Insights from Surgically treated Post Covid Acute Invasive Fungal Rhino-Orbital sinusitis in Chandrapur Study (SPAROS): A Population Based study of Coronavirus Associated Mucormycosis (CAM) characteristics in India.

Introduction: Coronavirus disease 2019 (COVID-19)-associated mucormycosis (CAM) became a public health problem in India in 2021. However, information about the incidence, presentation and prognosis of CAM remains sparse.
Methods: This study describes 100 cases from the Surgically treated Post COVID Acute invasive fungal Rhino-Orbital Sinusitis in Chandrapur (SPAROS) study, a prospective observational follow-up study of patients with CAM diagnosed in Chandrapur district, India. Two-step cluster analysis using four input variables - blood glucose on admission, diabetes status, glucocorticoid exposure and severity of COVID-19 - was used to define three distinct CAM clusters.
Results: The incidence of CAM in the general population was 7.1 cases/1000 patients hospitalized with COVID-19. Steroid exposure and pre-existing diabetes were present in 76% and 55% of cases, respectively. At median follow-up of 18 days, only two deaths had been recorded, while 93 cases were stable. Glucocorticoids, particularly methylprednisolone, seemed to precipitate CAM. Admission to the intensive care unit appeared to be predictive of less extensive surgery. Discussion: Three subtypes of CAM were identified: COVID-19-associated diabetes and mucormycosis, COVID-19-associated classical mucormycosis, and COVID-19-induced mucormycosis. A CAM hypothesis was proposed based on the dynamics of severe acute respiratory syndrome coronavirus-2 and glucose regulated protein.
Conclusion: The clinical characteristics, natural course and pathogenesis of CAM differ from mucormycosis in the pre-COVID era. It is hoped that this classification will be useful in CAM management.

Introduction

COVID19 pandemic in India over last two years has shown two distinct epidemiological waves (Jain et al 2021) The first wave was followed by increased incidence of Mucormycosis from October 2020 onwards During second wave mucormycosis was made notifiable disease by law and India reported over 45000 cases from 780 districts. (https://timesofindia.indiatimes.com/india/over-45000-cases-of-mucormycosis-reported-in-india-health-ministry-tells-rajya-sabha/articleshow/84585912.cmsaccessed 7th march 2022) Literature about incidence, presentation, prognosis of CAM and how it is different from non-covid mucormycosis is sparse. Our study reports CAM data from district registry in Chandrapur, Maharashtra, which is incidentally the region where covid19 outbreak by delta variant was first reported (Jain et al 2021).Our study is prospective follow up of cases from Mucormycosis registry of Chandrapur District between 11th may 2021 and 24th May 2021.

Methods and material

SPAROS study

All mucormycosis cases were reported in the district registry during notification period. SPAROS is a prospective observational study of all those who developed mucormycosis and had SARS CoV2 rt PCR positivity after 1st March 2021. Case of mucormycosis was defined as that having both clinical and radiological evidence of mucormycosis, alongwith fungal hyphae in debrided tissue on KOH mount and confirmed on biopsy . The fungal angioinvasion was assessed in histopathology slide and confirmed by oral pathologist. The follow-up period was till mucormycosis resolution or death. The study planned to enroll patients till 31st December 2021 or discontinuation of mandatory reporting whichever is earlier, and included a network primary and secondary hospitals (supplementary material 1) across Chandrapur district. Exclusion criteria is organ transplantation, active malignancy,those lost to follow up for more than a month and those refusing consent . The objective was to describe acute invasive rhino orbital fungal infection and factors determining morbidity and mortality .The information is collected based on standard case record form (supplementary material 2). The follow up information of the cases was collected on 3rd of june 2021. Mortality/unstable/stable condition was ascertained on follow up. Recovery status was categorised as stable or unstable /dead.

