*Address for Correspondence: Dr Bhargavi, Department of Pathology, Kurnool Medical College, Kurnool 518002. Andhra Pradesh, INDIA Email: [email protected] Received Date: 22/06/2021 Revised Date: 01/07/2021 Accepted Date: 25/07/2021 DOI: https://doi.org/10.26611/1052011 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

and Cunninghamella spp., which belong to the subphylum Access this article online Mucormycotina. Mucormycotina cause no harm to Quick Response Code: immunocompetent individuals but infect Website: immunosuppressed people resulting in mucormycosis. www.medpulse.in Major predisposing factors are neutropenia, use of corticosteroids, Diabetes mellitus, iron overload and breakdown of cutaneous barrier as a result of burns, 1 Accessed Date: surgical wounds and trauma . Severe coronavirus disease (COVID-19) in itself alters the innate immunity due to 04 October 2021 COVID-19-associated immune dysregulation characterized by decreased T cells, including CD4 and 2,3 CD8 cells. Mucormycosis in Severe coronavirus disease INTRODUCTION (COVID-19) during the first wave escaped unnoticed in Mucormycosis is an opportunistic infection caused by many health care centers while only a few cases were environmental fungi including mucor spp., Rhizopus spp., reported.3 Initially while COVID-19 associated pulmonary

How to cite this article: Sunil Kumar Katari et al. Study on a deadly uninvited guest during the historical second wave at a tertiary health care center in south India. MedPulse International Journal of Pathology. October 2021; 20(1): 01-05. https://www.medpulse.in/Pathology/ MedPulse International Journal of Pathology, Print ISSN: 2550-7605, Online ISSN: 2636-4697, Volume 20, Issue 1, October 2021 pp 01-05 aspergillosis was increasingly recognized, mucormycosis be sparse or fragmented6. In lesions exposed to air, thick- which was rare previously has now become the deadly walled spherical structures can form at the ends of the addition to this Covid 19 spectrum. All physicians hyphae9. Routine HandE stains may show only the cell including ophthalmologists should, therefore, be aware of wall with no structure inside. In immunosuppressed hosts, the probability of development of opportunistic fungal the hyphal elements will be found with abundant necrosis, infections such as mucormycosis in patients with COVID- hemorrhage, and blood vessel thrombosis10. Important 19 illness, especially in those with comorbidities and on diagnostic features include identification of fungal immunosuppressive agents in the coming future4. elements invading the blood vessel wall or inside their Mucormycotina are ubiquitous in the environment and can lumen6. Sparse neutrophilic inflammation can be found in be found in soil and decomposing matter5. Their spores are the periphery of the lesion. easily airborne, which can cause contamination of Pitfalls in morphological diagnosis: The major microbiological culture media6. So not all culture results morphological differentiation should be done among are reliable. As mucormycosis is an invasive fungal Mucorales genera, Aspergillus spp., and Candida spp5. infection, accurate identification and diagnosis of The presence of abundant septation and acute-angle Mucormycotina on histopathological examination assists branching should suggest the diagnosis of Aspergillus the clinician in further treatment. spp., while yeasts with pseudo-hyphae should suggest Aim of the study: To know the incidence of Candida spp. Poor staining of hyphae with GMS Mucormycosis in the histological specimens received. To (Grocott’s methenamine silver stain) should suggest know the Age, Sex and Site wise distribution of the same. mucormycosis. In order to specifically identify Mucorales To compare the present study with other global studies in tissues or to detect dual infections by Mucorales genera regarding incidence, age and gender wise distribution and and other fungi, it is important to use suspect : histopatholocally confirmed ratio. immunohistochemistry, in situ hybridization, or PCR15,16.

RESULTS MATERIAL AND METHODS With the approval of the ethics committee, this prospective Total no. of histopathological specimens received during 486 study was done in the department of Pathology, Kurnool the present study Medical College, Kurnool, on 162 biopsy specimens sent Total no. of specimens suspecting Mucormycosis 162 from patients of Severe coronavirus disease (COVID-19) Total no. of specimens histopathologically positive for 116 suspected with mucormycosis of the Rhino-orbito-cerebral Mucormycetes regions who underwent surgery in Government General Out of 486 biopsy specimens received in the Hospital, Kurnool, from 25th April 2021 to 20th June 2021. histopathology department during the period of present (56 Days) study, 162 were from patients of Severe coronavirus Inclusion criteria: All resected tissues of Rhino-orbito- disease (COVID-19) treated in GGH Kurnool, who were cerebral regions of Severe coronavirus disease (COVID- suspected to have developed Mucormycosis. These 19) patients suspected of mucormycosis were included in patients underwent surgical resection of affected tissues from the paranasal sinuses and orbital regions. the present study. Exclusion criteria: All resected tissues from non-covid patients were excluded from the present study. All the specimens were fixed in 10% neutral buffered formalin, processed in Automatic tissue processor into paraffin embedded sections and stained with routine Hematoxylin and Eosin stain, examined under light microscopy and correlated with clinical and radiological findings. The histopathological reports were analyzed with respect to age, sex and site wise distribution Graph 1: Proportion of Mucormycosis suspected specimens mucormycosis. The data was properly tabulated and analyzed. Results and Conclusion were derived from such Of the 162 biopsy specimens received, 116 were analysis. The data shown in charts was prepared using histopathologically confirmed positive for mucormycosis. Microsoft Excel Software. Identifying Mucormycosis on histopathology: Mucormycetes form non-septate hyphae of variable width (6-50µm) with frequent right-angle branching1. The hyphae may vary in width, appear folded or crinkled, and

