Aspergillus tubingensis Endocarditis: A Case Report and Review of the Literature

Aspergillus endocarditis is a rare infection that may affect immunocompetent patients following heart valve replacement or heart surgery. We report the case of a 39 year old woman with a history of intravenous drug use who developed endocarditis with direct examination of the resected valve and vegetation showing the presence of mycelia. Cultures were positive for an Aspergillus of section Nigri, which was subsequently identified as Aspergillus tubingensis by sequencing. The clinical course was favorable following surgery and prolonged antifungal therapy (8 months in total). Antifungal susceptibility testing showed good in vitro activity of amphotericin B, voriconazole and echinocandins against planktonic cells of this A. tubingensis isolate. However, only amphotericin B displayed significant activity against biofilms. In vitro combinations of voriconazole or amphotericin B with echinocandins did not meet the criteria of synergism. Our review of the literature identified 17 other cases of endocarditis attributed to Aspergillus of section Nigri with an overall mortality rate of 57% (100% in the absence of surgery). Endocarditis caused by Aspergillus niger and related cryptic species are rare events, for which surgical management appears to be crucial for outcome. While amphotericin B was the only antifungal drug displaying significant anti-biofilm activity, the type and duration of antifungal therapy remain to be determined.


Background
Aspergillus spp. are opportunistic mold pathogens causing invasive aspergillosis in patients with severe immunosuppression, such as hematologic cancer patients or transplant recipients [1]. While the lung represents the main port of entry, primary extrapulmonary infections are occasionally observed. Aspergillus endocarditis is a very rare entity, which has been reported not only in immunocompromised patients, but also in immunocompetent individuals with a history of valve replacement, open heart surgery, or intravenous drug use [2][3][4]. In this setting, direct inoculation of the fungus in blood may occur via contaminated material. Aspergillus endocarditis is notoriously difficult to treat in the absence of evidence-based recommendations regarding the type and duration of antifungal therapy [3]. Valve replacement is considered to be mandatory [3]. The mortality rate is very high and there is an important risk of relapsing infection among survivors, which usually poses the indication for prolonged suppressive therapy [3].
We report here a case of A. tubingensis prosthetic valve endocarditis in an apparently immunocompetent patient known for intravenous drug use, which was successfully treated with surgery and combined antifungal therapy. This work was completed by a review of the literature of Aspergillus section Nigri endocarditis and by in vitro experiments of anti-planktonic and anti-biofilm activity of the different antifungal drugs against the isolated pathogen.

