Dual Fungal Infection of Aspergillosis and Mucormycosis in a COvID-19 Patient: A Rare Case Report

Coronavirus disease 2019 (COvID-19) infections can be related to vast spectrum of co-existent bacterial and fungal infections. A 49-year-old diabetic male was admitted with a history of fever, cough and breathlessness since 5 days. He developed persistent headache with right sided purulent nasal discharge. Relevant histo-pathological, biochemical, microbiological and imaging studies were performed which proved it to be a dual infection of Aspergillosis and Mucormycosis. We present one such case in a COvID-19 patient to highlight its unusual clinical features along with the diagnostic and therapeutic challenges


INTRODuCTION
The (COVID-19) infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be interconnected with vast spectrum of disease patterns which ranges from Rhino-Orbital Cerebral Mucormycosis (ROCM) to life-threatening pneumonia along with superadded co-existent bacterial and fungal infections.These co-infections can be due to a prior co-morbid condition.Most common comorbidity associated with co-existent infections is Diabetes Mellitus (dM) and india has higher frequency of Type ii DM (8.9% of adults, that is around 77 million patients, which is a major risk factor for COVID-19 co-infections. 1

CASE REPORT
A 49-year-old male was admitted to our Hospital, with complaints of fever, cough and breathlessness since five days.He developed right sided headache which was gradually progressive, dull type of headache radiating to eyes, cheeks and forehead which occurred throughout the day.There was also history of right sided nasal discharge which was purulent, copious, non blood

DISCuSSION
Mucormycosis is universally known as black fungus with prevalence rate of 0.14 cases per one thousand of Indian population. 2 Mucormycosis is fatal deep fungal infection caused by the family Mucoraceae.There are six clinical forms of Mucormycosis, most common is the (ROCM) (44-48%) followed by the cutaneous type (10-19%), pulmonary (10-11%), disseminated (6-10%) and lastly the intestinal type (2-11%).Our case belongs to the ROCM form. 3Aspergillosis can manifest in two forms: Invasive Aspergillosis and pulmonary Aspergillosis. 4 Aspergillosis and Mucormycosis co-infection frequently occurs in immune-compromised individuals.Invasive Aspergillosis of ROCM form can extend to adjacent areas and present as yellowish to black necrotic ulcers. 5Some of the frequent factors implicated for development of fungal infection in COVID-19 are listed as follows: hypoxemia, hyperglycemia either due to DM or due to steroids, increased iron levels secondary to elevated ferritin levels, reduced phagocytic activity of WBC's (white blood cells) attributed to immunosuppressed state caused by virus or steroid therapy. 6[Refer Figure 7] COVID-19 infected patients commonly presents with hypoxemia which clinically present as breathlessness and cyanosis. 7DM) is one of the entrenched comorbid conditions of COVID-19 and it is noted in 10% of admitted patients.Diabetes increases the severity of COVID-19 infection and uncontrolled hyperglycemia can lead to increased   patients is still debatable.Deleterious effects of steroid therapy are supposed to be due to lack in management of steroid-induced hyperglycemia as steroids negates the immune-modulatory effect of steroids. 120][11] Diagnosis of dual infection presents as a challenging scenario.Pre-requisite for diagnosing fungal infection includes: high clinical suspicion of the disease, clinically patient presents with sinus pain, proptosis, peri-orbital swelling and all these symptoms are considered to be "red flag" symptoms.So, along with high index of clinical suspicion, judicious utilization of imaging techniques (CT, MRI) coupled with diagnostic modalities such as histopathology examination (HPE), microbiological and biochemical assessment which can help in early and accurate diagnosis. 13PE of Mucormycosis: demonstrates aspetate, irregular hyphae with branching angle of 90°.Characteristic finding is "tissue necrosis" which differentiates it from Aspergillus.The other differentiating points in favour of Aspergillus includes thinner hyphae, regular septations and 45° degree angle seen in Aspergillosis but HPE has certain limitation in differentiating Aspergillus and Mucormycosis. 14Fungal hyphae are noted only in 50% cases of HPE as hyphae are frangible in nature and can be damaged during manipulation of tissue so excessive tissue homogenization should be avoided.Mucorales grow rapidly on fungal culture media (SDA) incubated at temperature of 25°C to 30°C.Microaerophilic environment can augment the yield of fungus on culture media. 15,16Invasive Aspergillosis (IA) in Covid-19 remains a diagnostic challenge because of multiple factors.HPE using special stains such as: Periodic Acid Schiff (PAS) and Grocott-Gromori's methenamine-silver stains (GMS) can be also equally effective in demonstrating Aspergillus specimen, but it is difficult to conclusively rule out other filamentous fungi such as Fusarium specimen etc. Hence a more sensitive diagnostic method may be required for a conclusive opinion and thereby requiring the following interventions a) Direct microscopy using optical brightener methods (Calcoflour or Blankophor) to increase the diagnostic sensitivity and specificity b) Molecular assays targeting ribosomal DNA (rDNA) can also be utilized for definitive diagnosis.Further similar studies are required to standardize the laboratory procedure. 17Molecular based diagnostic methods: PCR (Polymerase chain reaction), RFLP (Restriction fragment length polymorphism) can also be utilized for detection of Mucorales species targeting mainly 18srRNA gene.The considerable drawback for molecular methods remains less number of subjects studied for the efficacy of molecular methods. 180][21][22][23][24] our case was a male diabetic suffering with severe COVID-19 disease, admitted in ICU care and was on parenteral steroids.0][21][22][23][24] Garg et al. 19 showed similar observation with an elderly male patient having combordities including Hypertension and Diabetes.Johnson et al. 23 similar observations were also noted as compared to the current study as it presented with 76 year old male being affected with Diabetes and coexistent infection of Aspergillus and Mucormycosis which was demonstrated by BAL examination (Bronchoalveolar lavage).Bellanger et al. 24 also identical findings with 55 year old male with coexistent infection of Aspergillus and Mucormycosis which was demonstrated on BAL and tracheal aspirate.

