Central nervous system Aspergillus quadrilineatus infection in a COVID‐19 patient, a case report and literature review

Abstract Background Viral pneumonia such as COVID‐19‐associated aspergillosis could increase susceptibility to fungal super‐infections in critically ill patients. Methods Here we report a pediatric case of Aspergillus quadrilineatus cerebral infection in a recently diagnosed COVID‐19‐positive patient underlying acute lymphocytic leukemia. Morphological, molecular methods, and sequencing were used to identify this emerging species. Results Histopathological examination showed a granulomatous necrotic area containing dichotomously branching septate hyphae indicating a presumptive Aspergillus structure. The species‐level identity of isolate growing on brain biopsy culture was confirmed by PCR sequencing of the β‐tubulin gene as A. quadrilineatus. Using the CLSI M38‐A3 broth microdilution methodology, the in vitro antifungal susceptibility testing demonstrated 0.032 μg/mL MIC for posaconazole, caspofungin, and anidulafungin and 8 μg/mL against amphotericin B. A combination of intravenous liposomal amphotericin B and caspofungin therapy for 8 days did not improve the patient's condition. The patient gradually continued to deteriorate and expired. Conclusions This is the first COVID‐19‐associated cerebral aspergillosis due to A. quadrilineatus in a pediatric patient with acute lymphocytic leukemia. However, comprehensive screening studies are highly recommended to evaluate its frequency and antifungal susceptibility profiles. Before being recommended as first‐line therapy in high‐risk patients, more antifungal susceptibility data are needed.


| INTRODUC TI ON
Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported as one of the highest risk factors for opportunistic fungal infections including disseminated Aspergillus infections. 1 Invasive aspergillosis (IA) is a life-threatening fungal infection associated with high morbidity and mortality in immunocompromised patients, especially in hematopoietic stem cells and solid organ transplant recipients, neutropenic patients, and those receiving intensive chemotherapy. 2In contrast to pulmonary aspergillosis, cerebral Aspergillus infections is a rare clinical demonstration, associated with high mortality and poor outcome despite extensive antifungal therapy and surgical interventions. 1 The incidence of cerebral aspergillosis is not clearly known and seems to be associated with the host's underlying conditions including primary immunodeficiencies. 3The overall prevalence is reported to be less than 7%, in patients with hematologic malignancies, and the frequency might be as high as 20%-40%. 4,5ong genera Aspergillus, A. fumigatus followed by A. flavus are the primary cause of IA in individuals with COVID-19 pulmonary infections; [6][7][8] however, other aspergilli are also documented, such as A. calidoustus, A. sublatus, and A. tubingensis. 9Aspergillus species are one of the most ubiquitously found saprophytic molds in soil and decaying vegetation, with the potential to cause opportunistic disease primarily in patients with defective immune systems and can be divided into 13 clinically important sections. 10The Nidulantes section has been subdivided into eight series, of which there are 25 species in the Nidulantes portion including A. quadrilineatus. 11Overall, members of A. nidulans complex have been associated with chronic granulomatous disease. 10re, we present a case of COVID-19-associated cerebral aspergillosis due to A. quadrilineatus in a pediatric patient with underlying acute lymphocytic leukemia (ALL).

| C A S E PR E S E NTATI O N
An 11-year-old female with no previous medical history was admitted to the emergency department of Children's Medical Center in Tehran, Iran on September 7th, 2021, demonstrating severe intermittent and generalized abdominal pain and non-blood vomiting which prolonged for a week.There was no history of fever, diarrhea, headache, and loss of consciousness.The hematological work-up showed pancytopenia: white blood cell count; 7.88 × 10 The abdominal sonography showed evidence of fatty liver grade I, splenomegaly, and bilateral kidney enlargement.Based on laboratory data and persistent vomiting, patient was diagnosed with acute kidney injury, and tumor lysis syndrome was considered as the first differential diagnosis.After 12 h, the patient underwent hemodialysis, and rasburicase (0.2 mg/kg/day), allopurinol (300 mg/m 2 /day), pack cell transfusion, and platelets transfusion were conducted.On the next day of hospitalization (day 1), a bone marrow (BM) sample was collected and submitted for cytology analysis, which showed a predominant blast population in about 30% of all the analyzed cells; remarkably positive for CD20, CD19, CD22, CD34, and HLA-DR.
However, it was negative for CD3, CD10, CD17, and myeloperoxidase.B-cell acute lymphoblastic leukemia (B ALL) was confirmed.
The first abdominopelvic and chest computed tomography (CT) scan showed mild splenomegaly and a few grand glass opacities in the subpleural region of both lower lobes in favor of COVID-19 infection which was confirmed by positive polymerase chain reaction for SARS-CoV-2.On the second day, the patient was moved to the COVID-19 isolation unit and started receiving antiviral therapy through intravenous remdesivir.The treatment included a loading dose of 5 mg/kg on day 1, followed by 2.5 mg/kg every 24 h for the next 5 days.By day 6, the uric acid level had reduced to 4.5 mg/dL, which led to the discontinuation of rasburicase and allopurinol administration in addition to remdesivir treatment.Throughout the 2 weeks, the patient received supportive care, such as supplemental oxygen, noninvasive ventilation, and fluid and electrolyte support while staying in the COVID-19 ward.
On day 15, her COVID-19 symptoms seemed to be resolved and recommended to initiate induction chemotherapy with Lasparaginase, vincristine, and dexamethasone in the oncology department.Twenty-eight days after the beginning of chemotherapy (day 43), the patient appeared lethargic and unable to speak comprehensive screening studies are highly recommended to evaluate its frequency and antifungal susceptibility profiles.Before being recommended as first-line therapy in high-risk patients, more antifungal susceptibility data are needed.