Case Presentation
A 39 year-old woman known for active intravenous drug use (mainly cocaine) and a biological aortic valve replacement for Enterococcus faecalis endocarditis more than 10 years ago was admitted to the emergency room for altered level of consciousness and septic shock. Blood cultures drawn at admission were positive for Proteus mirabilis (all four bottles from two distinct pairs). Her hemodynamic condition rapidly improved after initiation of broad-spectrum antibiotic therapy and subsequent switch for ceftriaxone and addition of gentamicin after identification of the bacterial blood pathogen. A transoesphageal cardiac ultrasound revealed an 11 mm motile element on the right coronary leaflet of the aortic valve ( Fig. 1, panel A). The patient underwent biological replacement of the aortic valve. A piece of the resected valvular tissue and vegetation was sent to the laboratory of microbiology. Direct examination did not reveal the presence of bacteria at gram staining, but thin septate and branched mycelial elements consistent with an Aspergillus spp. were visualized at silver staining ( Fig. 1, panel B). Conventional cultures of the resected valve did not reveal the presence of bacteria, but a filamentous fungus grew on all plates after 24 h of incubation, which was subsequently identified as an Aspergillus of section Nigri. The direct panfungal PCR (targeting the 18S rDNA) was positive for an Aspergillus of section Nigri. Of note, the direct eubacterial PCR (targeting the 16S rDNA) was also positive for P. mirabilis. Another piece of the cardiac valve was also sent for histopathological examination, which showed fibrino-leucocytic debris without mycelial elements at Grocott staining.
The galactomannan and 1,3-beta-D-glucan were positive in serum (optical density 2.34 and [ 500 pg/ ml, respectively). The diagnostic work-up was completed by a cerebral and thoraco-abdominal CT-scan that did not show evidence of secondary foci of the infection. A diagnosis of mixed bacterial (P. mirabilis) and fungal (A. niger) endocarditis secondary to intravenous drug injection was established. Intravenous liposomal amphotericin B (L-AMB, 5 mg/ kg/day) was started with addition of caspofungin (70 mg loading dose, followed by 50 mg/day from day 2). Three days later, L-AMB was substituted for intravenous voriconazole (6 mg/kg bid on day 1, followed by 4 mg/kg bid from day 2) and gentamicin was interrupted due to the development of acute renal failure. The clinical and biological course was favorable with serum galactomannan rapidly turning negative and a decline of serum 1,3-beta-D-glucan. Documentation of the clearance of the P. mirabilis bacteremia was obtained at day 5 from the start of antibacterial therapy and the patient was treated with ceftriaxone for a total of 6 weeks after surgery. The patient was discharged at post-operative day 10 and was followed at the outpatient clinic for monthly clinical evaluation and laboratory tests (hepatic tests, creatinine, complete blood count). Voriconazole was switched from intravenous to oral administration after 7 days and combination therapy of voriconazole and caspofungin was continued for a total of 14 days. The trough concentration of voriconazole measured at that time (day 14) was 4.5 mg/l. Voriconazole monotherapy was continued for a total of 47 days and was then substituted for oral isavuconazole (200 mg tid on days 1 and 2, followed by 200 mg qd) because of abnormal hepatic tests. The PET-CT performed 6 months after surgery showed no signs of recurrent infection. Isavuconazole was stopped after a total of 8 months of antifungal therapy because of shortage of the drug and multiple interactions with the patient's psychotropic medications (risperidone, pregabalin, clorazepate, trazodone) precluding administration of other azole drugs. About 6 months later, the patient developed a novel episode of endocarditis, which was suspected to be recurrent Aspergillus endocarditis, despite a negative galactomannan in serum. However, microbiological analyses of the resected valve established the diagnosis of Saccharomyces cerevisiae endocarditis without any evidence of recurrent Aspergillus infection [31].

Methods
The mold recovered by culture of the infected cardiac valve and identified as an Aspergillus of section Nigri by morphological examination was further ranching hyphae consistent with an Aspergillus spp characterized at the species level by beta-tubulin (BenA) sequencing using primers previously described [32]. Drug interactions were tested by the checkerboard dilution method using the broth microdilution protocol of the Clinical and Laboratory Standards Institute (CLSI) M38 (3rd edition) for MIC determination [33]. The fractional inhibitory concentration index (FICI) was calculated and interpreted as previously described [34]. In order to test the antifungal activity against the Aspergillus biofilms, we measured the reduction of the tetrazolium salt 2,3-bis(2methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2Htetrazolium-hydroxide (XTT) by metabolic active fungal cells within the biofilm, as previously described [35,36]. In brief, from a suspension of 2.

Review of the Literature
A systematic search was performed on PubMed (https://pubmed.ncbi.nlm.nih.gov/) using the terms: ''endocarditis'' or ''aortitis'' and ''Aspergillus niger'' or ''Aspergillus tubingensis'' or ''section Nigri''. Our search identified 17 cases of endocarditis or aortitis caused by Aspergillus spp. of section Nigri. Description of these cases with addition of the present case report (n = 18) is provided in Table 1. All patients except one had a past history of heart surgery and/or heart valve replacement or valvuloplasty. Only one patient was immunocompromised (acute leukemia). Septic embolisms were commonly observed. Mortality was 100% in the absence or surgical intervention and 45% among patients who underwent surgery of valve replacement and/or thrombus removal. Among the patients who survived without recurrence in follow-up, the duration of antifungal therapy ranged from 6 to 12 months.

