Rhino-orbital Fungal Invasion in Post COVID-19 Cases at a Tertiary Eye Care Centre in Hyderabad- A Cross-sectional Study

1. Professor, Department of Microbiology, Sarojini Devi Eye Hospital/Osmania Medical College, Hyderabad, Telangana, India. 2. Assistant Professor, Department of Microbiology, Sarojini Devi Eye Hospital/Osmania Medical College, Hyderabad, Telangana, India. 3. Postgraduate Trainee, Department of Microbiology, Sarojini Devi Eye Hospital/Osmania Medical College, Hyderabad, Telangana, India. 4. Postgraduate Trainee, Department of Microbiology, Sarojini Devi Eye Hospital/Osmania Medical College, Hyderabad, Telangana, India.

Abstract

Introduction: In India, especially in Southern part of the country during the second wave of the pandemic there has been a sudden surge of rhino-orbital invasion by various fungi like Fusarium, Mucor, Aspergillus etc., in post Coronavirus Disease-2019 (COVID-19) patients. Several risk factors have been attributed like lowering of cell-mediated immunity by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), lymphopenia and associated reduction in CD4+ (Cluster of Differentiation) and CD8+ cell population, Steroid-induced hyperglycaemia, immunosuppression, and uncontrolled diabetes.

Aim: To detect rhino-orbital fungal conquering post COVID-19 cases.

Materials and Methods: This was a cross-sectional study done at a tertiary eye care centre in Hyderabad, between May to June 2021 and the study subjects were post COVID-19 patients with fungal invasion reporting to Government SD Eye Hospital and Government ENT Hospital Hyderabad, India. The patients were evaluated for associated co-morbidities, location of fungal infection, use of steroids, oxygen therapy usage and microbiologically identified the causative fungi. Descriptive analysis was done and data was presented as numbers and percentages were calculated.

Results: Overall, 470 cases of post COVID-19 suspected fungal invasion cases were referred from the Ophthalmology and Ear, Nose, and Throat (ENT) Departments, of which 287 (61.06%) cases were culture positive for various fungal elements. Fusarium spp. was predominantly seen in 165 (57.49%) cases followed by Aspergillus spp. in 71 (24.74%) cases, Mucor was isolated in 42 (14.64%) cases and other fungi like Candida spp. were isolated in 5 (1.74%) cases and Bipolaris spp. was seen in 4 (1.39%) cases. Pre-existing Diabetes Mellitus (DM) was present in 233 (81.18%) cases, cases, corticosteroid intake for the treatment of COVID-19 was recorded in 203 (70.73%) cases and oxygen therapy was administered in 160 (55.74%) cases.

Conclusion: As per the evaluation especially in post COVID-19 patients there seems to be a nexus between diabetes, imprudent use of steroids, decreased immune status, inappropriate use of oxygen therapy and invasion of fungi, therefore cautious and prudent use of the steroids and oxygen among the COVID-19 treatment protocol is advised.

Aspergillus, Coronavirus disease-2019, Diabetes mellitus, Fusarium, Mucor, Steroid therapy

The disastrous pandemic of the century- COVID-19 has devastated the whole universe with its morbidity and mortality. With the uncertainty in the treatment, several options have been appropriated like use of systemic steroids which remarkably increased the survival rate, but also whose widespread use had a significant toll in causing secondary bacterial or fungal infections (1). In India, with the high prevalence of diabetes which is an alluding risk factor in the causation of fungal invasion (2),(3), other risk factors include underlying co-morbidities in these patients, like, haematological malignancies (i.e., acute myeloid leukaemia) end-stage kidney disease and organ transplant recipients (4),(5),(6). The optimal setting of raised glucose levels, low oxygen levels, high ferritin intake, decreased cell mediated immunity resulting in the failure of the first line of defence mechanisms like opsonisation, phagocytosis and mucociliary clearance and impaired second line defence mechanisms resulting in over expression of anti-inflammatory cytokines, T-helper cells (7) all these unitedly collaborated in the rise of the rhino-orbital mycosis co-infections in COVID-19 patients. Several cases of co-infection in people with COVID-19 with various fungi have recently been reported worldwide (8), but the actual incidence is unspecified and also in India (9) numerous cases of mycosis were reported during the second wave predominantly in post COVID-19 patients with diabetes and who were on steroid therapy, leading to severe complications. Mucormycosis, Fusarium, Aspergillus and Candida have all been reported from India in post COVID-19 patients (10). Hence, the present study was done to detect rhino-orbital fungal conquering post COVID-19 cases.