Covid 19 severity

Indian Council for Medical Research (ICMR) guidelines were used to clinically classify a case of covid19 and decide course of management . It divided cases into mild, moderate and severe . The guidelines recommended home isolation for mild cases and hospitalization for moderate/severe patients. (https://www.icmr.gov.in/pdf/covid/techdoc/COVID_Management_Algorithm_17052021.pdf assesed 22nd April 2021

We categorized those admitted after rt PCR positivity (moderate+severe) as group B, while those not admitted after rt PCR (mild) group A. In addition those requiring Oxygen in home isolation were deemed hypoxaemic were also included in B category. This was done as due to bed unavailability a number of moderate to severe COVID have to opt for home care . This binary classification effectively separated the subjects into those with hypoxemia (B) or those without hypoxemia. CT severity score, calculated from CT Chest done during COVID19 infection is widely used indicator of covid severity. (Saeed GA et al 2021) We obtained CT severity score of mucormycosis subjects from records.(supplementary material 2)

Post-operative morbidity

Length of hospitalization is commonly used as indicator of morbidity (Makino et al 2018). However in prevailing circumstances, the hospitalization length was more likely to be influenced by use/ availability of intravenous Amphotericin B infusion or oral Posaconazole rather than general condition. In contrast, a surgeon is likely to perform surgery depending on spread and disease burden. Also, extend of surgery as well as requirement of repeated surgeries is strong predictor of morbidity in Head neck face region (Wu et al 2018) Hence we used extent of surgery as surrogate for mucormycosis morbidity . A surgery was categorized as “extended” if the requirement of partial or total maxillectomy, ethmoidectomy, mandibular surgery, tooth extraction, repeated surgery, or orbital surgery. Rest all types of surgeries were categorized as “simple”.

Mucormycosis incubation period

Date of first SARS-CoV2 rt-PCR positive report and date of admission for mucormycosis was used to calculate CAM “incubation period”. If this period was less than 10 days the case was classified as active COVID19.

Hyperglycemia categorization

Diabetes was defined as pre-existing (D), if past history of hyperglycemia or anti-diabetic medication was available; Covid related (C) if hyperglycemia manifested anytime during or after onset of COVID19 symptom and no diabetes (N) if no history of hyperglycemia or anti-diabetic medications.

Baseline Covid epidemiological data

We also determined the number of rt-PCR confirmed COVID19 cases from 1st March to 31 may 2021 from the district official bulletin. In addition we determined total hospitalization due to COVID19 during the period. (https://covid19.nhp.gov.in/accessed 21st July 2021)Due to partial/ total lockdown during the period there was very less mobility out of Chandrapur district. Hence we take this number as a denominator to calculate incidence of mucormycosis per 1000 hospitalised COVID 19 cases . We also obtained blood glucose during admission of all COVID19 cases from COVID Care Centre Chandrapur in period from 11th May to 31st May 2021 .This data was used to compare blood glucose levels on admission with that of CAM cases. A total of 884 subjects blood glucose on admission thus, identified was de-identified. The study was approved by IEC vide GMCC/PSM/341/2021

Statistical analysis

Numerical variables are expressed as mean +/- 2SD. Pearson's correlation and spearman's correlation was used for parametric and non parametric data respectively.One way ANOVA was used to determine whether steroid exposure had any effect on incubation period .Tukey'S HSD was used as post hoc test used to find differences between the groups. Binary logistic regression was used to identify factors predicting simple or extended surgery. The factors analysed were blood glucose at presentation, ICU admission during COVID 19, presence of DM, presence of hypertension/CAD, history of GC exposure, type of steroid exposure, incubation period. As most of variables were nominal, Two step cluster analysis was used to determine CAM subtypes predefining a cluster quality of at least 0.5, (corresponding to “good fit”), each input factor importance of at least 0.4. and maximum of four cluster Following inputs were selected on clinical relevance - blood glucose at the time of admission, diabetes history (previous,diagnosed during covid or no diabetes), steroid exposure and Covid 19 severity. One way ANOVA was used to compare mean blood glucose at admission of each of the clusters with non-CAM subjects and Tukey HSD used for post hoc analysis . SPSS version 21 was used .

