A fungal epidemic amidst a viral pandemic: Risk factors for development of COVID-19 associated rhino-orbital-cerebral mucormycosis in India

ABSTRACT Purpose To study the risk factors for development of COVID-19 associated rhino-orbital-cerebral mucormycosis (ROCM) during the COVID-19 pandemic in India. Methods Multi-centric retrospective case-control study conducted from October 2020 to May 2021. Cases comprised of consecutive patients of COVID-19-associated ROCM (CA-ROCM) presenting at the participating ophthalmic institutes. Controls comprised of COVID-19-positive or COVID-19-recovered patients who did not develop ROCM. Comparative analysis of demographic, COVID-19 infection, treatment parameters and vaccination status between cases and controls performed. Clinical and imaging features of CA-ROCM analyzed. Results There were 179 cases and 361 controls. Mean age of presentation in cases was 52.06 years (p = .001) with male predominance (69.83%, p = .000011). Active COVID-19 infection at the time of presentation of ROCM (57.54%, p < .0001), moderate to severe COVID-19 (p < .0001), steroid administration (OR 3.63, p < .00001), uncontrolled diabetes (OR 32.83, p < .00001), random blood sugar >178 mg/dl were associated with development of CA-ROCM. Vaccination showed a protective effect (p = .0049). In cases with intracranial or cavernous sinus extension there was history of steroid administration (OR 2.89, p = .024) and orbital apex involvement on imaging (OR 6.202, p = .000037) compared to those with only rhino-orbital disease. Conclusion Male gender, active COVID-19 infection, moderate or severe COVID-19, uncontrolled diabetes, steroid administration during COVID-19 treatment are risk factors for developing rhino-orbital-cerebral mucormycosis. Vaccination is protective. Random blood sugar of >178 mg/dl in COVID-19 positive or recovered patients should warrant close observation and early detection of ROCM. Presence of ophthalmoplegia, blepharoptosis at first clinical presentation and orbital apex involvement on imaging are associated with intracranial extension in ROCM.


Introduction
The Coronavirus Disease 2019 (COVID-19) pandemic has devastated the entire world causing great loss to lives and economy. 1,2 Co-infections have added to the burden. 3 Fungal co-infections include aspergillosis, candidiasis, cryptococcosis and mucormycosis. 4 Aspergillosis, candidiasis and few cases of cryptococcosis have been reported largely from China, European nations and the United States 3,4 whereas mucormycosis has been reported mainly from India. 5 Mucormycosis is an invasive opportunistic disease caused by ubiquitous fungi of the phylum Zygomycota affecting different organ systems, rhino-orbito-cerebral mucormycosis (ROCM) being the commonest. 6,7 Infection in immunocompetent individuals is rare. 8,9 The largest number of cases in the immunocompetent has been reported from India followed by the United States. 7 Hematological malignancy and solid-organ transplant are the most common risk factors in the developed countries, 10 whereas it is uncontrolled diabetes in India. [9][10][11] There was a gradual rising trend of mucormycosis in India in the pre-COVID-19 era due to the rise in diabetes, from 12.9 cases per year in 1990-1999 to 89 cases per year in 2013-2015. 10,11 However, the COVID-19 era has seen a substantial rise in the incidence with 81% of the world's COVID-19 associated mucormycosis cases reported in India. 5 As of May 2021, over 14,000 cases were reported. 12 During the first wave, few reports of COVID-19associated ROCM (CA-ROCM) from India surfaced. [13][14][15][16][17] However, in the recent months, there has been a surge of CA-ROCM cases. 12,[18][19][20][21][22] This surge parallels the rise in COVID-19 during the second wave, 12,23 which has additionally burdened the health system and availability of essential drugs and resources causing significant morbidity and mortality. 12 Although CA-ROCM has been attributed to factors such as presence of uncontrolled diabetes, old age and steroid administration, [13][14][15] there are very few reports that have comprehensively analyzed the risk factors associated with CA-ROCM. This study was therefore performed to address this gap by analyzing data from multiple centers across the country.