Material and Methods

This was a descriptive, cross-sectional, microbiological evaluative study of patients with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) confirmed COVID-19, who presented to Government SD Eye Hospital and also those cases referred from Government ENT Hospital Hyderabad, India, between May to June 2021 and diagnosed as rhino-orbital mycosis. This study was approved by Institutional Ethics and Research Board- Ref no: IEC/OMC/2021/Acad-49. Written informed consent was taken from each patient. Patients were diagnosed by distinctive rhino-orbital clinica symptoms like swelling of eye, orbital pain, redness of eye, etc.

Inclusion criteria: All post COVID-19 (RT-PCR) patients with a high index of clinical suspicion of fungal invasion of both sexes presenting to the ophthalmology and ENT clinical department of Eye Hospital and ENT Hospital, were included.

Exclusion criteria: All post COVID-19 (RT-PCR) patients with bacterial invasion and those who did not give consent were excluded.

Sample size calculation: The following formula was used for calculating the adequate sample size in prevalence study n=Z²P(1-P)\d² Where ‘n’ is the sample size, ‘Z’ is the statistic corresponding to level of confidence, ‘P’ is expected prevalence (that can be obtained from same studies or a pilot study conducted by the researchers), and ‘d’ is precision (corresponding to effect size). As per the previous study and our previous laboratory observations (1), the average prevalence rate considered to be 12%.

Study Procedure

Specimens: The specimens that were included for processing are:

1. Biopsy tissue samples- obtained by debridement during Diagnostic Nasal Endoscopy (DNE) and after Functional Endoscopic Sinus Surgery (FESS).
2. Deep nasal swab- which includes palatal swab and after Regurgitation On Pressure over the Lacrimal Sac (ROPLAS) swab.

Sample processing: Samples were collected from all those cases with fungal invasion suspicion and their biopsy material was collected under aseptic precautions in a sterile container containing normal saline. Sterile cotton swabs (HiMedia) were used to collect samples from the deep nasal, palate and for ROPLAS and processed in the Department of Microbiology, Government SD Eye Hospital. Testing of samples included direct Potassium Hydroxide (KOH) mount examination, fungal culture, and finally Lactophenol Cotton Blue (LCB) mount examination. To determine diagnosis, the direct demonstration of fungal elements in the clinical sample is necessary. The microscopic examinations of specimen in KOH (10-20% KOH) wet mount were done to detect characteristic septa, hyphae their branching etc. The size, morphology, and quantity of any fungal elements were also noted. The tissue sample were cut into small pieces and inoculated without crushing in two tubes containing Sabouraud Dextrose Agar (SDA) with antibiotics (with chloramphenicol and gentamicin, without cycloheximide) and on two tubes without antibiotics, with one tube from each set incubated at 37°C and at 22°C. The sample was also inoculated in Brain Heart Infusion (BHI) broth and blood agar and incubated at 37°C. Cultures were examined for growth daily for the first week and twice a week for the subsequent period. The fungal isolates were finally identified by conventional techniques like LPCB mount.

Statistical Analysis

Descriptive analysis was done and data was presented as numbers and percentages were calculated.

Results

Sex ratio: Out of the 470 specimens that were processed, 287 (61.06%) specimens were fungal positive. The proportion of male patients 182 (63.41%) was higher than female patients 105 (36.59%) and the mean age of all patients was 45 years.

Risk factors: Various underlying risk factors and associated co-morbidities were DM, steroid use, in ardent oxygen therapy and lowered immune status state like Human Immunodeficiency Virus (HIV) (Table/Fig 1).