Results: A total of 100 cases were enrolled between 11th may 2021 and 24th May 2021. (figure 1 ) A majority of the cases (n=68) were contributed by the only tertiary hospital in the district - GMC and Civil Hospital Chandrapur. Rest 32 were contributed by 12 hospitals dispered all over the district. (supplementary material 1)A total of 52648 rt-PCR confirmed COVID19 cases were recorded in the aforementioned period. The total no of COVID 19 hospitalization due to covid19 was 13360 . The incidence of CAM, thus calculated, is 23.37/10000 rt PCR SARS-CoV2 positive cases. The incidence of CAM 7.1/1000 COVID hospitalization. The mean age was 49.3 +/- 11.1 years. There was male preponderance (68/96) as compared to females (28/100). Nineteen subjects had active covid. All had maxillary sinus involvement. 68 had involvememt of ethmoid and sphenoid sinuses while 24 had jaw and mandimular involvement. Only four had orbital involvement, two of these required exenteration, there was no case of rhino-orbito-cerebral mucormycosis and pulmonary mucormycosis. Headache was the most common presenting complaint affecting 58% of patients followed by oral complaints like toothache and gum swelling.Fever was conspicuous by its absence in all but four subjects (table 1 ). Post-surgery biopsy revealed Mucorales to be causative in 96 subjects and Aspergillus to be causative agent in four subjects.(Figure 2 &3 ) . None of the subjects had clinical diagnosis of pulmonary mucormycosis.Of 96 subjects, pre-existing diabetes was present in 53, diabetes diagnosed during covid in 37, while six had no history of diabetes. The mean blood glucose on admission for mucormycosis was 236+/- 82 mg/dl . Blood glucose was analysed with respect to extent of surgery, time to presentation, oxygen requirement, HRCT score severity.The mean glucose on admission was statistically correlated with CT severity score (Spearman rho=0.248,p=0.014). The steroid usage is mentioned in table 2 . Median duration of SARS CoV2 to mucormycosis diagnosis was 25 days (mean = 25+/- 14.1). This duration was shorter in those without steroid exposure (median 31 days) as compared to those who received steroids. The median duration was 18,25 and 24 days days for methylprednisolone, dexamethasone and dexamethasone + methylprednisolone combination respectively.This was statistically significant {F=2.9, p=0.037} . Post hoc analysis showed methylprednisolone intake during COVID 19 infection to be significantly associated with less time to mucormycosis onset, as compared to other groups (Tukey HSD mean 12.5 +/- 4.2 days p=0.029). (supplementary material 3) All subjects had maxillary sinus involvement on CT/MRI reports.(Figure 4 ) Of these 14 required simple surgery in the form of nasal debridement, while 81 required wider complete or partial maxillectomy, eyeball exenteration, and repeated debridement. The median follow up was 18 days. One patient could not be followed up. Hence we included 95 patients in statistical analysis of factors predicting extent of surgery . Of these two died, while rest were recovering and stable. Both were known cases of DM, required admission in ICU and had received dexamethasone + methylprednisolone combination during COVID 19 treatment. With only two deaths statistical analysis of factors predicting mortality was not possible. Hence we did COVID 19 Binary logistic regression to identify factors predicting morbidity. Of the factors analysed, only ICU admission during COVID 19 (p=0.031) negatively predicted extended surgery. Hyperglycemia, history of diabetes, steroid exposure was not associated with requirement of aggressive surgery. None of the cases presented with Diabetic ketoacidosis (DKA). The characteristics of clusters are presented in table 3 and table 4 Of the three clusters defined (Figure 5 ), cluster 1 (46.3 %) and cluster 3 (29.5%) had steroid exposure (100% in each) while cluster 2 (24%) had no steroid exposure (0%). Mild COVID19 cases were only placed in cluster 1. The mean blood glucose was 193 mg/dl, 286 mg/dl,266mg/dl respectively in clusters 1, 3 and 2. All those placed in cluster 3 had previous diabetes (100%), while majority in cluster 1 (71%) had diabetes diagnosed during COVID19. The mean blood glucose on admission of those without mucormycosis was 173.2=/-12 mg/dl. Tukey HSD post hoc analysis showed it to be lower than cluster 1 (173 vs 193 mg/dl) but not statistically significant (p=0.058). It was significantly lower than cluster 3 (173 Vs 286, p <0.001) and cluster 2 (173 Vs 266, p<0.001) (Figure 6 )

Figure 1

showing flowchart of the study

Table 1

CAM clinical presentation

Presentation	n= 95
Headache	56
Nasal congestion and pain/ blackening	18
Facial swelling and pain	15
Gum swelling and toothache	44
Eye swelling, red eye, visual complaints	18
Fever	4
Critical Illness/Diabetic Ketoacidosis (DKA)	0

Table 1 describing clinical features on presentation> The features were divided into, headache, nasal complaints, dental complaints, eye and sight complaints, fever,emergency conditions and hospitalization

Figure 2

showing biopsy slide of Mucorales 2A.40X Magnification- H&E stained sinus mucosa section showing thick long filamentous aseptate hyphae scattered in the connective stroma , these hyphae are 12-14 microns thick and branch in obtuse angles. The stroma also necrotic areas, chronic inflammatory cell infiltrate and dilated capillaries. 2B 40X Magnification- PAS positive sinus mucosa showing Mucor spp. fungal hyphae seen in connective tissue stroma, Hyphae are thick aseptate branching at right angles. Surrounding stroma is fibrocellular

Figure 3

showing biopsy slide of Aspergillus spp 40X Magnification, H&E stained section sinus mucosa showing Aspergillus spp. reveals, 3-5 microns in diameter, thin filamentous aseptate hyphae which branch in acute angles, scattered in the stroma along with lymphocytes, plasma cells, collagen fibers and fibroblasts.