Study design
This is a multi-centric retrospective case-control study conducted in ophthalmic institutes across India from October 2020 to May 2021 . The participating institutes included Narayana Nethralaya, Bangalore; Sunayan Eye Care and Aesthetic Clinic, Pune; Advanced Eye Hospital & Institute, Navi Mumbai; Lokmanya Tilak Municipal Medical College and General Hospital, Sion, Mumbai; Manipal Hospital, Bangalore; Advanced Eye Center, PGIMER, Chandigarh; Sankara Nethralaya, Chennai; Eyehub, Faridabad; and Aditya Birla Sankara Nethralaya, Kolkata. The study design, anonymised data collection and analysis was approved by the Institutional Ethics committees of all participating institutes. The study adheres to the tenets of Declaration of Helsinki. Consecutive patients of COVID-19 associated rhinoorbital cerebral-mucormycosis (CA-ROCM) presenting at these collaborative centres were included as cases. Controls comprised of patients presenting to the centres during the same period, who were COVID-19 positive or COVID-19 recovered and did not develop ROCM.
'Active COVID-19' was defined as a patient who has tested positive for SARS-CoV-2 virus either by RT-PCR (nasal and throat swab) or Rapid antigen test (nasal and throat swab) with or without symptoms of COVID-19 at the time of presentation to ophthalmic institute. 'COVID-19 recovered' was defined as a patient who had tested positive for COVID-19 infection (positive nasal and throat swab RT-PCR or Rapid antigen test), underwent treatment and eventually tested negative for SARS-CoV-2 on repeat RT-PCR testing within the previous 5-month period.
(2) Presenting with symptoms of ROCM to ophthalmic plastic surgeons from October 2020 to May 2021.

Exclusion criteria
(1) Patients of ROCM presenting during the study period not associated with COVID-19 i.e. patient with no prior/active COVID-19.
A standardized checklist was used to collect information from all participating institutions. This included sociodemographic (age, gender, city), clinical details at first presentation to the Oculoplastic surgeon (symptoms and duration of ROCM), imaging features [Computed Tomography (CT)/ Magnetic Resonance Imaging (MRI)] of ROCM, medical history of co-morbidities (history of Diabetes mellitus (DM), status of control, random blood sugar levels and HbA1c levels at presentation, other co-morbidities), details of COVID-19 (number of days from detection at presentation to Oculoplastic surgeon, severity of COVID-19, i.e. mild, moderate or severe), COVID-19 treatment details (steroid administration, antiviral therapy, IL-6 inhibitors, oxygen supplementation, ventilatory support), COVID-19 vaccination status, microbiology (direct microscopy on KOH mount, growth on culture media) and histopathology findings if present. For the purpose of this study, uncontrolled DM and hyperglycemia were defined as random blood sugar at presentation >178 mg/dl (after constructing a ROC curve described in results) or a HbA1c greater than 8%.
The diagnosis of ROCM was categorized as Possible, Probable or Proven ROCM based on the criteria customized to ROCM as adapted from ECMM-MSG ERC guidelines (European Confederation of Medical Mycology and the Mycoses Study Group Education and Research Consortium). 24 Quantitative variables were analyzed with descriptive statistics i.e. mean, median, standard deviation. Unpaired T test was applied to quantitative variables. Chi-square test was applied to qualitative variables, p value <.05 was considered significant. Odds ratio and Relative risk were calculated after building 2 × 2 tables. Multiple logistic regression analysis was performed to assess independent risk factors for development of ROCM (MedCalc v20.009).    OR 32.8313) at presentation, a strong risk factor in this study for developing CA-ROCM. More than one-fourth (n = 51, 28.49%) of the cases were newly diagnosed diabetics at the time of presentation for ROCM.

Diabetes mellitus and ROCM
Among the cases, a ROC curve analysis was carried out to determine the level of random blood sugar (RBS) as a risk factor for ROCM at the time presentation irrespective of the diabetes status ( Figure 1) which showed that RBS >178 mg/dl as a risk factor for CA-ROCM has a sensitivity and specificity of 87.3% and 90.3% respectively (P < .001). At this level, the positive likelihood ratio is 8.98. A higher RBS (Avg 291.19 mg/dl, median 275 mg/dl) and HbA1c (Avg 11.0008, median: 10.65%) at presentation were significantly associated with development of CA-ROCM (Blood sugar p < .0001, HbA1c <0.0001). Table 3 summarizes the treatment received for COVID-19. Antiviral therapy, IL-6 inhibitor administration, oxygen supplementation, ventilatory The diabetes status was analyzed separately in cases in which steroids were not administered during COVID-19 treatment (N = 50). There were 62% (31/50) cases who were known diabetics and 36% (18/50) cases who did not have a prior known diabetic status. Uncontrolled blood sugar was noted in 90.32% (N = 28/31; average RBS 311.6 mg/dl, range 157-542, median 306, SD 171.7) in the former and 88.8% (N = 16/18, average RBS: 330.3 mg/dl, range 100-780, median 306, SD 171.70) in the latter. In one case the DM status was unknown as per history but had a blood sugar level of 88 mg/dl. The relationship between a prior history of diabetes, administration of steroid, and RBS >180 mg/dl is illustrated in Figure 2.

