A case of COVID‐19‐associated mucormycosis due to Lichtheimia ramosa

Abstract Background Mucormycosis is a life‐threatening invasive fungal infection in immunocompromised and COVID‐19 patients. Case Report Here, we report a fatal rhino‐orbito‐cerebral mucormycosis caused by Lichtheimia ramosa, in a 79‐year‐old diabetic female. She was initially admitted to the hospital for COVID‐19 infection and received broad‐spectrum antibiotics and corticosteroids. After 1 month, she was admitted again because of persistent headaches and decreased right eye movement when the computed tomography scan showed mucosal thickening and opacification of paranasal sinuses. Microbiological investigations, including culture and direct microscopy, and histopathological findings confirmed the diagnosis of proven mucormycosis. The isolated causal agent was identified as Lichtheimia ramosa by sequencing the entire ITS region of nuclear ribosomal DNA. Despite surgical debridement and administration of liposomal amphotericin B 5 mg/kg/day, the patient's level of consciousness suddenly deteriorated; she was intubated and mechanically ventilated in the ICU and died on the same day. Conclusion To our knowledge, this is the first worldwide case of COVID‐19‐associated rhino‐orbito‐cerebral mucormycosis due to Lichtheimia ramosa.


| INTRODUC TI ON
Mucormycosis is a rare opportunistic fungal infection that mainly affects individuals with predisposing conditions such as uncontrolled diabetes mellitus, neutropenia, hematological malignancies, receiving steroids, and organ transplantation. 1, 2 The sinuses, brain, and lungs are the primary sites affected by mucormycosis. The major clinical forms of the infection are rhino-orbito-cerebral and pulmonary and rarely gastrointestinal, cutaneous, and disseminated. 3 The unprecedented mucormycosis outbreaks in India and some other parts of the world have highlighted COVID-19 as a significant risk factor 4 ; however, COVID-19-associated mucormycosis (CAM) or post-COVID-19 infections have been reported from almost all over the world. The most clinical manifestation of mucormycosis in COVID-19 patients has been reported as rhinosinusitis and rhino-orbito-cerebral forms. 2 The intertwined relationship between COVID-19 and mucormycosis worsens infection extension and mortality rates. Although diabetes has been the most frequent risk factor for mucormycosis before the COVID-19 outbreak, in diabetic COVID-19 patients and even nondiabetic ones, long-or even shortterm use of corticosteroids has often been associated with the deadly upsurge of mucormycosis. [4][5][6] Mucormycosis is an invasive fungal infection caused by the members of the order Mucorales. Rhizopus oryzae is the most common causative agent responsible for nearly 60% of infections globally, followed by the other Rhizopus species, Mucor species, and Lichtheimia (formerly Absidia, Mycocladus) species. Rhizopus species are mostly recorded in patients with diabetes mellitus, while Lichtheimia infections were primarily associated with hematological malignancies and presented as severe cutaneous and pulmonary infections. 6,7 Here, we report a post-COVID-19 rhino-orbito-cerebral mucormycosis in a 79-year-old diabetic woman. As far as we know, this is the first case of rhino-orbito-cerebral CAM caused by Lichtheimia ramosa worldwide, which could be an emerging Mucoralean fungus.

| C A S E REP ORT
On August 19, 2021, a 79-year-old female with a history of diabetes mellitus, ischemic heart disease, and hypertension was hospitalized with a possible diagnosis of COVID-19 in Shahid Beheshti Hospital, Kashan, Iran. Physical examination of her head and neck, chest, heart, abdomen, and limbs showed no abnormal findings.
Nasopharyngeal and oropharyngeal swab samples became positive in real-time reverse transcription polymerase chain reaction (rt-RT-PCR) test performed by Light Cycler 96 system (Roche) to detect SARS-CoV-2 targeting N and RdRp genes (Pishtaz Teb). 8 The patient was managed with antibiotics for suspected bacterial superinfection, and with corticosteroids, including prednisolone (125 mg/day) and dexamethasone (8 mg intravenously/daily) for a week until PCR for SARS-CoV-2 was negative.
The patient was admitted again to the hospital 1 month later, pre- Slide culture and staining with aniline blue revealed the microscopic morphology suggestive of Lichtheimia species ( Figure 2E). DNA of the colony was extracted using glass bead extraction followed by phenol-chloroform purification method as described previously, 9 the entire ITS region of nuclear ribosomal DNA was PCR-amplified using the universal primer pairs ITS1 and ITS4, 10

| DISCUSS ION
Mucormycosis is one of the most severe infections due to its rapid insidious onset, fast progression, and extremely high mortality. 11 Before the beginning of COVID-19, the overall mortality rate of mucormycosis was estimated to be 54%, and the disseminated form was about 96% fatal. 12 The frequency of Lichtheimia infections in Europe is significantly higher than in the USA and Asia. 19 Overall, the epidemiology of mucormycosis has shown different patterns worldwide. 20 According to morpho/physiological and molecular data, Lichtheimia contains at least five species: L. corymbifera, L. ramosa, L. ornata, L. hyalospora, and L. sphaerocystis, which are isolated from various body sites including the respiratory tract, paranasal sinuses, brain, heart, blood, and wound. Lichtheimia ramosa (syn. L. hongkongensis, formerly Absidia idahoensis) is the most common pathogenic Lichtheimia species, currently regarded as an emerging pathogen associated with pulmonary and cutaneous infections 18 and has been increased from 5% to 19% in the last decade. 21 It is reported in a variety of patients with different underlying diseases. 22 As they are very similar in morphology, a recent study has claimed that a significant proportion of L. corymbifera isolated from human infections is indeed L. ramosa. 23 L. ramosa has most often been isolated from cases of cutaneous and pulmonary mucormycosis 24  Accurate species identification of human pathogenic Mucorales is challenging, mainly due to the frequent lack of diagnostic morphological features, particularly the fact that germination and production of fungal spores are highly dependent on culture media and incubation conditions. The recent guidelines published by the Clinical and Laboratory Standards Institute recommend using ITS sequencing as the best method for identifying species within the Mucorales. 27 Thus, we used this strategy to identify the Mucorales we isolated from the patients.
Like other mucormycosis, the treatment of L. ramosa infection involves the combination of antifungal treatment by amphotericin B and posaconazole as well as surgical debridement. The MICs for amphotericin B were significantly lower for L. ramosa than L. corymbifera. 28

| CON CLUS ION
This is the first case of rhino-orbito-cerebral CAM due to Lichtheimia ramosa. As a fungal coinfection in the COVID-19 pandemic, mucormycosis is one of the most alarming concerns; therefore, clinicians should use corticosteroids for patients wisely, adopt early and precise diagnosis of cases, check glycemic levels, and apply timely treatment or/and surgical operations.

ACK N OWLED G M ENTS
This work is supported by Isfahan University of Medical Sciences, Isfahan, Iran (Grant number 1400180), which we gratefully acknowledge. Also, the authors thank the staff at Shahid Beheshti Hospital, Kashan, Iran.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are openly available in