Invasive Pulmonary Aspergillosis Due to Aspergillus Lentulus in a Liver Transplant Recipient: a Case Report and Literature Review

Background: Aspergillus lentulus, a new species discovered in 2005, is a rare fungal pathogen with reduced sensitivity to antifungal agents. Case presentation: Here we reported a 58-year-old male who underwent a liver transplantation on January 2020 and followed by maintenance immunosuppressive therapy. Then A.lentulus was cultured from his sputum twice and identied by using the MALDI-TOF-MS and conrmed by the reference of RUO database. In the susceptibility tests of the isolate, minimal inhibitory concentrations for amphotericin B, voriconazole, posaconazole, and caspofungin were found to be 0.5 mg/L, 1.0 mg/L, 0.5 mg/L and 8 mg/L, respectively. The patient was improved after treatment with a voriconazole 0.2g q12h orally plus caspofungin 50mg qd intravenously. Conclusion: A. lentulus is easily confused with A. fumigatus and its susceptibility to amphotericin B and azoles antifungal agents is reduced. Therefore, the identication of strain and drug susceptibility test are very important for clinical treatment. lesions, and the patient improved eventually. In conclusion, here we reported the rst case of probable invasive aspergillosis caused by A. lentulus after liver transplantation in our country, which showed a good clinical response to voriconazole and caspofungin. We exemplify the important role of accurate species identication and antimicrobial susceptibility testing due to the differential MICs of Aspergillus-related species. More clinical features and treatment of infection with A. lentulus should be discussed in the future.


Background
Aspergillosis have become a major problem among immunocompromised patients, and always led to life-threatening infection in patients with prolonged neutropenia, allogeneic hematopoietic stem cell transplant (HSCT), solid organ transplant (SOT), corticosteroid use, and others [1]. In SOT recipients,invasive fungal disease is now a major cause of postoperative death [2].
Aspergillus species are widely found in soil, air samples and decayed materials all over the world [3]. Human infection due to Aspergillus species was mainly caused by typical A. fumigatus isolates. However recent taxonomic studies showed that the Aspergillus genus includes at least eight subgenus, subdivided into 22 complexes or sections. The main sections of medical importance are Fumigati, Flavi, Terrei, Nigri, Usti, and Nidulantes, which can also cause invasive infections [4,5].
Aspergillus lentulus was rst found by Balajee and his colleagues[6] in a medical center in the United States, and subsequently, the species was also isolated from patients in other countries as well as environmental samples. The study showed this pathogen morphologically closely resembling A. fumigatus, once has been misidenti ed as a variant of A. fumigatus sensu stricto and was found to exhibit decreased antifungal susceptibilities [7].
Herein we report a probable pulmonary invasive aspergillosis caused by A. lentulus in an adult patient after liver transplantation, which was successfully treated with voriconazole plus caspofungin.

Case Presentation
A 58-year-old Chinese male with chronic kidney failure and a 20-year history of hepatitis B was submitted to the hospital because of decompensation liver cirrhosis, then he underwent liver transplantation on January 2020 and followed by maintenance immunosuppressive therapy consist of high-dose methylprednisolone, tacrolimus, and mycophenolate mofetil. No graft rejection occurred later. On post-transplantation day 5, severe anuria and hyperkalemia led to renal replacement therapy. At day 26 after transplantation, multi-drug resistant Klebsiella pneumoniae was isolated from sputum culture, which was treated with ZAVICEFTA (Ceftazidime/Avibactam) 0.94g every 12 hours for 14 days considered his renal function. His temperature was 36.8°C, laboratory investigation showed leucocyte count was 6.47*10^9/L, neutrophil percentage was 88.8%, C-reactive protein (CRP) was 71.2 mg/L and procalcitonin (PCT) was 1.96 ng/mL. An X-ray showed: Exudation in both lungs, thickness of blood vessels in both hilar, pleural effusion. At day 33 after transplantation, Enterococcus faecium was isolated from blood culture, then we treated with vancomycin started with a 1 g loading dose (LD) and 0.5 g maintain dose (MD) once daily. At day 36 after transplant, a chest x-ray performed because of his persistent signs of in ammation, but without fever, showed a new in ltrate in the right pulmonary. The lung computed tomography (CT) scan also demonstrated bilateral in ammatory in ltrate. Two respiratory samples (sputum) were obtained. At this time, the patient had no fever and his temperature was 36.8°C, his laboratory tests showed anemia (hemoglobin 7.6 g/dL), leukocytes 11.05*10^9/L, neutrophils 92.9% and thrombocytopenia (38*10^9/L), serum creatinine 134 µmol/L (normal range 44-115 µmol/L), and serum urea 23.4 mmol/L (normal range 2.9-8.2 mg/dL), CRP was 60.1 mg/L and PCT was 0.86 ng/mL. The (1-3)-β-D-glucan (BDG) level was 23.6 pg/ml and GM test was 0.808ng/mL. Then A. lentulus was isolated twice from both of these sputum specimens. Invasive pulmonary aspergillosis was diagnosed according to host factors (liver transplantation and immunosuppression), radiological, and microbiological ndings.
In order to recover Aspergillus species from the sputum specimens, 0each sample was inoculated onto two Sabouraud dextrose agar (Autobio Diagnostics, China). These plates were incubated at 25°C and 35°C for growth. The original colonies were then subcultured onto another SDA agar and incubated at 35°C for observation of their macroscopic and microscopic morphology. (Fig. 1,see Additional le Figure_1) The surface of the colonies was at rst white, then turning cottony and greenish with a white border. The reverse was white (Fig. 1A). Fluorescence microscopy was used to determine the microscopic morphology of the isolates. Branched conidiophore structures and reduced sporulation were observed at 400x magni cations (Fig. 1B). MALDI-TOF-MS (bioMerieux, France) was used for the identi cation of the isolates which revealed the identity of 75.9% to the A. lentulus reference structure according to the RUO database.
Antifungal drug susceptibility toward itraconazole, voriconazole, posaconazole and amphotericin B was tested on the isolate according to Clinical Laboratory Standards Institute guideline M61 using Sensititre TM YeastOneTM YO10 AST plate (ThermoFisher Scienti c, USA), which revealed minimum inhibitory concentrations (MIC) of 0.5 mg/L, 1.0 mg/L, 0.5 mg/L and 8 mg/L, respectively. Antifungal therapy with a voriconazole LD of 0.4g q12h followed by a MD of 0.2g q12h orally plus caspofungin 70 mg (LD) followed by 50mg MD intravenously was started immediately. Four days after start of voriconazole, the steadystate serum trough level was 2.1mg/L. Thirteen days after initiation, sputum culture turned negative and laboratory tests showed a reduction in leukocytes 7.2*10^9/L, neutrophils 82.7%, CRP 48.3 mg/L and PCT 1.27 ng/mL, but an increase in BDG level (53.5 pg/ml). However repeated CT scan showed a reduction of the lesions, and the patient's condition improved gradually and recovered eventually.