K E Y W O R D S
acute lymphoblastic leukemia, Aspergillus quadrilineatus, cerebral aspergillosis, COVID-19 coherent sentences.She developed bizarre movements and loss of consciousness, so immediately, she was transferred to the intensive care unit (ICU) and was intubated.The patient was re-examined, and according to fever, petechiae, and purpura on the lower limb, she was suspected of meningococcemia; the blood culture samples were collected.Also, wide-spectrum antibiotics (Clindamycin and Ceftriaxone) and acyclovir were initiated.The blood culture was negative.
However, the brain's magnetic resonance imaging (MRI) revealed two lesions in the right parietal and left frontal lobes (Figure 1).The

| Morphological and molecular identification
The fungal growth was maintained on SDA, and a stock culture was deposited into the Invasive Fungi Research Center (IFRC) culture collection of Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran.Macroscopic and microscopic studies were conducted and tentatively identified as Aspergillus species (Figure 3B,C).Morphological characteristics of Aspergillus quadrilineatus consist of short, columnar and biseriate conidial heads.

| Literature review
A comprehensive search of various databases (PubMed, Web of Science, ScienceDirect, and Scopus) was conducted to find relevant articles about fungal infections caused by A. quadrilineatus.The search was limited to published literature in English up to May 31, 2023.
Several studies also showed that SARS-CoV-2, via a spike protein, binds to angiotensin-converting enzyme 2 receptors expressed on endothelial cells of blood vessels in the brain and epithelial cells of pneumocytes, resulting in neurovascular damage and pulmonary infection, respectively. 1Neurovascular inflammation provides an arbitrary and suitable environment for colonizing Aspergillus species.Following involvement and invasion of the neurovascular system, brain abscesses, vasculitis, thrombosis, granuloma, meningitis, ventriculitis, and cerebritis occur. 16rebral aspergillosis has been primarily reported among children with ALL more frequently than acute myeloid leukemia and chronic myeloid leukemia. 17To the best of our knowledge, this is the first COVID-19-associated cerebral aspergillosis due to A. quadrilineatus in a pediatric patient with acute lymphocytic leukemia.The patient was in the high-risk group because of using corticosteroids, an intravenous catheter, and broad-spectrum antibiotics while undergoing intensive chemotherapy.These situations are essential risk factors for IFI.

| DISCUSS ION
patient underwent left frontal osteoplastic craniotomy on day 44.The brain was edematous, and the lesion had a cortical presentation in some places.The medial side of the left frontal lobe resection was performed, and the brain biopsy sample was sent for microbiological and histopathological examination.On day 44, histopathologic hematoxylin and eosin staining demonstrated septate hyphae with dichotomous branching, resembling molds, which suspected the presence of fungal infections.Fungal culture also becomes positive 2 days after inoculating the Sabouraud dextrose agar plate (day 46), showing growth of filamentous fungi morphologically (Conidiophore, vesicle, and phialide) consistent with Aspergillus species.For the management of the case, antifungal therapy started with a combination of intravenous liposomal amphotericin B (5 mg/kg/ day) and caspofungin (100 mg/IV/day), and concomitant antibacterial therapy (clindamycin, ceftazidime, and vancomycin).During follow-up, a few days later, the Glasgow Coma Scale reached stage III, and there was no clinical improvement.The patient's condition continued to deteriorate and finally expired on day 54 post-admission (Figure2).
mycob ank.org/page/Pairw ise_align ment), and isolate (accession number OP244415) was identified as A. quadrilineatus by showing 100% similarity with the type strains of that species (accession numbers OL625674 and AB248335), which had been initially isolated from environments.In addition, the maximum likelihood method and Tamura-Nei model Tree, created by MEGA-11 from β-tubulin F I G U R E 1 Brain MRI showing two lesions in the right parietal lobe and left frontal lobe (A-D).

COVID- 19 -
positive patients are highly susceptible to opportunistic fungal infection, including invasive candidiasis, aspergillosis, mucormycosis, and pneumocytosis due to overexpression of antiinflammatory cytokines, dysregulation of T-helper cell differentiation, and impaired cell-mediated immune response. 1,6SARS-CoV-2 uses different mechanisms to suppress and compromise the human immune system like reducing T-lymphocytes and increasing interleukins (IL-1 and IL-6) production.
[18][19][20]A.quadrilineatus belongs to the section Timeline of disease progression in the course of COVID-19 pneumonia, acute lymphocytic leukemia, and proven aspergillosis caused by A. quadrilineatus.Demographic characteristics, clinical data, and treatment profiles of reported cases due to Aspergillus quadrilineatus.Acute non-lymphoblastic leukemia; CGD, Chronic granulomatosis disease; ALL, Acute lymphoblastic leukemia; AmB, Amphotericin B; ND, Not determined.
25r study showed that amphotericin B had high MIC against A. quadrilineatus which is similar to previous data reported by Verweij et al.They showed that A. quadrilineatus was less susceptible than A. nidulans against caspofungin (MICs: 0.32 μg/mL), but triazoles had low MICs against both species.25TheearlierreportsF I G U R E 2 TA B L E 1