Clinical profile: The (Table/Fig 2) shows distribution of 470 patients with some of the common rhino-orbital clinical symptoms like swelling of eye, orbital pain, redness and facial swelling.

Specimens processing: Out of the 470 specimens that were processed, 287 (61.06%) specimens were fungal positive (Table/Fig 3).

Microbiological tests: On combining all the microbiological investigations (KOH and culture), out of total 470 cases, there was positivity by KOH in 245 (52.12%) cases, and culture in 287 (61.06%) cases. 245 cases were positive for both KOH and culture. All isolates have been identified microbiologically by 10% KOH wet mount, culture on SDA and final diagnosis by morphological evaluation by LPCB mount.

Microbiological profile: Out of 287 culture isolates, Fusarium spp. was the most commonly identified in 165 (57.49%) cases (Table/Fig 4), (Table/Fig 5). Aspergillus spp. The (Table/Fig 4), (Table/Fig 6), (Table/Fig 7), (Table/Fig 8) was found in 71 (24.73%) cases followed by 42 (14.63%) cases of Mucor (Table/Fig 4), (Table/Fig 9), 5 (1.74%) cases of Candida spp. (Table/Fig 4), (Table/Fig 10), 4 (1.39%) cases of Bipolaris spp (Table/Fig 4), (Table/Fig 11).

Discussion

The COVID-19, caused by SARS-CoV-2, is rapidly spreading across the globe causing scads of morbidity and mortality. The aggressive feature of COVID-19 infection as cited in one of the previous study are its complications which subsist as acute respiratory distress syndrome and respiratory failure (83.3%), secondary bacterial and fungal infections (27.3%), acute cardiac injury (9.1%), hypoxic encephalopathy (18.2%), acute kidney injury (18.2%), shock (9.1%), and acute liver injury (9.1%) (11). With the ongoing second wave of COVID-19 pandemic there has been a dramatic increase in the occurrence of invasive fungal co-infections observed worldwide and also in India- there has been an increase in fungal infections, predominantly rhino-orbital mycosis largely as a result of complex nexus of factors like pre-existing diseases which includes uncontrolled diabetes (especially in developing and tropical countries), use of steroid therapy, alteration in immune status as a result of COVID-19 infection causing increased cytokine levels (Interleukin-6 (IL-6), IL-10, and Tumour Necrosis Factor (TNF)-?), lymphopenia (in CD4+ and CD8+ T cells), and decreased Interferon (IFN)-? expression in CD4+ T cells, hospitalisation in Intensive Care Units (ICU), immunocompromised states like HIV and other previous chronic diseases like Koch’s, Chronic Kidney Disease (CKD) etc (9),(12). To evaluate the incidence of fungal co-infections in COVID-19 patients, authors searched various channels unfortunately we could find very few articles reporting on fungal co-infections. In a study conducted by Garg D et al., (PGIMER), Chandigarh, India the median (range) age was 57.5 (22–86) years, with male preponderance and DM was the most common predisposing condition (5). In the present study, the mean age was 45 years and was most common in males (63.41%) as compared to females (36.59%). As previously documented, there is a strong association between poorly controlled DM and rhino-orbital invasive fungal infection as evidenced by the study done by Chakrabarti A et al., who reported an incidence of nearly 50% in patients with uncontrolled DM among the rhino-orbital cerebral fungal invasion (13). In present study, DM was found in 233 (81.18%) patients. In a study done by White PL et al., on a strategy to diagnose COVID-19 associated invasive fungal disease in the Intensive Care Unit (ICU) they could find that 56.1% of the patients were on corticosteroid’s therapy (14). In the present study, also 70.73% of the patients were on steroid therapy which is therefore a significant contributory factor. As regards the oxygen therapy’s role in causing fungal infection in these patients, it is controversial and search did not yield any result, but since some of the patients were on oxygen therapy in non medical voluntary establishments where proper sterilisation might not have been maintained resulting in contamination of the pipes used for oxygen, and the cylinders, therefore there may be a definite risk of fungal invasion. With regards to the studies for the secondary fungal infections in COVID-19 cases, White PL et al., screened about 135 adults with COVID-19 infection and reported an incidence of invasive fungal infections of 26.7%, aspergillosis (14.1%), Candida (12.6%) (14). Chowdhary A et al., from New Delhi, India reported bloodstream Candida infection in 10 critically ill COVID-19 admitted patients (15). In a Chinese study by Chen N et al., on 91 COVID-19 patients they reported five cases of fungal association (one case of Aspergillus flavus, one case of Candida glabrata and three cases of Candida albicans (12). In a German study by Koehler P et al., found COVID-19 associated invasive pulmonary aspergillosis in 5 (26.3%) of 19 critically ill patient (16). There have been several reports from India triggering a rise in COVID-19 associated rhino-orbito-cerebral mucormycosis since March 2020 (17),(18). There are reports of cases of rhino-orbital mucormycosis in COVID-19 disease (7). Sen M et al., reported six cases of COVID-19 disease with rhino-orbital mucormycosi (19). Sarkar S et al., also reported 10 cases of clinically diagnosed orbital mucormycosis with concurrent COVID-19 infection (20).