Discussion and Conclusions
We reported the case of a patient who developed A. tubingensis endocarditis on a biological prosthetic valve, probably following direct inoculation of the pathogen in the blood via a contaminated drug injection, in the absence of other primary focus of infection. The patient was successfully treated by surgery and 8 months of antifungal therapy consisting of initial L-AMB therapy for 3 days followed by a combination of voriconazole and caspofungin for 14 days and then voriconazole and isavuconazole monotherapy.
Aspergillus endocarditis is a rare event and represents the second cause of fungal endocarditis after Candida spp. [37,38]. Prosthetic heart valves, structural heart disease or past history of endocarditis were the most frequent underlying conditions [37,38]. History of active intravenous drug abuse has also been reported [37,39], although it is unclear whether the fungus originates from the drug or its adjuvant, or from the contaminated material of injection.
Our review of the literature identified 17 case reports of endocarditis caused by Aspergillus spp. of section Nigri. Most of these cases were attributed to A. niger by phenotypic identification, but were not characterized at the species level by molecular analysis. Our case represents the first report of endocarditis attributed to A. tubingensis, which is actually one of the most frequent cryptic species of section Nigri in Europe [24,25,27]. This species may exhibit some decrease of azole susceptibility compared to A. niger sensu stricto, according to previous in vitro studies [24][25][26]. Our strain had a voriconazole MIC of 1 mg/l, which actually does not exceed the epidemiological cut-off value (i.e. 2 mg/l) of A. niger [29]. Data about the in vitro interactions of voriconazole with caspofungin (or other echinocandins) against Aspergillus spp. are controversial and suggest an occasional synergistic effect, which may be strain-dependent [40][41][42]. Data for A. niger are scarce, but the interaction was classified as indifferent for a majority of isolates [41]. Similar observations have been reported for the interaction of amphotericin B and caspofungin against Aspergillus spp. and A. niger [43]. Our checkerboard testing with the present A. tubingensis strain showing indifferent interactions for these antifungal combinations are consistent with these previous observations. Data on antifungal activity on biofilms are more scarce. One study assessed the  While the optimal antifungal therapy of Aspergillus endocarditis remains to be determined, our review of the literature shows that surgery is a key determinant of therapeutic success, which is in accordance with experts' recommendations [3,4]. The crucial role of surgery is not surprising as secondary embolic events represent a very frequent complication and the major cause of death among these patients. Fortunately, our patient did not experience any symptom or radiological evidence of thrombo-embolic complications before surgery, which was probably a good prognostic factor.
Another debated question in the management of Aspergillus endocarditis is the duration of antifungal therapy. In our literature review, all patients who survived without recurrence received antifungal therapy for at least 6 months. Whether this duration can be shortened is unknown. The use of PET-CT in followup may help in determining the duration of treatment as it was the case here.
In conclusion, this case of A. tubingensis endocarditis is the first one formally attributed to this cryptic species and among the rare ones attributed to Aspergillus of section Nigri that have been reported until now. Our in vitro analyses suggest that amphotericin B is the only antifungal drug displaying significant anti-biofilm activity, which may support its use as first-line therapy. We did not observe any benefit of the addition of an echinocandin on the basis of our in vitro data for the present case, but clinical data are lacking to determine the optimal antifungal regimen for such rare infections. Funding Open access funding provided by University of Lausanne. This work was done as part of our routine practice without any specific funding.
Data Availability The dataset of this case report is available upon reasonable request of the editors and with respect of the confidential rules of our institution and anonymity of the patient.
Code Availability Not applicable.

Declarations
Conflict of interest F. Lamoth has received research funding from the Swiss National Science Foundation, the Santos-Suarez Foundation, Novartis, MSD and Pfizer outside of the scope of the present work, and has contributed to advisory boards for Gilead.
Ethical Approval Not applicable, according to the local ethics requirements about case reports.

Consent to Participate Not applicable.
Consent for Publication Written informed consent was obtained from the patient for the publication of this case report.
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