Table 2

Use of steroids in CAM

Type of steroid	Number of patients (n=95)	Median duration (days)
Methylprednisolone only (M)	20	14
Dexamethasone only (D)	8	7
Both Methylprednisolone & Dexamethasone (DM)	44	14
No Glucocorticoid exposure (N)	23	-

Table 2 showing steroid exposure in CAM cases. A substantial proportion (27%) had no steroid exposure.

Figure 4

showing CAM MRI 4a T1WI axial section showing hypointense right maxillary sinus and right nasal cavity 4b post contrast MRI axial section showing enhanced inflamed mucosal lining of right maxillary sinus and nasal cavity 4c post contrast MRI coronal section showing retro-orbital involvement . 4d post contrast MRI coronal section showing inflamed mucosal lining of right frontal sinus.

Table 3

Cluster phenotype description

	Cluster 1 / CADM (Covid19 associated mucormycosis and diabetes)	Cluster 3 / CACM (Covid19 associated classical mucormycosis)	Cluster 2 /CIM (Covid19 induced mucormycosis)
Steroid exposure	+++	++	-
Previous Diabetes	+	+++	+
Blood glucose (mg/dl)	150-200	200-400	200-300
Covid 19 severity	++	+++	++++
Atherosclerosis	++	+	++
Pathophysiology	Likely to have active COVID19/ steroid use/excessive dosage/	Use of antibiotics/ immunosuppression due to critcal illness	Covid 19 associated small vessel thrombosis
Prognosis	Good	Bad, still better than non covid mucormycosis	Good

Table 4

Characteristics of different cluster groups

	Total CAM (n= 95)	CADM (n=44)	CACM (n=28)	CIM (n=23)
Mean Age (years)	49.3	49.73	51.71	45.7
M/F	68/28	32/12	20/8	15/8
Clinical presentation
Headache	56	20	17	19
Nasal congestion and pain/ blackening	18	7	2	9
Facial swelling and pain	15	4	9	2
Gum swelling and toothache	44	23	14	7
Eye swelling, red eye, visual complaints	18	4	11	3
Fever	4	0	4	0
Critical Illness/Diabetic Ketoacidosis (DKA)	0	0	0	0
Site of involvement
Maxillary sinus involvement	96	44	28	23
Ethmoid and/or sphenoid sinus involvement	68	34	23	11
Jaw and mandibular involvment	24	9	3	12
Orbital involvement	4	1	3	0
Hypertension present	10	3	5	2
Covid severity (mild/moderate Vs severe)	23/72	21/23	0/28	2/21
Mean CT severity score	5.05	5.5	6.07	4.1
Diabetes/diagnosed after covid/non diabetic	40/50/5	7/32/5	28/0/0	5/18/0
Mean Blood glucose on admission (mg/dl)	236	193	286	266
Use of steroids
No steroids	23	0	0	23
Methylprednisolone (M	20	12	8	0
Dexamethasone (D)	8	5	3	0
Both Methylprednisolone and dexamethasone (DM)	44	27	17	0

Figure 5

showing cluster analysis

Figure 6

shows comparison of blood glucose on admission of three clusters of mucormycosis (CADM, CACM,CIM) with that of 884 covid19 patients admitted in Covid Care Hospital Chandrapur during the same period.