In this study Fusarium spp. 165 (57.49%) was the most common fungi that have been isolated (Table/Fig 4). The predisposing risk factors for the mycosis progression in Fusarium species was diverse- DM as seen in 82 (49.70%) cases, while 77 (46.67%) had a history of treatment with corticosteroids, HBsAg was positive in 3 (1.82%) cases, CKD was seen in one (0.60%) patient and one (0.60%) of the patients was HIV positive and one (0.60%) was on anti-TB treatment.

The extensive use of steroids and antibiotics as therapy against COVID-19 may lead to the development of rhino-orbital fungal diseases therefore the use of these agents should be monitored to achieve a therapeutic effect at the lowest dose and shortest durations and also the logical benefit of administering steroids in patients with COVID-19 need continuous review to avoid the increasing number of fugal invasions in these patients. Patients with DM and immunocompromised state should also be precisely monitored for fungal infection as they have an added risk of rhino-orbital fungal invasion.

Limitation(s)

While, it is true that there are limitations of conventional diagnostic tests and not all tests are available due to lack of adequate infrastructure in intra hospital laboratories authors had tried to overcome the challenges in present setup with the available microbiological techniques and subjected all the samples first on to KOH mount and cultured in SDA and finally LPCB mount. The final diagnosis was based on the morphology in the KOH mount, cultural characteristics on SDA, and finally as per details revealed in LPCB mount.

Conclusion

A vigilant assess of the risk factors, identify the types of invasive mycosis, judge the strengths and limitations of diagnostic methods and an early effective diagnosis through tissue biopsy and culture, with immediate and aggressive collaborative and individualised treatment, judicious use of antifungal drugs, surgical debridement, which all play a crucial role in reducing the high mortality and morbidity associated with mycosis.

Acknowledgement

Authors would like to acknowledge with deep gratitude the services of all the healthcare providers around the world for their dedication in fighting the COVID-19 pandemic. Authors are thankful to Dr. V Rajalingam, Superintendent, Sarojini Devi Eye Hospital, Dr. P Modini. Head, Department of Ophthalmology, Sarojini Devi Eye Hospital, all residents and staff members of the Department of Ophthalmology and Microbiology, Sarojini Devi Eye Hospital for their care to the patients involved in the study and their help and input on the research project.

Author contributions: BMSV designed and planned the study, analysed the data; wrote the manuscript and supervised the whole study. PGB extracted data, contributed in the processing, under the guidance of BMSV KM and SS performed data collection and processing, and also contributed in preparing the tables and photographs under the guidance of BMSV.

DOI and Others

DOI: 10.7860/JCDR/2022/52209.16003

Date of Submission: Sep 01, 2021
Date of Peer Review: Oct 22, 2021
Date of Acceptance: Jan 03, 2022
Date of Publishing: Feb 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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