Aspergillus flavus endocarditis and meningitis in a child with marfan syndrome

Background and Purpose: Aspergillus species are implicated as the etiology of approximately 26% of endocarditis cases. Central nervous system aspergillosis is a life-threatening condition that has a mortality rate of 80%. Case report: Herein, we report a four– year- old female who was admitted to the pediatric infectious ward due to a fever of unknown origin in January 2020. She was a known case of Marfan syndrome with a family history of this syndrome in her mother. The species was identified using (PCR) and the antifungal susceptibility test was performed using four antifungal agents based on the Clinical and Laboratory Standards Institute M38 3rd edition. Fluconazole-resistant Aspergillus flavus was identified to be responsible for endocarditis and meningitis as well as fever of unknown origin. Conclusion: The clinicians should be aware and consider fungal endocarditis in blood culture-negative endocarditis even in patients with no significant risk factor when antibiotic therapy fails.


Introduction
ncidence of invasive fungal infections has progressively increased over the past few decades [1]. Invasive aspergillosis (IA) is associated with the highest rate of morbidity and mortality in the immunocompromised population [2]. Endocarditis and meningitis secondary to systemic Aspergillus infection constitute the severe manifestations of IA which bear a poor prognosis. Fungal endocarditis results in echogenic large bulky valvular mass and may present with acute left ventricular failure and dyspnea [3].
Nearly 5% of the reported fungal endocarditis belong to the pediatric age group [4]. Aspergillus species (spp.) are the etiology of approximately 21% of Aspergillus endocarditis cases in neonates [5]. Aspergillus fumigatus is known as the most common etiologic agent (8%), followed by Aspergillus flavus (2%), Aspergillus niger (2%), and Aspergillus terreus (1%) [5]. The aortic valve is the most common site of cardiac involvement (e.g., the aortic ring abscesses); however, the mitral valve (MV) involvement is rare. The survival rate depends on the underlying diseases, site of involvement, type of organism, and treatment [6]. The central nervous system (CNS) aspergillosis is a life-threatening condition that has amortality rate of more than 80% [7].However, little is known about this serious infection in the pediatric age group [8].The CNS-invasive aspergillosis might be manifested with meningitis, cerebritis, abscesses, granulomas, or mycotic aneurysms [9]. Moreover, it is mainly caused by A. fumigatus and A. flavus and rarely by A. terreus, Aspergillus oryzae, Aspergillus granulosus, and Aspergillus candidus [10,11]. Delayed or missed diagnosis, long duration of symptoms before hospitalization, as well as extra cardiac and CNS manifestations usually lead to death [2].
Herein, the authors report the case of a four-yearold female who was admitted to the pediatric infectious ward with a fever of unknown origin in January 2020.

