Aspergillus alliaceus and Aspergillus flavus co-infection in an acute ...

Medical Mycology November 2010, 48, 995–999

Case Report

Aspergillus alliaceus and Aspergillus flavus co-infection in an acute myeloid leukemia patient BETIL OZHAK-BAYSAN*, ANA ALASTRUEY-IZQUIERDO†, RABIN SABA‡, DILARA OGUNC*, GOZDE ONGUT*, AYSEN TIMURAGAOGLU§, GOKHAN ARSLAN#, MANUEL CUENCA-ESTRELLA† & JUAN LUIS RODRIGUEZ-TUDELA† *Akdeniz University Medical Faculty, Department of Medical Microbiology, Antalya,Turkey, †Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain, ‡Akdeniz University Medical Faculty, Department of Infectious Diseases and Clinical Microbiology, Antalya,Turkey, §Akdeniz University Medical Faculty, Department of Hematology, Antalya, Turkey, #Department of Radyology, Faculty of Medicine, Akdeniz University, Antalya,Turkey

We report a case of a pulmonary infection caused by Aspergillus flavus and Aspergillus alliaceus in an acute myeloid leukemia patient. These isolates were identified using traditional and sequencing-based molecular methods. Keywords acute myeloid leukemia, Aspergillus flavus, coinfection, Aspergillus alliaceus, pulmonary infection

Introduction Invasive mold infections are found frequently in patients with hematologic malignancies, particularly those with acute myeloid leukemias [1,2]. Among those with invasive fungal infections (IFI), invasive aspergillosis (IA) is one of the major clinical problems in these patients [3]. Despite significant progress in the treatment of fungal infections, IA is associated with an extremelly high rate of mortality. In high risk leukemia patients and allogeneic bone marrow transplant recipients mortality may be as high as 80–90% [3,4]. Although Aspergillus fumigatus accounts for the majority of IA cases, a number of such infections are caused by non-A. fumigatus Aspergillus species, such as Aspergillus flavus and Aspergillus terreus. The later etiologic agents have been increasingly reported to have low susceptibilities to antifungals [5,6]. A. flavus is the second most important Aspergillus spp. causing human infections and appears to be more virulent and more resistant to antifungal drugs than other Aspergillus spp. [7]. Aspergillus alliaceus is the anamorphic species belonging to Aspergillus section Flavi. This section includes three main clades, i.e., Received 12 November 2009; Received in final revised form 15 February 2010; Accepted 4 March 2010 Correspondence: Dilara Ogunc, Akdeniz University Medical Faculty, Department of Clinical Microbiology, 07070 Antalya, Turkey. Tel: 90 242 2496914; Fax: 90 242 2272535; E-mail: [email protected] © 2010 ISHAM

A. flavus, A. tamari and A. alliaceus. A. flavus is a member of A. flavus clade, whereas Aspergillus alliaceus and Petromyces albertensis together with Aspergillus lanosus are assigned to A. alliaceus clade [8]. Although human infections caused by A. alliaceus are rare, this species has been previously isolated from a case of chronic otitis externa after surgery [9]. Similarly, Balajee et al. reported a case of invasive pulmonary aspergillosis caused by P. alliaceus (teleomorph of Aspergillus alliaceus) [10].

Case report A 64-year-old male was admitted to the Emergency Department of Akdeniz University Medical Faculty with a 10-day history of abdominal pain, cough, shortness of breath and fever. On physical examination, he had a blood pressure was 120/70 mmHg, a pulse of 92 beats/minute, temperature of 38.5°C, and a respiration rate of 22 breaths/minute. There were rales at bilateral lower lobes at auscultation and the liver was palpable 2 cm from the mid clavicular region. The results of his blood tests were: hemoglobin, 88 g/l; platelet count, 41109/l; white blood cell (WBC) count, 4109/l; and C-reactive protein (CRP), 98 mg/l. Ninety percent of the leukocytes were blasts in the peripheral blood smear. He was hospitalized because of fever, neutropenia and de novo leukemia. Cefepime (32 g/day) and clarithromycin (20.5 g/ day) were started empirically after samples for blood cultures DOI: 10.3109/13693781003749418

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were taken for the first time. The latter were negative for bacteria and fungi. A diagnosis of de novo acute myeloid leukemia (AML M-0) was made after the peripheral blood and bone marrow examinations (bone marrow aspirate smear, Sudan Black B staining, alpha-naphthyl acetate esterase activities, immunophenotyping). After diagnosis, the patient was treated with cytosin arabinosid (200 mg/m2 for 7 days) and idarubicin (12 mg/m2 for 3 days). Highresolution computed-tomography (HRCT) was performed and showed bilateral consolidation and diffuse ‘ground glass’ opacities of the lower lobes which were interpreted as infiltration due to infection or leukemia. After 3 days of therapy, the patient became afebrile. The antibiotic regimen was switched to oral levofloxacin 0.5 g/day at day 14. At that time, his WBC count was 0.4109/l, absolute neutrophil count (ANC) was 0.08109/l and CRP level was 8 mg/l. One week later the patient became febrile again, had a productive cough, was deeply neutropenic (ANC 0.03109/l) and his CRP was 170 mg/l. A second set of blood cultures along with initial sputum cultures were taken and piperacillin/tazobactam 44.5 g/day and amphotericin B deoxycholate 0.7 mg/kg/day were started empirically. There was

