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Azole-resistant Aspergillus fumigatus due to TR46/ Y121F/T289A mutation emerging in Belgium, July 2012 E Vermeulen1, J Maertens2, H Schoemans2, K Lagrou ([email protected])1,3
1. Catholic University of Leuven, Department of Microbiology and Immunology, Leuven, Belgium 2. University Hospitals Leuven, Department of Hematology, Leuven, Belgium 3. University Hospitals Leuven, Department of Laboratory Medicine, Leuven, Belgium Citation style for this article:
Vermeulen E, Maertens J, Schoemans H, Lagrou K. Azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012. Euro Surveill. 2012;17(48):pii=20326. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20326 Article submitted on 16 November 2012 / published on 29 November 2012 A new azole resistance mechanism in Aspergillus <0.5). Galactomannan detection in broncho-alveolar lav- fumigatus consisting of a TR46/Y121F/T289A altera- age (BAL) fluid tested positive as well (index 5.8), and tion in the cyp51A gene was recently described in the Aspergil us fumigatus was cultured from BAL fluid. A Netherlands. Strains containing these mutations are diagnosis of probable pulmonary invasive aspergillosis associated with invasive infection and therapy failure. (IA) was made following revised European Organization This communication describes the first case of fatal for Research and Treatment of Cancer/Mycoses Study invasive aspergillosis caused by TR46/Y121F/T289A Group (EORTC/MSG) criteria [1]; the patient agreed to outside the Netherlands, in the neighboring country participate in a double-blinded phase III clinical trial of Belgium, suggesting geographical spread. TR46/ comparing two azoles with anti-Aspergil us activity. Y121F/T289A leads to a recognisable phenotypic sus- However, serum galactomannan levels did not decrease ceptibility pattern which should trigger cyp51A geno- while he received azole therapy and the clinical condi- tion of the patient deteriorated rapidly. Case report
Meanwhile, the isolate was tested for azole suscep- A 57-year-old male, diagnosed with stage IIIA multiple tibility, following Clinical and Laboratory Standards myeloma (IgG kappa) in 2009, received a fully matched, Institute (CSLI) protocols and showed an azole-resist- unrelated haematopoietic stem cell transplantation ant phenotype, with high-grade resistance to voricona- following reduced-intensity conditioning (fludarabine- zole (minimal inhibitory concentration (MIC) >16 mg/L) melphalan-ATG) in May 2012. Prior treatment regimens and less pronounced resistance to itraconazole (MIC included multiple lines of chemotherapy, autologous =4 mg/L) and posaconazole (MIC =1 mg/L). Formal transplantation, proteasome inhibitors (bortezomib), clinical breakpoints have not been established by CLSI immunomodulatory agents (lenalidomide) and high- for Aspergil us susceptibility testing. Based on the epi- dose corticosteroids. At the time of transplantation, demiological cut-off values (wild-type distributions), the patient had achieved a very good partial response resistance to itraconazole is defined as an MIC >1 (<10% residual monoclonal paraprotein). The post- mg/L, to voriconazole as >1 mg/L and to posaconazole transplantation course was complicated by grade III as >0.5 mg/L [2]. An excellent essential agreement (EA) hyperacute graft-versus-host disease (GVHD), involv- between CLSI and EUCAST methods was described in ing mainly the skin and the gastro-intestinal tract. susceptibility testing of A. fumigatus to these azoles Methylprednisolone was started at 2 mg/kg and slowly [3] and EUCAST already established breakpoints for tapered over the following weeks. However, high-dose itraconazole and posaconazole (itraconazole ≤1 mg/L corticotherapy needed to be re-installed in June 2012 is considered susceptible and >2 mg/L resistant; posa- because of a relapse of grade III acute GVHD. The conazole ≤0.12 is considered susceptible and >0.25 patient was receiving fluconazole 400 mg daily since resistant) [4]. A recent report, using an in vitro dynamic May 2012 as prophylaxis, but was never exposed to model of pulmonary IA that enabled simulation of human voriconazole pharmacokinetics, proposed CLSI breakpoints for voriconazole as ≤ 0.5 mg/L for suscep- One month later, in July 2012, the patient presented with dyspnea, pleuritic-type chest pain and fever, up to 39.9°C. Thoracic computed tomography (CT)-scan imag- Given this new finding of azole resistance and the rapid ing showed multiple ill-defined lesions surrounded by clinical decline, the investigators decided to withdraw ground glass opacities, suggestive of angio-invasive the patient from the clinical study. Nine days after the pulmonary mold infection. Serum galactomannan test- start of azole therapy, liposomal amphotericin B was ing was repeatedly positive (maximum index 5.2; norm started at a dose of 3 mg/kg. Nevertheless, the patient developed widespread IA with eye and brain involve- the Netherlands. TR46/Y121F/T289A-bearing strains ment. A brain magnetic resonance imaging (MRI) scan have also been found throughout the environment in taken 15 days after the initial diagnosis of invasive Belgium, among azole-resistant A. fumigatus isolates aspergillosis showed multiple nodular non-contrast- cultured from outdoor air sampling, which was per- enhancing lesions suggestive of cerebral aspergillosis; formed from June to September 2012 (data not shown). this was confirmed by positive galactomannan testing This case confirms the geographic spread of this new in cerebrospinal fluid (index 4.8). The patient died 19 resistance mechanism, possibly following the same days after his first presentation with dyspnea. Azole path as TR34/L98H, which now causes therapy-resist- resistance in the strain affecting the patient was shown ant infections across Europe and even outside Europe to be due to cyp51A mutation TR46/Y121F/T289A.