Discussion

Epidemiology of CAM

The population incidence of mucormycosis in pre-covid era has shown an increase over decades. It has been variously reported to be 1.2 to 1.7 per million western countries. (Prakash et al 2019). In Chandrapur and adjoining regions, cases of mucormycosis have been few and mostly in those with severe immunosuppression (Quraishi et al 2020)(Sagar et al 2021). Tertiary care autopsy prevalence of mucormycosis has been reported to be 6.3 to 33 per million hospitalization(Prakash et al 2019)(Gunea et al 2020). Compared to this,we report mucormycosis prevalence of 710 per million COVID hospitalisation. This points to factors related to covid management and the disease itself as being causative. The CAM prevalence in our study is 2300/ million of COVID 19 infections, which is at least ten fold higher than those reported in transplant recipients. The incidence of CAM in COVID 19 is higher than that of mucormycosis in general population . Its also several fold higher than organ transplant recipients/or Diabetes Mellitus. We report a much lower prevalence (0.07% Vs 0.27%), and mortality rate (2% Vs 45%) than previously described from India(Patel et al 2021) The differences can be attributed to referral bias in the study as the concerned study was a retrospective in design and carried out in tertiary hospitals. (Patel et al 2021) Our finding of increased male preponderance is the same as described in non-covid mucormycosis and CAM studies previously (Singh et al 2021) (Patel et al 2021) (Prakash et al 2019), and is likely due to the fact that testosterone increases expression of GRP78, a stress protein crucial for hyphal invasion by Mucorales. (Ebrahim et al 2020)

Clinical Presentation

Headache and orbital complaints were common while fever was rare. Previous case series on mucormycosis have described fever as a common presentation. (Quraishi et al 2020)(Sagar et al 2021) (Hingnikar et al)The difference is possibly because of a debilitated general state in former while a peculiar interaction of COVID19, steroids and hyperglycemia inducing fungal invasion in later. SARS CoV2 induced transient T-cell suppression in presence of broad spectrum antibiotics, and use of steroids might be some potential explanation for lack of fever. All of our patients had maxillary involvement. This is in contrast to previous CAM series (Singh et al 2021) (Patel et al 2021) (Sharma et al 2021), where substantial ethmoid, orbital and pulmonary mucor cases are described, which is likely referral bias. Maxillary sinus is the most common site of non-invasive fungal infections and a likely conduit of fungal spores in otherwise healthy states.

Role of steroids

While inappropriate GC use has been previously implicated in CAM, we found a sizable percentage (29.5%) without steroid exposure. Those without steroid exposure were likely to present late. Glucocorticoids predispose to fungal angioinvasion by impaired neutrophil margination and repression of adhesion factors (Reusch et 2021) In particular methylprednisolone was found to be associated with earlier CAM presentations. Methylprednisolone impairs conidial phagocytosis by neutrophils and predispose to invasive fungal infection (Simitsopoulou et al 2015). Due to perceived better clinical efficacy against covid 19, methylprednisolone has been widely used as compared to dexamethasone in India (Ranjbar et al 2021) Whether use of methylprednisolone per se could have contributed to increased CAM in India remains speculative.

Role of hyperglycemia

In line with previous studies (Lu et al 2021), we found a strong correlation between CT severity score and blood glucose at presentation. The mean blood glucose (mean = 236 mg/dl) was higher than that described after severe covid (mean = 170 mg/dl) (Bode et al 2019). But it was much lower that the levels at which we saw mucormycosis in pre-covid times. (Quraishi et al 2020)(Sagar et al 2021) (Hingnikar et al) In fact, blood glucose of hospitalised COVID19 subjects who did not develop mucormycosis was not different from one of CAM cluster- CADM (see below). In remaining two CAM cluster CACM and CIM antecedent hyperglycemia seemed to have clear role in mucormycosis causation. Elevated blood glucose can exponentially increase viral replication by increasing glucose in pulmonary airway surface liquid. Both prevalence of undiagnosed diabetes and poor glycemic control among diagnosed diabetics is higher in India. Even in diagnosed diabetes, glycemic control is poorer . The non CAM cases in our study demonstrated higher blood glucose levels that those described in other countries. However, our findings of CADM subtype calls for recognizing factors over and above hyperglycemia in CAM pathogenesis. None of our patients had DKA, which was in contrast to our experience in pre-covid era

Role of critical illness

Those with ICU requirement were likely to undergo less extensive surgery and this was independent of blood glucose and steroid exposure .However on factor analysis we did not find ICU requirement of COVID to be the defining factor in cluster. It is likely ICU patients were better cared for, mucormycosis picked up in the early stage, antifungal administered early and anticoaugulated with LMWH . All these factors may have reduced severity of mucormycosis and contributed to less extensive surgery.