no growth on cultures and concomitant Aspergillus galactomannan antigens tests (Platelia Aspergillus; Biorad) were negative. HRCT of the chest showed pleural fluid and progression of consolidation of the lower lobes, as well as new consolidation areas. Peripheral ‘ground glass’ opacities at the upper lobes of the lung were detected. Since his high fever continued and his clinical condition deteriorated, the antimicrobials were changed to imipenem 40.5 g/day) and liposomal amphotericin B (LAmB) 3 mg/kg/day. On day 32, the patient was still neutropenic and had fever. Bone marrow aspiration was performed and blast percentage was found to be 10%. Re-induction chemotherapy was restarted with the same protocol. A second sputum sample was taken on the patient’s 40th day of hospitalization. Microscopic examination revealed numerous hyphae suggestive of Aspergillus spp. and Aspergillus flavus and Aspergillus alliaceus were recovered in culture. On the following day, bronchoalveolar lavage (BAL) fluid was taken for culture which yielded the same molds (Fig. 1). Hence, LAmB was changed to voriconazole (loading dose, 6 mg/kg/day, followed by 4 mg/kg/day intravenously every 12 h).

Fig. 1 (a) Aspergillus alliaceus colonies, pale buff to ginger-brown, with grey-to-black sclerotial bodies and (b) the microscopic appearance of Aspergillus alliaceus. (c) Aspergillus flavus colonies, green and goldish to brown, and (d) the microscopic appearance of Aspergillus flavus.

© 2010 ISHAM, Medical Mycology, 48, 995–999

A. alliaceus and A. flavus coinfection

On the 48th day of hospitalization, the patient developed cardiopulmonary failure and required intubation. The patient continued to deteriorate and died on the 50th day. An autopsy was not performed. Microscopic and macroscopic characters were used for the morphological identification of clinical isolates. For comparative purposes, colonies grown on Sabouraud dextrose agar (SDA) were subcultured to Czapek-Dox, 2% malt extract agar and potato-dextrose agar (PDA). Colonies were pale buff to ginger-brown, with black sclerotial bodies. Conidiophores were smooth-walled and hyaline. Vesicles were spherical and phialides were biseriate (Fig. 1), consistent with A. alliaceus. Molecular identification was performed in order to confirm the identification at the species level. The Aspergillus isolates were cultured in GYEP medium (0.3% yeast extract, 1% peptone, Difco, Soria Melguizo S.A., Madrid, Spain) with 2% glucose (Sigma Aldrich Quimica, Madrid, Spain), for 24 to 48 h at 30ºC. Genomic DNA was extracted by using previously described procedures [11]. DNA segments comprising a region of the beta tubulin and the internal transcribed spacers (ITS) were amplified with primers βtub3 (5′-TTCACCTTCAGACCGGT-3′), βtub2 (5′-AGTTGTCGGGACGGAATAG-3′), ITS1 (5′-TCCG TAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCT TATTGATATGC-3′) in a GeneAmp PCR System 9700 (Applied Biosystems, Madrid, Spain) [12]. The reaction mixtures contained 0.5 μM of each primer, 0.2 μM of each deoxynucleoside triphosphate, 5 μl of PCR 10 buffer (Applied Biosystems), 2.5 U Taq DNA polymerase (Amplitaq; Applied Biosystems), and 25 ng of DNA in

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a final volume of 50 μl. The samples were amplified in a GeneAmp PCR System 9700 (Applied Biosystems) by using the following cycling: one initial cycle of five min at 94°C, followed by 30 cycles of 30 s at 94°C, 45 s at 55°C (beta tubulin) or 56ºC (Internal transcribed spacer), and two min at 72°C, with one final cycle of five min at 72°C. The reaction products were analyzed in a 0.8% agarose gel. Sequencing reactions were done with 2 μl of a sequencing kit (BigDye Terminator cycle sequencing, ready reaction; Applied Biosystems), 1 μl of the primers (βtub 3, βtub 2, ITS1 and ITS4) and 3 μl of the PCR product in a final volume of 10 μl. Sequences were assembled and edited using the SeqMan II and EditSeq software packages (Lasergene: DNAstar, Inc., Madison, WI, USA). Identification was performed by comparison of the sequences with 10 ITS and β tubulin sequences of Aspergillus flavus (CBS100927 type strain) and Aspergillus alliaceus (NRRL 1206, NRRL 315, NRRL 5108, CBS 511.69) strains obtained from the GenBank database. The analyses were conducted with InfoQuest FP software, version 4.50 (BIORAD Laboratories, Madrid, Spain). The methodology used was maximum parsimony clustering. Tree stability was assessed by parsimony bootstrapping with 2000 simulations. Aspergillus fumigatus CNM-CM237 was used as an outgroup to root the trees. The two strains isolated in the present work were identified as A. alliaceus and A. flavus (Fig. 2). The in vitro susceptibilities of A. alliaceus and A. flavus to antifungal drugs were performed using a broth dilution method following the European Committee for Antimicrobial