Case report
A four-year-old female patient was admitted to the pediatric infectious ward due to a fever of unknown origin in January 2020. She was a known case of Marfan syndrome with a family history of this syndrome in her mother. Both of them had a history of anterior lens dislocation that led to surgery. She also had a history of congenital heart disease, including atrial septal defect type 2 (ASD2), mitral regurgitation (MR), and MV prolapse regarding fever, headache, and meningeal signs a lumbar puncture was performed. She was treated for probable bacterial meningitis since the result of her cerebrospinal fluid (CSF) test was positive and active; moreover, the white blood cell was observed in CSF fluid.
After five days and secondary to the changing of heart murmur intensity that revealed progressive severe MR, she was referred to a tertiary referral center for the evaluation of cardiac involvement. Transthoracic echocardiography (TTE) revealed a large dense heterogeneous and oscillating mass (18 mm×12 mm in size) attached to the atrial septum closed to the hinge point of the anterior leaflet of MV with independent movement towards the heart valve (an argument for the fungal endocardial vegetation) (Figures 1,2,3).
Results of color Doppler flow imaging revealed an abnormal regurgitation flow at the areas of A3 anterior leaflet of mitral suggestive of perforation of MV. In addition, valvular destruction, perforation of the anterior leaflet at the site of the lesion, as well as the existence of perivalvular infection and severe eccentric MR reduced left ventricular ejection fraction. Subsystemic pulmonary hypertension was found with mild pericardial effusion due to acute heart failure, previous history of congenital heart disease, and ASD2.
Neurologic physical examination for the evaluation of meningeal involvement contains clinical examination and paraclinical data. The cerebrospinal fluid analysis showed meningeal irritation and brain magnetic resonance imaging revealed a small subcortical focus of involvement probably due to post-endocarditis embolic lesions.
Infective endocarditis (IE) was diagnosed according to the DUKE criteria. Regarding the clinical and paraclinical criteria, one major criterion and two minor criteria were observed. Persistent fever and the presence of refractory heart failure due to severe acute The results of the present study showed in vitro resistance to fluconazole (minimum inhibitory concentration> 16 µg/mL). The TTE in the outpatient clinic after two weeks, as well as one, three, and six months after discharge showed no recurrence except mild eccentric non-significant MR. Tables 1 summarize the  The peripheral blood sample was taken and inoculated into an anaerobic blood culture medium (manufactured in BC, USA) at 30ºC for five days in an automated blood culture system (manufactured in BC, USA); at 30ºC for five days; however, no growth was observed. The resected vegetation was inoculated into formalin and normal saline solution. The pathology result revealed a fibrohyalinized tissue with an area of fibrinoleukocytic exudate, mixed inflammatory cell infiltration, and neovascularization consistent with  Initially, the biopsy specimen of the MV was taken and a sub-culture was made on Sabouraud Dextrose Agar (SDA) (manufactured in Merck, Germany) at 35ºCfor seven days. After seven days, the colonies of Aspergillus spp. were grown in the SDA medium. The PCR for the amplification of the β-tubulin gene was conducted to identify the species precisely. Genomic DNA was obtained from the colonies cultured on the Czapek's Agar (manufactured in Merck, Germany) forseven days at 35ºC using the genomic DNA extraction kit (manufactured in Roche Life Science, Germany).
In this study, the amplification of the β-tubulin gene was performed using the β-tubulin forward and βtubulin reverse primers [12]. The PCR amplicon was subjected to sequence using the same primers. Subsequently, the sequences were compared with reference data available from the Gene Bank database using the BLAST sequence search tool (http://www.ncbi.nlm.nih.gov/BLAST).
The antifungal susceptibility test was performed by the broth microdilution methodusing four antifungal agents, including voriconazole, itraconazole, fluconazole, and amphotericin B (manufactured in Sigma-Aldrich, USA). Based on the Clinical and Laboratory Standards Institute (CLSI) M38 3rd ed [13], Candida parapsilosis (ATCC 22019) was chosen as a quality control strain in every run.
The culture on SDA revealed green and powdery surface colonies. The microscopic examination of the colonies was compatible with A.flavus. The sequencing result was interpreted and deposited in the GenBank under accession no: MW195498.
The patient was first treated with vancomycin and ceftriaxone which was later switched to the combination of meropenem, ciprofloxacin, and vancomycin when the fever continued and the clinical status deteriorated. The patient was treated with liposomal amphotericin B regarding the result of cultures and the diagnosis of IA. She was discharged after four weeks of treatment with IV amphotericin B. It is worth mentioning that the antifungal treatment was continued with oral voriconazole for an additional four weeks after the performance of the susceptibility test.
This case report was performed in compliance with the Declaration of Helsinki and written informed consent was obtained from the legal guardians of the patient. The details will be included in the manuscript for publication.

Discussion
The IA typically advances subsequent to long and severe neutropenia which commonly affects the lungs and rarely influences paranasal sinuses, CNS, skin, and soft tissues. Medical signs may be nonspecific (i.e., long-lasting fever in acute neutropenia not treated with spectrum antibiotic therapy). Despite the significance of this infection, there are limited data on the epidemiology, value of diagnostic tests, and therapeutic approaches in the pediatric population when the heart and CNS are involved.
Early and accurate diagnosis of IA affects the therapeutic strategies, which are mainly derived from clinical studies on adults. When fungal meningitis is suspected, the choice of the antifungal drug should be restricted to agents that sufficiently pass the bloodbrain-barrier, such as the lipid formulations of amphotericin B (e.g., amphotericin B lipid complex and liposomal amphotericin B) or the broad-spectrum triazole voriconazole [14,15].
Given the results of a randomized controlled trial on adults, voriconazole is a superior choice for the treatment of IA, including cardiac and CNS infections [16]. No pediatric randomized trial has assessed the outcome of cardiac and CNS infections treated with voriconazole. Some studies have reported the successful outcome of IA management with voriconazole in the pediatric age group [17]. Our patient was successfully treated with combination Curr Med Mycol, 2020, 6(4): 70-74 73

Conclusion
According to the results of this case report, the optimization of both diagnosis and treatment leads to a better outcome of IA in children. Based on the findings of this study and the aforementioned studies worldwide, after obtaining approved antifungal test results, fluconazole can be regarded as a suitable firstline choice for the treatment of IA.