Other co-morbidities
Concurrent systemic co-morbidities were noted in 35 cases and 29 controls. Chronic kidney disease (CKD) was the second most common among cases (n = 10) after Hypertension (HTN, n = 12). There were 2 cases each of post organ transplant (renal = 1, liver = 1) and asthma. One each with Pulmonary Tuberculosis (PTB), post-partum status, ischemic heart disease and AIDS.

Analysis of ROCM cases
The mean duration of symptoms of CA-ROCM was 5.815 days (range 1-30, median: 5). Patients who presented early at an average of 4.3 days had a visual acuity better than 6/60 and those who presented later at a mean of 6.39 days had a visual

Subgroup analysis of cases
Based on imaging features, cases were further categorized. Cases with severe CA-ROCM, i.e. evidence of intracranial extension with or without cavernous sinus extension were categorized under Group 1 and cases with only sinus with/without orbital disease under Rhino-Orbital Mucormycosis, i.e. Group 2. Table 4 summarizes the significant risk factors for development of severe CA-ROCM. Presentation in the 5 th decade of life, active COVID-19 infection, the clinical features of ophthalmoplegia and blepharoptosis at first presentation, evidence of orbital apex involvement on imaging and steroid administration for the treatment of COVID-19 were significant risk factors for development of severe disease.

