Spectrum of fungal infections in COVID patients: The double whammy!

Dear Editor,

Recently a sudden surge of fungal infections has been alarmingly noted in cases of COVID-19 pneumonia admitted at our centre. A spike was seen in samples received at the Mycology section of Microbiology department for detection of fungal elements. The samples received were those sent from the COVID ICU and were primarily from patients readmitted after having recovered recently from COVID. Similar reports of a surge in fungal coinfections in COVID patients were being received from other centres in the country during the same time., The spectrum of fungal infections seen at our centre in a cluster of cases is highlighted in this communication to bring to notice the alarming increase in fungal infections occurring in this pandemic.

Twenty samples were received (repeat samples not included) from previously diagnosed COVID -19 reverse transcriptase-polymerase chain reaction (RT-PCR) positive patients. The investigation forms accompanying these samples had few common clinical features mentioned. All these patients were diabetic, post COVID (RT PCR confirmed) and had been treated at multiple facilities for COVID before being admitted in this hospital. Majority had red flag signs and complaints of headache or nasal secretions/discharge. Samples received included nasal scrapings, pus discharge, biopsy tissue following debridement and nasal swabs obtained during diagnostic nasal endoscopy. All necessary biosafety precautions were taken into consideration while processing the samples.

A direct mount to look for fungal elements was prepared with 10% Potassium hydroxide (KOH) quickly within minutes of receiving the sample. The treating surgeon was informed immediately if fungal hyphae were seen on direct microscopy. These, if appearing broad, without septae and with wide angle branching presumptively could be Mucorales (Fig. 1). Thin septate hyphae with acute angle branching would presumptively point towards Aspergillus. The biopsy tissue was cut into small pieces with a sterile surgical blade with due precaution not to mince it too fine to prevent fungal hyphae from getting disrupted. Direct mount revealed inflammatory cells and fungal hyphae invading the tissue (Fig. 2). For culture, the tissue bits were inoculated onto Sabourauds Dextrose Agar (SDA) and incubated at 37 °C. Microbiological diagnosis of fungal etiology was proven on culture in ten samples. Six of them were identified as Rhizopus, one as Mucor and the other three as Aspergillus. Further speciation of Aspergillus, carried out on the basis of slide-culture as Aspergillus. niger, Aspergillus. flavus, and Aspergillus. terreus.

Fig. 1

Direct wet mount from nasal scraping showing unstained broad aseptate hyphae.

Fig. 2

Direct wet mount from nasal scraping showing unstained fungal hyphae with numerous inflammatory cells in the background.

In our experience, direct microscopy was useful for rapidly screening samples suspected to be having fungal elements. In addition to requiring some degree of expertise, direct microscopy is not useful in identifying the fungus up to the genus or species level. The further identification was done on culture when growth started appearing on SDA slants within 24–48 h. The growth was woolly, cottony, whitish grey in colour (Fig. 3). Growth on other slants was a black growth identified as Aspergillus niger, a yellowish powdery growth identified as Aspergillus. flavus and brownish velvety growth as Aspergillus. terreus. The slide culture technique helped to speciate the fungal growths by displaying the morphology on lactophenol cotton blue (LCB) mounts (Fig. 4, Fig. 5). Majority of the fungi belonged to order Mucorales.

Fig. 3

Sabourauds Dextrose Agar (SDA) slant with woolly, cottony growth in 24 h of Mucor.

Fig. 4

Lactophenol cotton blue mount from slide culture showing fungal hyphae with typical umbrella-like collapsed columella suggestive of Rhizopus.

Fig. 5

Lactophenol cotton blue mount of slide culture growth showing fungal hyphae and sporangiophore bearing sporangium.

Culture of specimens is essential for the diagnosis of mucormycosis as it allows accurate identification and antifungal susceptibility testing. Mucorales are thermotolerant and grow rapidly at 37 °C on any carbohydrate containing media, colonies appearing within 24–48 h. These are identified on microscopic and colonial morphology and optimal temperature supporting growth. There are some important precautions to be taken for an optimal yield; these include proper sampling and handling of the specimens. A major concern about culture is its low sensitivity, cultures may not yield any growth and may be falsely negative in up to 50% of mucormycosis cases. This can be attributed to a number of reasons, such as grinding or homogenization of tissue specimens, which may destroy the delicate hyphae of mucormycetes, dry swabs affecting viability of fungi, recent or ongoing antifungal therapy, or even a lack of expertise. Therefore, upon suspicion of a case, good communication and close collaboration between clinicians and the microbiology laboratory is necessary.

A positive fungal culture from a sterile site confirms the diagnosis, while a positive culture from a non-sterile site should keep the possibility of a contaminant and thus both the clinical and radiological data should be reviewed to establish the diagnosis. Histopathology helps to eliminate false positive results and provides a tissue diagnosis. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) identification of cultured Mucorales is a promising method in “difficult-to-characterize growths”.

Direct microscopy of fresh material is an inexpensive, yet invaluable method to rapidly give a presumptive diagnosis which can help in a prompt therapeutic intervention. It is recommended, along with histopathology, by experts of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium (ECMM/MSG ERC). Communication between the lab and the clinician with proper sampling and transport are key to accurate and immediate diagnosis.

Disclosure of competing interest

The authors have none to declare.