A 65 year-old hypertensive and non-diabetic male presented with fever, cough and dyspnoea for 3 days in the Department of Pulmonary Medicine. Physical examination showed the presence of bilateral rhonchi and crepts. RT-PCR assay for COVID-19 (using the Q-Line Molecular ER nCOV-19 RT-PCR kit) was found to be positive (Ct-value 21) on the day of admission. Further, high-resolution computed tomography (HRCT) of lung showed peripheral ground glass opacity along a major fissure in bilateral lung parenchyma, focal areas of subpleural consolidation in the right middle, right lower and left lower lobes, subpleural band opacity in all the lobes (lower lobe>upper lobe) and vascular prominence in the bilateral lower lobe.
A diagnosis of moderate COVID-19 pneumonia with CO-RADS 4, CT severity score 15/25 was made. Among other laboratory tests, viral markers (HIV, HBsAg and HCV) were negative, haemoglobin was 11.4 g dl-1 (13.3-16.2 g dl-1), platelet count was 2.66 lac/cmm (1.65-4.15 lac/cmm), total erythrocyte count was 4.06 million/cmm (4.3-5.6 million/cmm) and total leucocyte count was 18910/cmm (4000-11000/cmm), with raised neutrophil and decreased lymphocyte count. Uric acid was 4.5 mg dl-1 (3.1-7.0 mg dl-1), serum sodium was 129 mmol l-1 (136-146 mmol l-1), serum potassium was 3.9 mmol l-1 (3.5-5.0 mmol l-1) and serum ionized calcium was 5.3 mg dl-1 (4.5-5.3 mg dl-1). Renal function tests were within normal limits, whereas in liver function tests, the SGPT level was 154 U l-1 (7-41 U l-1). C-Reactive protein (CRP) was 37.6 mg l-1 (0.2-3.0 mg l-1), the serum ferritin level was 1102.24 ng ml-1 (29-248 ng ml-1), the IL-6 level was 37.26 pg ml-1 (0-16.4 pg ml-1) and the plasma d-dimer level was 0.88 microg ml-1 (0.22-0.74 microg ml-1).
The patient was put on intravenous steroids (dexamethasone) with supplemental oxygen. Subcutaneous enoxaparin was started along with IV piperacillin-tazobactam and anti-pyretics. During further intensive care unit (ICU) stay, the clinical condition of patient deteriorated from the third day, with laboratory parameters showing raised total leucocyte count (24450/cmm), raised neutrophil count (89%), raised CRP (46 mg l-1), raised serum ferritin (1112.89 ng ml-1), raised LDH (310.3 U l-1), raised SGPT (232 IU l-1) and raised SGOT (56 IU l-1). Random blood sugar was found to be raised (310 mg dl-1), which might have been due to the ongoing IV steroid administration.
With the onset of sepsis and suspicion of COVID-associated mucormycosis, blood culture and sputum culture were ordered for detection of any possible bacterial or fungal aetiology. The blood culture was negative after 5 days of aerobic incubation by the BacT/ALERT automated blood culture system. Sputum culture was also not helpful as only mixed oral commensals were obtained in culture. Ziehl-Neelsen staining of the sputum sample and its culture using the mycobacteria growth indicator tube (MGIT) technique was negative for   Mycobacterium tuberculosis   complex (MTC) and non-tuberculous mycobacteria (NTM). The fungal culture of sputum performed on Sabouraud dextrose agar (SDA) medium showed growth of A. flavus but no anti-fungals were started at this juncture, as the finding was considered to be a laboratory contaminant by the treating physicians.
Because of the deteriorating clinical condition, the patient was switched to meropenem, clindamycin and moxifloxacin, which continued for 2 weeks, with some improvement in the clinical condition. After 2 weeks, the patient again developed cough and severe dyspnoea. Repeat HRCT of the chest showed multiple thick-walled peripheral cavities in bilateral lung parenchyma, raising the possibility of bacterial and/or superadded fungal infection. Further, there was diffuse patchy consolidation in bilateral lower lung lobes and hydropneumothorax in the right pleural space (Fig. 1a).
A repeat sputum culture was performed. This time, the sputum culture, performed on blood and MacConkey agar, revealed pure growth of E. cloacae, which was further confirmed by the VITEK-2 automated identification and sensitivity testing system. The organism was sensitive to amikacin, gentamicin, imipenem, meropenem, cefotaxime, cefepime, aztreonam, piperacillin-tazobactam, cotrimoxazole, ciprofloxacin, levofloxacin and tetracycline, whereas it was resistant to cefazolin, cefoxitin, ampicillin and co-amoxiclav. Accordingly, levofloxacin and cefepime were started. The sputum sample was also sent for GeneXpert analysis for   Mycobacterium tuberculosis   (MTB) and repeat fungal culture. GeneXpert analysis was negative for MTB but fungal culture on SDA medium containing chloramphenicol, incubated at 25  C, revealed the growth of A. flavus on the fourth day of incubation, with a velvety yellow to green colony having a granular texture on the obverse side (Fig. 1b), and a cream-coloured colony on the reverse side. The culture isolate was confirmed phenotypically using the slide culture technique and the lactophenol cotton blue (LPCB) mount. On the LPCB wet mount, the presence of uniform, septate fungal hyphae with parallel walls and branching at acute angles was observed. Rough, pitted and spiny conidiophores with a biseriate arrangement of phialides covering the upper two-thirds or the entire vesicle were observed. (Fig. 1c).
The identification of A. flavus was further confirmed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instrument.
As far as serological tests are concerned, it was only possible to determine the serum galactomannan level by enzyme immunoassay, and this was found to be negative.
Based on the above findings, voriconazole 200 mg every 12 h orally was immediately added to the ongoing therapy. After 7 days of meticulous treatment, the patient showed signs of clinical improvement and was subsequently discharged with advice to continue the same dose of antibiotic and anti-fungal treatment for 2 and 5 more weeks, respectively, with further follow-up.