Fig. 2 A phylogenetic tree of the subset of isolates included in the study obtained by using maximum parsimony phylogenetic analyses and 2000 bootstrap simulations based on beta tubulin (a) and ITS (b) sequences. Aspergillus fumigatus CNM-CM 237 were used as the outgroup to root the trees (CM 5089 A. flavus, CM 5090 A. alliaceus).


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Susceptibility Testing (EUCAST) procedures [13]. This standard method is similar to the one published by the CLSI (formerly NCCLS) reference method for broth dilution antifungal susceptibility testing of filamentous fungi, but with the following minor modifications; (i) RPMI 1640 was supplemented with glucose to reach a 2% concentration and (ii) inoculum size was between 1.0 105 to 5.0 105 CFU/ml [2]. Aspergillus fumigatus ATCC 2004305 and Aspergillus flavus ATCC 2004304 were used as quality control strains. The antifungal agents used in the study were amphotericin B (range 16–0.03 mg/l; Sigma Aldrich Química), itraconazole (range 8–0.015 mg/l; Janssen Pharmaceutica S.A., Madrid, Spain), voriconazole (range 8–0.015 mg/l; Pfizer S.A., Madrid, Spain), posaconazole (range 8– 0.015 mg/l; Schering-Plough Research Institute, Kenilworth, NJ, USA), terbinafine (range 16–0.03 mg/l; Novartis, Basel, Switzerland), caspofungin (range 16–0.03 μg/ml; Merck & Co, Inc., Rahway NJ, USA), micafungin (range 16–0.03 μg/ml; Astellas Pharma Inc, Tokyo, Japan) and anidulafungin (range 16–0.03 μg/mL; Pfizer S.A.). The plates were incubated at 35ºC for 48 h in a humid atmosphere. Visual readings were performed with the help of a mirror. The endpoints for amphotericin B, itraconazole, voriconazole, ravuconazole, posaconazole and terbinafine were the antifungal concentration that produced a complete inhibition of visual growth at 48 h. For the echinocandins (caspofungin, micafungin and anidulafungin) the endpoint was the antifungal concentration that produced a visible change in the morphology of the hyphae compared with the growth (minimum effective concentration; MEC) [14,15]. Antifungal susceptibility results were as follows (MIC and MEC values in mg/l): for A. alliaceus; amphotericin B 16, itraconazole 8, voriconazole 0.25, posaconazole 8, terbinafine 16, caspofungin 16, micafungin 16, and anidulafungin 16. Results found with the A. flavus isolate were; amphotericin B 0.5, itraconazole 0.25, voriconazole 1, posaconazole 0.12, terbinafine 0.12, caspofungin 16, micafungin 16, and anidulafungin 0.03.

Discussion Invasive aspergillosis has emerged as a common and often fatal opportunistic fungal infection in patients with hematologic malignancies. Although traditional microbiological methods such as direct microscopy and culture serve as the cornerstone for definitive diagnosis of mycoses, rare or atypical fungi can be difficult to identify [16,17]. To our knowledge, this is the first report of IA caused by Aspergillus flavus in combination with A. alliaceus. After isolation of two different molds, presumptive identification was performed using traditional culture-based methods.

Identification at species level was achieved by sequencebased molecular methods. Prompt and accurate antifungal susceptibility testing is crucial, especially with emerging opportunistic fungi. In the present case, the antifungal susceptibility test results of the two etiologic agents were not similar. For A. flavus, MICs of the tested antifungals were low except for caspofungin and micafungin. In the case of A. alliaceus, only voriconazole showed a low MIC, demonstrating that voriconazole is a better choice for treatment of both strains. In 2007, Balajee et al. reported a clinical isolate of A. alliaceus which had reduced in vitro susceptibility to amphotericin B and caspofungin [10]. Optimal treatment regimen has not been established in infections caused by A. alliaceus because they have been rarely encountered in clinical cases. More studies addressing antifungal susceptibility of these organisms are needed. As stated by Balajee et al., conventional methods are essential not only for identification, but also for in vitro susceptibility testing of fungi [10]. Traditional culture methods seem equally important for detection of mixed infections as demonstrated in our case. However, identification of unusual pathogens based on morphology is difficult and unreliable. The use of morphology in combination with sequence data tends to provide more reliable results.

Acknowledgements This study was supported by Akdeniz University Scientific Project Unit. None of the authors has any conflicts of interests. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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