[8,12,13,15-17]. The phenotype of the TR46/Y121F/ T289A strains consists of a very high MIC to voricona- Characterisation of the Aspergillus
zole (>16 mg/L), and an itraconazole MIC which is often isolate derived from the patient
multiple dilutions lower. In contrast, in TR34/L98H The Aspergil us isolate, cultured from BAL fluid, was mutated strains, itraconazole MICs are typically higher identified as Aspergil us fumigatus complex based than voriconazole MICs. This finding (MIC voricona- on microscopic and macroscopic characteristics. This zole >16 mg/L and voriconazole MIC ≥MIC itraconazole) identification was confirmed to the species level with should raise awareness of this new TR46/Y121F/T289A beta-tubulin sequencing, as described previously [6]. resistance mechanism in other centres and countries. The isolate was tested for susceptibility with broth microdilution following the CLSI M38-A2 protocol [7]. Susceptibility testing should not delay initiation of Genotypic identification of the resistance mechanism therapy. Culture has a low sensitivity and takes about was performed by sequencing of the cyp51A gene, as 48h to become positive; susceptibility testing takes at least another 48h. Resistance is therefore often a late finding in the management of the individual patient. Discussion and conclusion
Molecular techniques are a promising tool to rapidly Invasive aspergillosis is an important infectious com- provide information about resistance genotype, but cli- plication in haematologic patients [9], but also in nicians should be aware that they are often designed to other groups of immunocompromised and intensive detect known resistance mechanisms and can therefore care patients [10]. Triazoles are the mainstay of ther- miss new mutations. On the other hand, not all muta- apy, with voriconazole the first-line therapy for IA [11]. tions necessarily lead to a resistant phenotype [19]. However, reports of azole resistance have emerged, not Surveillance programs are crucial to monitor the local only after long-term azole exposure [12], but also after epidemiology of azole resistance, to correctly assess short-term exposure and in azole-naïve patients [13]. the risk of resistance associated with current treatment In the Netherlands, over 90% of the resistant clinical strategies. Susceptibility testing in individual patients strains were attributable to the same resistance mech- with invasive aspergillosis should not be delayed until anism [13]. Therefore, an environmental route of resist- treatment failure because of the life-threatening char- ance development is assumed and this is suspected acter of this disease which is illustrated by this case.
to be related to the selective pressure of azole fungi- cides in the environment [14]. This predominant resist- ance mechanism is mediated by a tandem repeat of 34 Acknowledgments
bases (TR34) in the promoter region of the cyp51a gene EV receives a grant from Research Foundation Flandres and a substitution at position 98 (TR34/L98H), which (Fonds Wetenschappelijk Onderzoek Vlaanderen). An epide- encodes a residue of the azole target, sterol 14-alpha- miological study including typing and susceptibility testing demethylase. This resistance mechanism, conferring of clinically relevant Aspergillus isolates has been approved pan-azole resistance, has to date spread across Europe by the local Ethics Committee University Hospitals Leuven, as well as to China and India [8,12,13,15-17]. Because of the widespread and abundant consumption of azole fungicides in agriculture, there is a risk that other Conflicts of interest
resistance mechanisms might emerge in the environ- ment as well [14]. Recently, in October 2011, a new Potential conflicts of interest are listed as follows. JM has served as consultant to Schering-Plough, Gilead Sciences, resistance mechanism due to a 46 base tandem repeat Merck, Sharp & Dohme, Pfizer Inc., Bio-Rad, Fujisawa (TR46) in the promoter of the cyp51a gene and two healthcare, Inc., Astellas, Nextar and Zeneus (Cephalon). JM point mutations (TR46/Y121F/T289A) was described in has received research funding from Bio-Rad, Merck, Sharp persons with IA who failed therapy in the Netherlands & Dohme, and Pfizer Inc. JM has been on the speaker’s bu- reau for Schering-Plough, Gilead Sciences, Merck, Sharp & Dohme, Pfizer Inc., Bio-Rad, Fujisawa healthcare, Inc, Astellas and Zeneus (Cephalon). KL has received research To our knowledge, the case described in this report grants from Gilead Sciences, Pfizer Inc. and Merck, Sharp is the first case of azole resistance in A. fumigatus & Dohme and served on the speakers’ bureau of Pfizer Inc. due to TR46/Y121F/T289A outside the Netherlands. and Merck, Sharp & Dohme. HS has served as consultant to The patient lived about 60 kilometres from the Dutch border and did not have any recent travel history to References
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