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Graph 2: Percentage of Mucormycosis positive confirmed cases

Table 1: Site wise and Sex wise Distribution of Mucormycosis Graph 4: Age wise and Sex wise Distribution of Mucormycosis Site Males Females Cerebral 2 - Gender Distribution Occular 6 - In the present study out of 116 positive cases, males were Paranasal sinuses 87 21 95 were males and 21 were females. Total 95 21

Graph 5: Gender distribution in Mucormycosis cases

Graph 3: Site wise and sex wise distribution

Table 2: Age and Sex wise Distribution of Mucormycosis Age Male Female Total Percentage 0-10 2 - 2 1.72% 11-20 - - - - 21-30 2 - 2 1.72% 31-40 29 2 31 26.72% Figure 1: H&E Slender non-septate hyphae dividing at right angles 41-50 25 7 32 27.6% Figure 2 Gross. Eyeball affected by Mucormycosis 51-60 23 9 32 27.6% 61-70 12 3 15 12.92% >70 2 - 2 1.72% 95 21 116 100

Figure 3: H&E Angioinvasion by Mucormycetes (40X) Figure 4and5 MRI of ocular and PNS invasion by Mucormycosis DISCUSSION The present study which was done during the second wave of Covid 19 pandemic was compared to the studies11,12,13 conducted prior to the Covid 19 pandemic to know the influence of Covid 19 on the incidence of mucormycosis during the pandemic. Authors Petrikkos G et al11 Parisa Badiee et al12 Kiran Bala et al13 Present study (Greece) (Iran) (India) (India) Period of study 1993-2003 2010-2011 1st Jan 2010- 30th June 25th Apr 2021 to 20th June 2011 2021 Duration 10 Years study 2 years study 18 months study 56 days No of Suspects (Retrospective) 28 31 162 Histopathologically confirmed cases 24 7 (25%) 3 116 (71.6%)

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According to the study done by Petrikkos G et al11. (in In the present study, the peak incidence was observed in Greece), which was a retrospective study for a period of 10 the age groups 31-60 years which correlates with Parisa years from 1993 to 2003, only 24 cases of Mucormycosis Badiee et al12 and Kiran Bala et al13 studies. The mean age were reported. In the study by Parisa Badiee et al12 (in Iran) of incidence was 47 years in Parisa Badiee et al12 and in out of 28 suspected cases, only 7 were histopathologically Kiran Bala et al study it was 40.43 years while in the confirmed for Mucormycosis which accounts to 25% of present study it was 45.75 years. tissue samples. Study done by Kiran Bala et al13 (in India) shows out of 31 suspected samples, only 3 cases were histopathologically confirmed for Mucormycosis. But the present study shows that out of 162 suspected samples sent, 116 were positive by histopathology.

Graph 8: Mean age of incidence of mucormycosis in comparative studies

Nature of Mucormycosis: Mucorales cause a wide range of predominantly Angio-invasive disease in immunosuppressed patients7. COVID 19 and Mucormycosis: Most of the predisposing factors for mucormycosis are fulfilled in COVID 19. Normal host defense against the Mucorales genera is mainly by macrophages that inhibit germination of spores by nonoxidative killing. But once the Graph 6: Suspects vs Confirmed cases of Mucormycosis germination of spores occurs, the neutrophils take over In both the above charts a clear peak of suspects and the host defense by using oxidative burst to kill confirmed cases of mucormycosis can be seen in the proliferating hyphal elements directly. Thus, patients who present study which ranged just for a span of 56 days, have diseases affecting the function of these two cell compared to other studies which ranged from 1.5 years to types will be at risk for infection5. As the reports suggest, 10 years where only negligible counts of mucormycosis in patients of Covid 19, the Absolute Neutrophil Counts cases were observed, clearly confirming the effect of are decreased promoting easy infection by Covid 19 (whether Corona virus itself as pathogen or due Mucormycotina. COVID 19 affects more commonly to various other reasons like comorbidities or its treatment individuals with comorbidities like diabetes. Diabetic protocols) on the higher incidence of mucormycosis. In the ketoacidosis causes dysfunction of macrophages and is present study Male to Female ratio correlated with study the most frequent risk factor for sinusitis and done by Petrikkos G et al11 but defers with Parisa Badiee et rhinocerebral infection with mucormycosis6. Moreover, al study. In the study made by Parisa Badiee et al., there these fungi thrive with the availability of free iron. was a slight increase in female incidence while in Petrikko Reports suggest abnormal rise in Serum Ferritin values G et al study and the present study there is a remarkably during COVID 19, definitely one of the reasons for the high incidence of mucormycosis in males compared to probability of mucormycosis. In diabetes, increased free females. iron is mainly due to ketoacidosis and/or glycosylation- induced poor iron affinity1. Course of Mucormycosis: During the initial phases of the infection there is edema, but as the hyphae invade blood vessels the tissue undergoes necrosis and has a characteristic black color6 and hence the name black fungus. Loss of vision and smell is attributed to this angio-invasive feature of mucormycosis. If its progress is not arrested by timely treatment, it can lead to septicemia and death with an overall mortality rate of 54%8. Graph 7: Male : Female ratio in comparative studies.

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