Mycopathologia https://doi.org/10.1007/s11046-018-0271-8 SHORT COMMUNICATION TR34/L98H Mutation in CYP51A Gene in Aspergillus fumigatus Clinical Isolates During Posaconazole Prophylaxis: First Case in Korea . . . . Hyeon-Jeong Lee Sung-Yeon Cho Dong-Gun Lee Chulmin Park Hye-Sun Chun Yeon-Joon Park Received: 25 January 2018 / Accepted: 12 May 2018 The Author(s) 2018 Abstract Azole resistance in Aspergillus fumigatus F495L mutation in the CYP51A gene of A. fumigatus is an emerging problem, especially in immunocom- clinical isolate obtained from bronchial washing ﬂuid. promised patients. It has been reported worldwide, Minimal inhibitory concentrations for itraconazole, including in Asia, but has not yet been reported in voriconazole, and posaconazole were [ 16, 1, and Korea. Here, we report a case of invasive pulmonary 4 lg/mL, respectively. While IPA improved partially aspergillosis (IPA) caused by azole-resistant A. fumi- under voriconazole treatment, the patient died from gatus that developed in a hematopoietic stem cell carbapenemase-producing Klebsiella pneumoniae transplantation recipient during posaconazole prophy- bacteremia. Further epidemiological surveillance laxis for immunosuppressive therapy of graft-versus- studies are warranted. host diseases. We identiﬁed TR34/L98H/S297T/ Keywords Aspergillus fumigatus Azoles Posaconazole CYP51A Handling Editor: Sudha Chaturvedi. H.-J. Lee S.-Y. Cho (&) D.-G. Lee S.-Y. Cho D.-G. Lee Division of Infectious Diseases, Department of Internal The Catholic Blood and Marrow Transplantation Center, Medicine, The Catholic Blood and Marrow Seoul St. Mary’s Hospital, College of Medicine, The Transplantation Center, Vaccine Bio Research Institute, Catholic University of Korea, 222, Banpo-daero, Seocho- Seoul St. Mary’s Hospital, College of Medicine, The gu, Seoul 06591, Republic of Korea Catholic University of Korea, 222, Banpo-daero, Seocho- gu, Seoul 06591, Republic of Korea Y.-J. Park e-mail: email@example.com Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of H.-J. Lee Korea, 222, Banpo-daero, Seocho-gu, Seoul 06591, e-mail: firstname.lastname@example.org Republic of Korea D.-G. Lee e-mail: email@example.com e-mail: firstname.lastname@example.org H.-J. Lee S.-Y. Cho D.-G. Lee C. Park H.-S. Chun Vaccine Bio Research Institute, Seoul St. Mary’s Hospital, The Catholic University of Korea, 222, Banpo- daero, Seocho-gu, Seoul, Republic of Korea e-mail: email@example.com H.-S. Chun e-mail: firstname.lastname@example.org 123 Mycopathologia Introduction blood cell counts, and ALL again relapsed after the second HSCT was conﬁrmed. Aspergillus infections including invasive aspergillosis On the 7th day of hospitalization (HD 7), the patient (IA) mainly affect immunocompromised patients, suddenly complained of fever (maximum body tem- such as hematopoietic stem cell transplantation perature 38.6 C) and dyspnea, and then, his blood (HSCT) recipients or patients undergoing immuno- pressure dropped to 84/43 mmHg. Oxygen demand suppressive therapy. Due to the increased number of was gradually increased, and tracheal intubation was immunocompromised patients, the incidence of IA has performed. At that time, it was the seasonal inﬂuenza increased over the past three decades. Among epidemic, and rapid inﬂuenza antigen test resulted in Aspergillus species (spp.), Aspergillus fumigatus inﬂuenza A positive and chest X-ray showed inﬁltra- remains the most common species in all pulmonary tions in the right lung ﬁelds (Fig. 1). Therefore, the syndromes. Although diagnostic advances and new ﬁrst impression was inﬂuenza A pneumonia with triazole antifungal drugs have now been established, septic shock. Considering the possibility of combined mortality rates associated with IA remain high and other nosocomial bacterial pneumonia or atypical range between 28.5 and 50% . pneumonia, not only peramivir (600 mg once), but As triazole drugs have been the mainstay of both also cefepime (2g q12 h), levoﬂoxacin (750 mg once treatment and prevention of IA since the 1990s, there daily), and teicoplanin (400 mg q12 h for 3 doses and were concerns about the increasing possibility of that then 400 mg once daily) were administered. On the azole-resistant Aspergillus spp. or azole breakthrough chest, low-dose computed tomography (LDCT) per- IA by expanding the usage of triazoles . Since the formed, and multifocal ground-glass opacities (GGOs) ﬁrst azole-resistant A. fumigatus was found both in the accompanied by peribronchial consolidations and ill- Netherlands and in Italy in 1998 , azole resistance deﬁned centrilobular nodules in both lungs were in A. fumigatus isolates has been found in almost every observed (Fig. 2). On the 3rd day of fever onset (HD European country, the Middle East, Asia, Africa, 10), bronchoscopy was performed. His condition Australia, and most recently, North and South Amer- recovered rapidly and intubation was removed on the ica. Substitution of leucine 98 with histidine in the 4th day. Blood and sputum cultures, Streptococcal CYP51A gene in combination with a 34-bp tandem pneumoniae and Legionella urinary antigen tests, sequence in the promoter gene (TR34/L98H) is a Mycoplasma serum IgM/IgG tests, and serum galac- dominant resistance mechanism thought to be tomannan assay were all negative. acquired from the environment [4–6]. Reported rates On the 4th day after bronchoscopy (HD 11), of azole-resistant A. fumigatus vary from 0.6 to 27.8% Aspergillus spp. was cultured from the bronchial [5, 7]. However, there has been no evidence that azole- washing ﬂuid specimen. It was thought to be a true resistant A. fumigatus is a problem in Korea. Case Report A 27-year-old man who received second matched unrelated donor HSCT for relapsed acute lymphoblas- tic leukemia (ALL) (D ? 210) was hospitalized for treatment of aggravated grade IV skin graft-versus- host disease (GVHD). The patient started high-dose steroid therapy ([ 1 mg/kg per day of prednisolone) for GVHD and continued taking posaconazole (PCZ) tablets (300 mg q12 h for 2 doses and then 300 mg once daily) which had been administered for 77 days from the outpatient clinic for fungal prophylaxis in severe GVHD. During the high-dose steroid treatment, Fig. 1 Chest X-ray showed inﬁltrations in right upper and more than 10% of blasts were detected in peripheral lower lung ﬁelds 123 Mycopathologia Fig. 2 Low-dose chest computed tomography. a Ill-deﬁned centrilobular nodules in both lungs (white arrows). b Peribronchial consolidation that accompanied by ground-glass opacities pathogen because chest LDCT revealed consolida- fumigatus revealed high minimal inhibitory concen- tions with surrounding GGO and nodules consistent trations (MICs) to both itraconazole (ITZ) and PCZ with fungal pneumonia that developed during the (MICs were [ 16 and 4 lg/mL), while VCZ MIC course of long-term maintenance of high-dose steroid revealed a susceptible upper limit (1 lg/mL). Chest treatment. According to revised European Organiza- X-ray showed steady improvement, and intravenous tion for Research and Treatment of Cancer/Mycosis VCZ was changed to VCZ tablets (200 mg q12 h) and Study Group (EORTC/MSG) criteria , the patient maintained. Therapeutic drug monitoring of VCZ was was diagnosed as a culture-positive invasive pul- performed every week, and it was maintained within monary aspergillosis (IPA) with probable category. At therapeutic range between 1.2 and 4.2 lg/mL. There- the time of diagnosis of IPA, the patient had been after, the patient’s absolute neutrophil counts were receiving PCZ for 87 days, maintaining a therapeutic declined, and he experienced repeated neutropenic range of PCZ serum concentrations (1048–2232 ng/ fever. On the 69th day of hospitalization (HD 69), the mL). Therefore, this case was considered as PCZ patient died due to KPC-producing carbapenem- breakthrough IA. resistant Klebsiella pneumoniae bacteremia which PCZ was changed to intravenous voriconazole was thought to be associated with concurrent gut (VCZ) (loading dose 6 mg/kg q12 h, then 4 mg/kg GVHD. Previous lesions of IPA were decreased in size q12 h). Other antibiotics were also discontinued (from 5.4 to 1.3 cm) at that time. Subsequently, the A. because there was no evidence of other bacterial fumigatus isolate was analyzed for any known azole- pathogens. Eight days later, Aspergillus spp. was resistant mutations in CYP51A gene. The ampliﬁca- ﬁnally conﬁrmed as A. fumigatus by internal tran- tion and sequencing of CYP51A promoter were scribed spacer (ITS) sequencing and PCR of the b- performed using AFTR-F (5 -TAATCGCAGCAC- 0 0 tubulin gene as follows. Their entire ITS regions were CACTTCAG-3 ) and AFTR-R (5 -GCCTAGGA- 0 0 ampliﬁed using the primers of ITS1-F_KYO2 (5 - CAAGGACGAATG-3). Their CYP51A and 0 0 TAGAGGAAGTAAAAGTCGTAA-3 ) and ITS4 (5 - promoter sequences were compared to that of an TCCTCCGCTTATTGATATGC-3 ), as previously azole-susceptible A. fumigatus strain (GenBank acces- described . b-tubulin PCR was performed by bt2a sion no. AF338659). Including TR34/L98H, multiple 0 0 (5 -GGTAACCAAATCGGTGCTGCTTTC-3 ) and mutations including S297T in the CYP51A gene were 0 0 bt2b (5 -ACCCTCAGTGTAGTGACCCTTGGC-3 ) identiﬁed as shown in Table 1. . Amplicons of ITS and b-tubulin were sequenced and then identiﬁed using the BLASTN. Antifungal susceptibilities were determined using the broth Discussion dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 This case is that of culture-positive IPA with probable (2008) . Antifungal susceptibility testing of the A. category in an HSCT recipient, which developed 123 Mycopathologia Table 1 In vitro antifungal susceptibility proﬁle and CYP51A amino acid substitutions in A. fumigatus isolates in this case Source of PCZ exposure Antifungal MIC (lg/mL) MEC CYP51A amino acid substitutions isolate (days) ITZ VCZ PCZ AMB CAS TR34 Codon98 Others Bronchial 87 [ 16 1 4 0.5 0.06 ? L98H Y46F, V172 M, T248 N, E255D, washing ﬂuid S297T, K427E, F495L PCZ posaconazole, MIC minimum inhibitory concentration, MEC minimum effective concentration, ITZ itraconazole, VCZ voriconazole, AMB amphotericin B, CAS caspofungin during PCZ prophylaxis due to GVHD. Isolated A. not clearly characterized with phenotypic relation- fumigatus showed a high MIC for ITZ and PCZ, and ships, a recent report has suggested that S297T CYP51A gene analysis identiﬁed a TR34/L98H muta- substitution in TR34/L98H strains might represent a tion. In Korea, antifungal susceptibility epidemiology compensatory mutation showing this low VCZ MIC studies in Aspergillus spp.  have been reported, but . Although there have been no data on the azole no known gene mutation has been found. This is the resistance of A. fumigatus environmental and clinical ﬁrst case of IPA caused by TR34/L98H azole-resistant isolates in Korea, our hypothesis on the resistance A. fumigatus in Korea. mechanism of the ITZ- and PCZ-resistant strain in this Based on a systematic study of azole resistance case is a combination of environmental acquisition rates and mechanisms conducted by the Nijmegen and the acquired resistance from the patient, probably group in the Netherlands, TR34/L98H is the most due to long-term PCZ exposure. common mutation found in clinical and environmental In a national survey conducted on aspergillosis in azole-resistant A. fumigatus isolates across Europe the Netherlands, where epidemiology data are rela- [7, 14]. Environmental isolates (soil samples from tively well established, azole resistance rates have gardens, plant seeds, hospital surroundings, aerial been reported between 5 and 10%, and up to 30% in samples of hospitals) harboring TR34/L98H exhibit high-risk wards . VCZ is still the ﬁrst drug of resistance to azole fungicides, which are chemically choice of IA, even in the clinical setting of high similar to medical triazoles, showing cross-resistance resistance. There are no clear guidelines for azole- . Therefore, azole fungicides used in the environ- resistant and/or breakthrough IA . There are only ment have been suggested to induce azole resistance of expert opinions that change the antifungal class to Aspergillus spp. clinical isolates [4, 15]. Furthermore, another or the combination of VCZ plus echinocandin close genotypic accordance between A. fumigatus when the resistance threshold exceeds 10% or in the environmental and clinical isolates has been demon- case of azole breakthrough IA [1, 18, 19]. strated, and many patients harbor a single dominant Interestingly, this is an IPA case accompanying resistance mechanism, even though they are azole- inﬂuenza A pneumonia, either. Inﬂuenza has been naive or epidemiologically unrelated [4, 6, 15]. There- established a risk factor for IA since 2009 pandemic fore, it has been suggested that the main route of inﬂuenza A/H1N1 based on many case reports resistance is an acquisition from environmental source [20–24]. Inﬂuenza-associated IA has also been . Strains with the TR34/L98H mutation of CYP51A reported in Korea, even in immunocompetent patients gene also frequently harbor non-synonymous muta- [22, 23]. The interval between the diagnosis of tions in the CYP51A gene. Some of them are presented inﬂuenza and that of IA varies, and some cases have alone or in combination, which are known to be been diagnosed almost simultaneously as in this case. associated with higher MICs than the wild type [5, 7]. Although a clear mechanism between inﬂuenza and IA In this case, isolated A. fumigatus showed relatively has not yet been elucidated, cell-mediated defects, higher PCZ MIC (4 lg/mL) and lower VCZ MIC disruption of normal ciliary clearance, or virus- (1 lg/mL) compared to previously reported MICs of induced host adaptive immunity deﬁciency model TR34/L98H strains . Also, multiple amino acid has been proposed [22–25]. Inﬂuenza-associated IA substitutions including S297T (Table 1) were found in showed high mortality rate of 47–61% in a recent our isolate. Although most of these substitutions are study . However, the impact of concomitant 123 Mycopathologia clinical use of mold-active antifungal azoles? Clin Infect inﬂuenza pneumonia with IPA on mortality is not clear Dis. 2016;62(3):362–8. in this case, since the direct cause of death appears to 4. Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, be the K. pneumoniae bacteremia lasted until the day Samson RA, et al. Emergence of azole resistance in before death. Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 2008;5:e219. Mortality rates of patients with culture-positive 5. Chowdhary A, Sharma C, Meis JF. Azole-resistant azole-resistant IA have been reported as 50–100%, aspergillosis: epidemiology, molecular mechanisms, and higher than those of azole-susceptible IA [4, 5, 26]. In treatment. J Infect Dis. 2017;216:S436–44. addition, there are only limited treatment options in 6. Chowdhary A, Kathuria S, Xu J, Meis JF. Emergence of azole-resistant Aspergillus fumigatus strains due to agri- cases where azole breakthrough IA is increasing as in cultural azole use creates an increasing threat to human the present case. Currently, A. fumigatus susceptibility health. PLoS Pathog. 2013;9:e1003633. tests including MICs and CYP51A mutations are not 7. Hagiwara D, Watanabe A, Kamei K, Goldman GH. Epi- routinely available in most clinical settings. Therefore, demiological and genomic landscape of azole resistance mechanisms in aspergillus fungi. Front Microbiol. azole resistance prevalence surveillance studies are 2016;7:1382. needed for establishing the best choice of treatment. In 8. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards Korea, the ﬁrst case has been reported, and further JE, Calandra T, et al. Revised deﬁnitions of invasive fungal epidemiological surveillance studies are warranted. disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Coopera- tive Group and the National Institute of Allergy and Infec- Acknowledgements This study was supported by a Grant tious Diseases Mycoses Study Group (EORTC/MSG) from the Korean Health Technology R&D Project, Ministry of Consensus Group. Clin Infect Dis. 2008;46:1813–21. Health and Welfare, Republic of Korea (Grant No. HI15C2259). 9. Toju H, Tanabe AS, Yamamoto S, Sato H. High-coverage ITS primers for the DNA-based identiﬁcation of ascomy- Compliance with Ethical Standards cetes and basidiomycetes in environmental samples. PLoS ONE. 2012;7:e40863. Conﬂict of interest The authors declare that they have no 10. Glass NL, Donaldson GC. Development of primer sets conﬂict of interest. designed for use with the PCR to amplify conserved genes from ﬁlamentous ascomycetes. Appl Environ Microbiol. Ethical Standards This study was approved by the Institu- 1995;61:1323–30. tional Review Board of Seoul St. Mary’s Hospital at the 11. Clinical and Laboratory Standards Institute. Reference Catholic University of Korea with a waiver of informed consent Method for Broth Dilution Antifungal Susceptibility Test- (IRB No. KC16SISI0307) due to anonymous and retrospective ing of Filamentous Fungi; Approved Standard—Second design of the study. Edition. CLSI document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. Open Access This article is distributed under the terms of the 12. Morio F, Aubin GG, Danner-Boucher I, Haloun A, Sac- Creative Commons Attribution 4.0 International License (http:// chetto E, Garcia-Hermoso D, et al. High prevalence of tri- creativecommons.org/licenses/by/4.0/), which permits unre- azole resistance in Aspergillus fumigatus, especially stricted use, distribution, and reproduction in any medium, mediated by TR/L98H, in a French cohort of patients with provided you give appropriate credit to the original cystic ﬁbrosis. J Antimicrob Chemother. 