Prognosis

Mucormycosis associated with Diabetes esp DKA has far better prognosis that those associated with neutropenic states like hematological malignancies. This is likely because underlying conditions like DKA and hyperglycemia can be rapidly optimised. (Deutsche et al 2019) Similarly, we explain better than expected CAM prognosis in our study .The severity of covid 19 is likely to decrease by a couple of weeks in most survivors. This along with stoppage of steroids may be responsible for excellent overall prognosis in our patients.

Classification and implication

Exploratory analysis for CAM classification was based on factors implicated as necessary for fungal mucosal invasion- Diabetes, hyperglycemia during mucormycosis presentation, glucocorticoid receptor agonist use and COVID 19. We have taken the type of diabetes and hyperglycemia at presentation separately. A history of diabetes is suggestive of presence of long standing atherosclerosis, while hyperglycemia at presentation is suggestive of metabolic milieu necessary for fungal proliferation, use of glucocorticoids is representative of qualitative defect in neutrophils (Table 3)

First cluster consists of the majority of cases . This cluster consists of subjects who had steroid administered during covid followed by hyperglycemia and diabetes precipitation, uncontrolled hyperglycemia for some days. There was no predilection for orbit or lower jaw. Clinically, we termed it COVID19 associated diabetes and mucormycosis (CADM) as this cluster consists of cases with diabetes diagnosed during COVID19 and glucocorticoids, followed by mucormycosis. It consists of mild to moderate COVID in which aggressive glucocorticoid therapy has precipitated diabetes and invasive fungal sinusitis.Most active covid19 with mucormycosis seen in this subgroup. These require single debridement and have excellent prognosis . Control of hyperglycemia in this group-even for a few hours- can yield favorable results.Notably, this is the only subgroup in which mild cases were seen. Perhaps this points to inappropriate steroid usage.

The third cluster consists of the majority of those with previous diabetes history.This cluster consists of subjects with long standing uncontrolled diabetes, severe covid followed by mucormycosis which required extensive surgery. The subjects were likely to have systemic involvement, orbital involvement and likely mortality.Blood glucose during mucormycosis admission is highest of three sub-groups (around 300 mg/dl), suggestive of uncontrolled diabetes. Clinically this resembles mucormycosis described previously in covid and non-covid cases. Hence we term it COVID19 associated with classical mucormycosis (CACM). CACM is likely to be overrepresented in studies from tertiary care hospitals. We found mortality only in this subgroup. Aggressive surgery and antifungal therapy should be mostly directed to this group. The second cluster (CIM) consists of those in whom high SARSCoV2 load seemingly precipitated mucormycosis. This group consists of those without steroid exposure and previous history of diabetes and no risk factor for mucormycosis. Even the blood glucose was not statistically different from those without mucormycosis. However, all subjects had severe covid and there was predilection for jaw (table 4), including osteonecrosis of jaw.Thrombotic response to SARS CoV2 possibly plays a central role in causing fungal invasion in this group.

Our classification has practical implications in CAM management. Current guidelines on CAM management bundle it in one entity. The implication for the highly heterogeneous nature of CAM is in appropriate triaging. CADM and CIM may not require aggressive management apart from hyperglycemia control and simple debridement, while CACM should be managed aggressively. Antiplatelet /anticoagulation are likely to be optimum preventive strategies for CIM.

Postulated CAM Hypothesis

Identification of a phenotype without steroid exposure and associated with increased COVID19 severity points to the role played by the disease itself in causation of mucormycosis. Expression of Glucose Regulated Protein (GRP78) levels are exponentially increased in COVID19 (Sabirli et al 2021). Also, GRP78 itself plays a role in SARS-CoV2 internalisation by interaction with spike protein (Ibrahim et al 2020) Thus GRP78- SARS CoV2 interaction occurs in feed forward manner. GRP 78 expression is increased four fold in hypoxia. Thombosis of maxillary artery branches may induce intense mucosal hypoxia and acidosis. Delta variant has propensity for arterial thrombosis (Vaidyanathan 2021) Combined with steroid induced neutrophil dysfunction, this may lead to fungal angioinvasion. This hypothesis also explains excellent CAM prognosis. Once COVID resolves,GRP 78 levels come down, blood supply and immunosuppression recovers fast, and there is rapid clinical improvement. A certain level of hyperglycemia seems essential for mucormycosis, however it has only minimal role in recovery and final outcome. Steroid exposure is facilitatory in earlier precipitation and causation in those susceptible, but it is not essential in CAM pathogenesis. Methylprednisolone use may accelerate and possibly precipitate CAM. It's tempting to speculate that methylprednisolone may lead to increased GPR 78 expression as GC receptor agonists differ in transactivation profiles .