Logistic regression analysis
The multiple logistic regression analysis of risk factors is described in Table 5. Male gender, known A literature search carried out on PubMed from April 1 2020 to June 7 2021 with the terms 'COVID-19', 'Orbit' and 'mucormycosis,' 'rhino orbito cerebral mucormycosis' was performed. Review articles and reports with no mention of orbital involvement were excluded.
Almost 70% of cases were males in this study, which as a risk factor was statistically significant (p = .000011, OR 2.33). This is consistent with most series. 13,14,[25][26][27] The mean age at presentation was 52.06 years in the cases group which is comparable with other reported CA-ROCM series. 13,19 A large multicentric study on COVID-19 associated Mucormycosis (CAM) from India reported 56.9 years as the mean age and 80% males. The series included all systemic manifestations of CAM with 161 CA-ROCM cases (32). The age group of presentation with intracranial ROCM was younger compared to those with only rhinoorbital disease in our study. One possible explanation for this may be that systemic co-morbidity is not expected in the younger age group, and screening for DM has been considered only in individuals above 50 years of age traditionally. In the pre-COVID-19 era, almost a quarter of Indians with DM remained undiagnosed until presenting with mucormycosis. 32 Our study showed that active COVID-19 infection, moderate and severe COVID-19 are associated with developing CA-ROCM. Ravani et al reported COVID-19 positivity as one of the risk factors but did not elaborate on severity of COVID-19. 19 Moderate and severe COVID-19 infections have been noted in other CA-ROCM series. 14,22,25 Leukopenia, leukocytosis or lymphopenia can occur in COVID-19 with lymphopenia being more common in severe disease. 33 Function and regulation of T cells, antigen presenting cells, neutrophils and other immune cells is also impaired along with increased inflammatory cytokines. 33 SARS-CoV-2 induced impaired host immunity along with factors such as prolonged hospital stays, ventilatory support, additional immunosuppression due to steroid or IL-6 administration and co-morbidities like DM were risk factors for development of fungal co-infections. 4 Steroid administration and DM have been noted to be the underlying conditions in development of CAM. 13,14,[16][17][18][19]21,22,[25][26][27][28]30,31,34 Steroids were administered in 72% (108/158) of cases in our study (OR 3.63, RR 1.83). Though recommended only in severe disease, 35 patients with mild and moderate disease were documented to have history of treatment with steroids. High doses of glucocorticoids can result in secondary infections, prolonged shedding of SARS-CoV-2, glucose intolerance, apart from worsening of atheromatous vascular diseases, fluid retention and dyslipidemia etc. 35 Uncontrolled DM was noted to be a very strong risk factor for development of CA-ROCM in this study (OR 32.83, RR 3.23, p < .00001), the average HbA1c in CA-ROCM group in our study being 11% as compared to 7.45% in the control group. Ravani et al found HbA1c >8% to be significant predictor of mortality at 75 days along with cerebral involvement. 19 In our study, amongst the patients without steroid administration (50/158), hyperglycemia was noted in 88% (44/50) and in 32% (16/50) of patients who had no steroid administration nor prior known status of DM. More than a fourth (28.5%, 51/179) of the CA-ROCM cases were found to have new onset diabetes at presentation. This indicates the possible role of SARS-CoV-2 in causing new onset DM. Patel et al reported newly detected DM in 20.9% among CAM which was higher than that noted in the non-CAM. 34 The ROC analysis in our study was done taking into account the blood sugar levels in all cases and controls. The selected RBS value (178 mg/dl) yielded the best combination of sensitivity (87.3%), specificity (90.3%) as well as the highest likelihood ratio. Above this level, patients were labelled to have hyperglycemia.
DM, hyperglycemia, SARS-CoV-2 and the pathophysiology of mucormycosis are intricately interlinked. The direct damage to pancreatic beta cells by SARS-CoV-2 has been implicated in new onset Diabetes in COVID-19 positive/ recovered patients. 36,37 Hyperglycemia-secondary to steroids, uncontrolled DM or new onset DMencourages growth of fungi. 6,32 The hyperglycemic state and acidic state in diabetic ketoacidosis (DKA) affects the function of macrophages and neutrophils decreasing phagocytosis and iron binding by transferrin. 32 Free iron in circulation is increased in both DKA and COVID-19. 32,38 SARS-CoV-2 attacks hemoglobin and releases the iron from the porphyrins causing iron overload and increasing serum ferritin. 38 Mucorales have siderophore permeases and other mechanisms to acquire iron ions from their environment for their growth. 6 In addition, vascular endothelium is damaged by both mucorales and COVID-19. 6,38 Our study shows that prior COVID-19 vaccination had a protective effect against the development of CA-ROCM. It is now established that COVID-19 vaccination prevents severe disease and death to a significant extent. 39 Vaccination in the control group indirectly prevented development of severe disease, thereby reducing the need for hospitalization, administration of medications such as steroids, IL-6 inhibitors which compromise the body's immune system. This is the first study highlighting the potential role of vaccination in preventing CA-ROCM. Further studies are required to explore its additional role in the innate mucosal immunity and effect on molecular pathways on the pathophysiology of fungal infection.
A large majority of the patients (78%, 131/166) presented with low vision or loss of vision, and the long-term impact of such morbidity on the survivors can be anticipated. Low visual acuity at presentation is in concordance with other series. 13,14,16,[18][19][20][21][22][25][26][27][28] Patients who retained their vision presented significantly earlier in the course of the disease. An earlier identification and earlier therapy will help prevent loss of vision. Intra cranial extension, orbital apex syndrome and frozen globe have been associated with >70% mortality in ROCM. [40][41][42] All cases reported in a series of ROCM with intracranial involvement presented with orbital apex syndrome. 41 We found that involvement of the orbital apex on imaging (OR 6.2, RR 1.89) independently increases the risk of intra-cranial and cavernous sinus spread. Presence of blepharoptosis and ophthalmoplegia on clinical examination, orbital apex involvement on imaging should raise a strong suspicion of intracranial involvement of CA-ROCM.
In the pre-COVID-19 era, common risk factors for mucormycosis included DM, hematological malignancy, solid organ transplant and trauma. Chronic kidney disease, HIV and pulmonary tuberculosis were the other emerging conditions. 21 In contrast, our study highlights the risk factors responsible for the alarming rise of ROCM during the COVID-19 pandemic which include conditions other than just DM and steroid administration.
Limitations of this study include the retrospective nature. The information available only at one time point was documented and analyzed i.e. at the patient's first visit to the Ophthalmic Plastic surgeon. Thus, not all information on all parameters for all patients were available. Only the known comorbidities from the past history of the patients were documented. Elaborate details of sinus and cranial involvement, CA-ROCM treatment aspects and outcome were not included in this study. The strength of the study lies in the multi-city, multicentric case-control design. The direct comparison of development of CA-ROCM in COVID-19 infected/recovered individuals between those who developed ROCM and those who did not throws light on the additional risk factors with COVID-19 being common in both groups. The analysis of the data yields a cut off value of RBS greater than 178 mg/dl predictive of greater risk for developing CA-ROCM.
In conclusion, as India braces in anticipation of the third wave of COVID-19, a surge in COVID-19 associated secondary infections are also expected. Recommendations based on our study for preventing CA-ROCM in future waves include -effective screening and strict control of DM during and after recovery from COVID-19 for up to 2 months. An RBS of more than 178 mg/dl along with early symptoms of ROCM such a as facial or periocular pain at presentation warrants early imaging. Adherence to guidelines for steroid use in COVID-19 treatment and avoiding overuse of steroids, increasing awareness on the symptoms of ROCM among health workers and the general public to encourage early detection and adequate referral and promoting vaccination against COVID-19. Clinicians should be watchful for development of CA-ROCM in patients presenting in the early 5 th and 6 th decade of age with active COVID-19 infection, moderate to severe COVID-19, of male gender and be cognizant of new onset diabetes in COVID-19 patients. The visual acuity is worse in delayed clinical presentation. Ophthalmoplegia, blepharoptosis at first presentation and orbital apex involvement on orbital imaging are ominous signs indicative of intracranial extension of CA-ROCM.

Disclosure statement
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Funding
The author(s) reported there is no funding associated with the work featured in this article.