2012;67:1870–3. author(s) and the source, provide a link to the Creative Com- 13. Heo MS, Shin JH, Choi MJ, Park YJ, Lee HS, Koo SH, et al. mons license, and indicate if changes were made. Molecular identiﬁcation and amphotericin B susceptibility testing of clinical isolates of Aspergillus from 11 hospitals in Korea. Ann Lab Med. 2015;35:602–10. References 14. Koehler P, Hamprecht A, Bader O, Bekeredjian-Ding I, Buchheidt D, Doelken G, et al. Epidemiology of invasive aspergillosis and azole resistance in patients with acute 1. Verweij PE, Ananda-Rajah M, Andes D, Arendrup MC, leukaemia: the SEPIA Study. Int J Antimicrob Agents. Bru¨ggemann RJ, Chowdhary A, et al. International expert 2017;49:218–23. opinion on the management of infection caused by azole- 15. Snelders E, Huis In ‘t Veld RA, Rijs AJ, Kema GH, resistant Aspergillus fumigatus. Drug Resist Updat. Melchers WJ, Verweij PE. Possible environmental origin of 2015;21–22:30–40. resistance of Aspergillus fumigatus to medical triazoles. 2. Kim SB, Cho SY, Lee DG, Choi JK, Lee HJ, Kim SH, et al. Appl Environ Microbiol. 2009;75:4053–7. Breakthrough invasive fungal diseases during voriconazole 16. van Ingen J, van der Lee HA, Rijs TA, Zoll J, Leenstra T, treatment for aspergillosis: A 5-year retrospective cohort Melchers WJ, et al. Azole, polyene and echinocandin MIC study. Med Mycol. 2017;55:237–45. distributions for wild-type, TR34/L98H and TR46/Y121F/ 3. Verweij PE, Chowdhary A, Merlchers WJ, Meis JF. Azole T289A Aspergillus fumigatus isolates in The Netherlands. Resistance in Aspergillus fumigatus: can we retain the J Antimicrob Chemother. 2015;70:178–81. 123 Mycopathologia 17. Deng S, Zhang L, Verweij PE, Tsui KM, Hagen F, Hou- 22. Kim MJ, Kim MK, Kang CK, Jun KI, Bang JH, Park SW, braken J, et al. Triazole phenotypes and genotypic charac- et al. A case of acute cerebral aspergillosis complicating terization of clinical Aspergillus fumigatus isolates in inﬂuenza A/H1N1pdm 2009. Infect Chemother. China. Emerg Microbes Infect. 2017;6(12):e109. 2013;45(2):225–9. 18. Lestrade PP, Meis JF, Arends JP, van der Beek MT, de 23. Kwon OK, Lee MG, Kim HS, Park MS, Kwak KM, Park Brauwer E, van Dijk K, et al. Diagnosis and management of SY. Invasive pulmonary aspergillosis after inﬂuenza a aspergillosis in The Netherlands: a national survey. Myco- infection in an immunocompetent patient. Tuberc Respir ses. 2016;59:101–7. Dis Seoul. 2013;75(6):260–3. 19. Patterson TF, Thompson GR 3rd, Denning DW, Fishman 24. van de Veerdonk FL, Kolwijck E, Lestrade PP, Hodiamont JA, Hadley S, Herbrecht R, et al. Practice guidelines for the CJ, Rijnders BJ, van Paassen J et al. Inﬂuenza-associated diagnosis and management of aspergillosis: 2016 update by aspergillosis in critically ill patients. Am J Respir Crit Care the infectious diseases society of America. Clin Infect Dis. Med. 2017 Apr 7. https://doi.org/10.1164/rccm.201612- 2016;63:e1–e60. 2540le (Epub ahead of print). 20. Garcia-Vidal C, Barba P, Arnan M, Moreno A, Ruiz-Camps 25. Bermejo-Martin JF, Martin-Loeches I, Rello J, Anto´nA, I, Gudiol C, et al. Invasive aspergillosis complicating pan- Almansa R, Xu L, et al. Host adaptive immunity deﬁciency demic inﬂuenza A (H1N1) infection in severely immuno- in severe pandemic inﬂuenza. Crit Care. 2010;14(5):R167. compromised patients. Clin Infect Dis. 2011;53(6):e16–9. 26. van der Linden JW, Snelders E, Kampinga GA, Rijnders BJ, 21. Lat A, Bhadelia N, Miko B, Furuya EY, Thompson GR 3rd. Mattsson E, Debets-Ossenkopp YJ, et al. Clinical implications Invasive aspergillosis after pandemic (H1N1) 2009. Emerg of azole resistance in Aspergillus fumigatus, The Netherlands, Infect Dis. 2010;16(6):971–3. 2007–2009. Emerg Infect Dis. 2011;17:1846–54.
– Springer Journals
Published: Jun 1, 2018