Limitations:

The data from registry has inherent disadvantages like lack of uniformity in clinical assessment and documentation. We do not have information of type of antibiotics exposure, exact dose steroids and inflammatory markers specifically ferritin which could have had bearing on CAM classification. More importantly we do not have data of enviormental exposure, which might explain why many diabetic patients do not develop mucormycosis aka CIM cluster

Conclusion

CAM is heterogeneous in terms of clinical presentation, phenotype and likely etiology. Exposure to steroids is associated with early presentation. Use of methylprednisolone is a precipitating factor . There are three CAM sub-types, of which only CACM resembles mucormycosis described in literature. Future studies on CAM management should focus on conservative management and role of antithrombotic therapies.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contribution statement

AK, NR and RS designed the study. NR and JN recruited the patients and obtained clinical data. DM and AD analysed biological and radiological samples. RS and DM did statistical analysis. RS, JN and AK wrote article. All authors reviewed the manuscript and approved

Data Availability statement

The raw data is available and deposited in OSF. It is accessible on the following link upon request: shukla, ravindra. (2021, June 23). COVID 19 Associated Mucormycosis. Retrieved from osf.io/7fp2j

Ethics approval

This study was reviewed and approved by the ethics committee of Government Medical College, Chandrapur (No. GMCC/PSM/341/2021). This study was an observational study. Informed consent was obtained from all patients in our study vide

References

1.Bode B, Garrett V, Messler J, et al. Glycemic Characteristics and Clinical Outcomes of COVID-19 Patients Hospitalized in the United States [published correction appears in J Diabetes Sci Technol. 2020 Jun 10;:1932296820932678]. J Diabetes Sci Technol. 2020;14(4):813-821. doi:10.1177/1932296820924469

2.Deutsch PG, Whittaker J, Prasad S. Invasive and Non-Invasive Fungal Rhinosinusitis-A Review and Update of the Evidence. Medicina (Kaunas). 2019;55(7):319. Published 2019 Jun 28. doi:10.3390/medicina55070319

3.Guinea J, Escribano P, Vena A, et al. Increasing incidence of mucormycosis in a large Spanish hospital from 2007 to 2015: Epidemiology and microbiological characterization of the isolates [published correction appears in PLoS One. 2020 Feb 12;15(2):e0229347]. PLoS One. 2017;12(6):e0179136. Published 2017 Jun 7. doi:10.1371/journal.pone.0179136

4.Hingnikar P, Bhola N, Jadhav A, Sharma A. Mucormycosis of maxillary sinus in a newly diagnosed case of diabetes mellitus. J Datta Meghe Inst Med Sci Univ 2019;14:397-400

5.Ibrahim IM, Abdelmalek DH, Elfiky AA. GRP78: A cell's response to stress. Life Sci. 2019;226:156-163. doi:10.1016/j.lfs.2019.04.022

6.Ibrahim IM, Abdelmalek DH, Elshahat ME, Elfiky AA. COVID-19 spike-host cell receptor GRP78 binding site prediction. J Infect. 2020 May;80(5):554-562. doi: 10.1016/j.jinf.2020.02.026. Epub 2020 Mar 10. PMID: 32169481; PMCID: PMC7102553

7.Jain VK, Iyengar KP, Vaishya R. Differences between First wave and Second wave of COVID-19 in India. Diabetes Metab Syndr. 2021;15(3):1047-1048. doi:10.1016/j.dsx.2021.05.009

8.Lu, X., Cui, Z., Pan, F. et al. Glycemic status affects the severity of coronavirus disease 2019 in patients with diabetes mellitus: an observational study of CT radiological manifestations using an artificial intelligence algorithm. Acta Diabetol 58, 575–586 (2021).

9.Makino Y, Ishida K, Kishi K, Kodama H, Miyawaki T. The association between surgical complications and the POSSUM score in head and neck reconstruction: a retrospective single-center study. J Plast Surg Hand Surg. 2018 Jun;52(3):153-157. doi: 10.1080/2000656X.2017.1372288. Epub 2017 Sep 7. PMID: 28880703.