Conclusions
To our knowledge, this is the rst case of invasive pulmonary aspergillosis after liver transplantation in China caused by A. lentulus. In 2004, Balajee[6] rst reported a distinct variant of A. fumigatus that causes invasive infection in four HSCT recipients and demonstrated decreased susceptibilities to multiple antifungal drugs. The isolates sporulated slowly and needed prolonged incubations to be similar to typical isolates. Sequencing showed the mitochondrial cytochrome b gene sequences of all the new isolates were unique, which suggested the potential presence of a genetically unique. Phenotypic methods revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48°C, which was then designated A. lentulus [7]. From then on, this new species has been found in soil, environmental samples [8] and was reported in patients in America, Japan, Spain, Brazil and some other countries, described as a new sibling species of A. fumigatus due to their morphological resemblance.
According to the literature [9], approximately 4-5% of "A. fumigatus" isolated from patients have later turned out to be the related species, due to their morphological resemblance, which makes the accurate identi cation of A. lentulus di cult for clinical microbiology laboratories. Therefore, the reported prevalence of A. lentulus might be underestimated. Hence, molecular analysis is required for the identi cation of A. lentulus. Tamiya and his colleagues [10] used liquid chromatography/time-of-ight mass spectrometry to identify the secondary metabolites secreted as virulence factors by A. lentulus. Among all the secondary metabolites, auranthine in conidia and culture ltrate was only found in A. lentulus, but not in A. fumigatus or the other related species. In culture ltrate, the A. fumigatus isolates produced signi cantly higher amounts of gliotoxin compared with other species including A. lentulus.
Since the rst documented description, only 11 cases (except the present one) of IA caused by A. lentulus have been reported around the world, related to 17 patients. The characteristics and details of the cases in the literature are shown in Table 1. (see Additional le Table_1) The median age at diagnosis was 50 years (range, 12-82 years). Except two cases with incomplete information, nine patients (77.8%) were male in the remaining 9 cases. Among the 17 patients, 8 (47%) had a history of HSCT, 5 (29.4%) experienced SOT, and 4 patients had other underlying diseases (COPD, cystic brosis, refractory nephrotic syndrome, ANCA-associated vasculitis). A. lentulus and A. fumigatus were isolated simultaneously in one patient with COPD. All SOT patients received immunosuppressive therapy and three patients with no history of transplantation, also received immunosuppressive therapy including glucocorticoids. 6 (35.2%) patients had a history of antifungal drug usage before diagnosis.
As we all know, A. fumigatus are intrinsically sensitive to azoles and amphotericin B, but A. lentulus isolates usually have higher MIC values for these drugs and caspofungin, and even show primary resistance to azole drugs. Nowadays, the most common acknowledged mechanism concerning azole resistance in A. lentulus is mutations in the cyp51A gene. Mellado [11] performed heterologous expression in an A. fumigatus cyp51A de cient strain, con rming that cyp51A is responsible for the differences in A. lentulus-azole drug resistance. Since there is no breakpoint for A. lentulus according to CLSI and EUCAST, and the breakpoint of A. fumigatus cannot be referred to, so we did some therapeutic drug monitoring to keep the voriconazole trough concentration above 1.0 mg/L.
According to other researches, Tamiya [10] tested drug susceptibility for A. fumigatus and all three related species including A. lentulus, revealed that amphotericin B was effective against all four species. In contrast, A. lentulus had higher MICs to azoles such as voriconazole and itraconazole. Environmental isolates had decreased susceptibility to voriconazole and itraconazole than clinical isolates. As pointed out by Mortensen [12], susceptibility tests indicated In conclusion, here we reported the rst case of probable invasive aspergillosis caused by A. lentulus after liver transplantation in our country, which showed a good clinical response to voriconazole and caspofungin. We exemplify the important role of accurate species identi cation and antimicrobial susceptibility testing due to the differential MICs of Aspergillus-related species. More clinical features and treatment of infection with A. lentulus should be discussed in the future. Ethics approval and consent to participate This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Approval No. B2021-054).

Consent for publication
Written informed consent was obtained from our patient to publish this case report and its accompanying images.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This report did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.

Authors' contributions
All authors contributed to the study conception. YYZ performed the microbiological cultures, microscopic identi cation, molecular analyses and antifungal susceptibility testing; YS and XPS contributed to data collection and literature search; TW and XPS helped to treat the patient; YS was a major contributor in writing the manuscript and all authors commented on previous versions of the manuscript; YS, XPS and JX contributed to the nal manuscript review. All authors have read and approved the nal manuscript.