Limitations
The limitation of present study is that limited reporting of such cases and not many dual fungal infection cases are published in English literature.However, with the increasing number of such cases it is essential to provide satisfactory explanation for the increase in fungal co-infections in COVID-19 infected patients for providing better health care and decrease associated mortalities and morbidities.

CONCLuSION
Our case report highlights the fact that a high index of clinical suspicion, proper assessment of risk factors, diagnostic methods, advantages and disadvantages of various diagnostic methods and clinical settings in COVID-19 subjects needs to be followed for immediate management in order to significantly reduce the morbidity and mortality occurring from these fungal co-infections.
India has faced the first wave of Covid-19 pandemic on 11 th March 2020 and second wave of COVID-19 pandemic on 1 st March 2021 and is currently undergoing third wave of COVID-19 pandemic.During this pandemic several cases of ROCM were noted.However, reports of combined dual infection of Mucormycosis and Aspergillosis are very rare and the exact incidence of such dual infection remains unknown.Hence current study highlights a case of combined Mucormycosis and Aspergillosis in a Covid-19 patient for its rare occurrence.
Figure 1a.Sabourauds dextrose agar culture (SDA): demonstrates clear cotton wool appearance suggestive of Mucormycosis Figure 1b.SDA culture: demonstrates Salt and pepper like growth suggestive of Aspergillus
hospitalization in COVID-19 positive patients and simultaneously increasing the mortality rate of these patients. 8Various factors are attributed for increased mortality among COVID-19 patients with uncontrolled DM include: glycosylation of complement proteins, impaired binding of Oligosaccharides (C-type leptin), decreased Tumor necrosis factor alpha (TNF-a), Interleukin 10 (IL-10), and Interferon gamma(IFN-γ). 9-11Hyperglycemia doubles the risk of co-infection in COVID-19 subjects.Role of Steroid therapy in COVID-19

Figure 7 .
Figure 7. Various risk factors implicated for combined fungal infection in COVID-19 patients