10.Patel A, Agarwal R, Rudramurthy SM, Shevkani M, Xess I, Sharma R, Savio J, Sethuraman N, Madan S, Shastri P, Thangaraju D, Marak R, Tadepalli K, Savaj P, Sunavala A, Gupta N, Singhal T, Muthu V, Chakrabarti A; MucoCovi Network3. Multicenter Epidemiologic Study of Coronavirus Disease-Associated Mucormycosis, India. Emerg Infect Dis. 2021 Jun 4;27(9). doi: 10.3201/eid2709.210934. Epub ahead of print. PMID: 34087089.

11.Prakash H, Chakrabarti A. Global Epidemiology of Mucormycosis. J Fungi (Basel). 2019;5(1):26. Published 2019 Mar 21. doi:10.3390/jof5010026

12.Quraishi AH, Peshattiwar A, Umare G, Bannerji A. Perforation peritonitis secondary to intestinal mucormycosis in a boy with type I diabetes mellitus. J Indian Assoc Pediatr Surg 2020;25:118-20

13.Ranjbar, K., Moghadami, M., Mirahmadizadeh, A. et al. Meth

ylprednisolone or dexamethasone, which one is superior corticosteroid in the treatment of hospitalized COVID-19 patients: a triple-blinded randomized controlled trial. BMC Infect Dis 21, 337 (2021). https://doi.org/10.1186/s12879-021-06045-3

14.Reusch N, De Domenico E, Bonaguro L, et al. Neutrophils in COVID-19. Front Immunol. 2021;12:652470. Published 2021 Mar 25. doi:10.3389/fimmu.2021.652470

15.Sabirli R, Koseler A, Goren T, Turkcuer I, Kurt O. High GRP78 levels in Covid-19 infection: A case-control study. Life Sci. 2021;265:118781. doi:10.1016/j.lfs.2020.118781

16.Saeed GA, Gaba W, Shah A, Al Helali AA, Raidullah E, Al Ali AB, Elghazali M, Ahmed DY, Al Kaabi SG, Almazrouei S. Correlation between Chest CT Severity Scores and the Clinical Parameters of Adult Patients with COVID-19 Pneumonia. Radiol Res Pract. 2021 Jan 6;2021:6697677. doi: 10.1155/2021/6697677. PMID: 33505722; PMCID: PMC7801942

17.Sagar, V. V. S. S., Yelne, P., Mathurkar, S., & Gaidhane, S. (2021). Mucormycosis in RT-PCR negative Covid 19 patient with newly diagnosed Diabetes Mellitus: A Case Report. Asian Journal of Medical Sciences, 12(7), 149–153.

18.Sharma S, Grover M, Bhargava S, Samdani S, Kataria T. Post coronavirus disease mucormycosis: a deadly addition to the pandemic spectrum [published online ahead of print, 2021 Apr 8]. J Laryngol Otol. 2021;1-6. doi:10.1017/S0022215121000992

19.Simitsopoulou M, Walsh TJ, Kyrpitzi D, Petraitis V, Kontoyiannis DP, Perlin DS, Roilides E. Methylprednisolone impairs conidial phagocytosis but does not attenuate hyphal damage by neutrophils against Exserohilum rostratum. Med Mycol. 2015 Feb 1;53(2):189-93. doi: 10.1093/mmy/myu034. Epub 2014 Jul 23. PMID: 25056961.

20.Singh AK, Singh R, Joshi SR, Misra A. Mucormycosis in COVID-19: A systematic review of cases reported worldwide and in India [published online ahead of print, 2021 May 21]. Diabetes Metab Syndr. 2021;doi:10.1016/j.dsx.2021.05.019

21.Vaidyanathan G. Coronavirus variants are spreading in India - what scientists know so far. Nature. 2021 May;593(7859):321-322. doi: 10.1038/d41586-021-01274-7. PMID: 33976409

22.Wu V, Cusimano MD, Lee JM. Extent of surgery in endoscopic transsphenoidal skull base approaches and the effects on sinonasal morbidity. Am J Rhinol Allergy. 2018 Jan 1;32(1):52-56. doi: 10.2500/ajra.2018.32.4499. PMID: 29336291.

23.https://timesofindia.indiatimes.com/india/over-45000-cases-of-mucormycosis-reported-in-india-health-ministry-tells-rajya-sabha/articleshow/84585912.cms

24.https://www.icmr.gov.in/pdf/covid/techdoc/COVID_Management_Algorithm_17052021.pdf

24. https://covid19.nhp.gov.in/accessed 21st July 2021

Conflict of interest

Authors declare no conflict of interest relevant to present work