Abstract
The aims of the study were to investigate the prevalence of azole resistance among Aspergillus fumigatus clinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates.
TEXT
Aspergillus fumigatus is generally susceptible to the triazole antifungals, such as itraconazole, voriconazole, and posaconazole; over the past decade, however, azole resistance was emerged throughout the world (1). Resistant isolates have been found both in patients undergoing azole treatment and in azole-naive patients, as well as in the environment. In the prospective Surveillance Collaboration on Aspergillus Resistance in Europe (SCARE) study, the rate of azole resistance among A. fumigatus clinical isolates was 3.2% (range, 0 to 26.1%) (2).
Several resistance mechanisms have been identified, most caused by point mutations in the cyp51A gene, which encodes the azole target enzyme (i.e., lanosterol 14-α-demethylase) required for the biosynthesis of ergosterol. Some mutations are responsible for multiazole resistance, and this resistance has a great impact on the outcomes for patients with both invasive aspergillosis and chronic disease, because it reduces the few therapeutic options available (3). The most frequently reported substitution is in codon 98, consisting of substitution of a histidine with a leucine always in association with a 34-bp tandem repeat in the promoter region (TR34/L98H), resulting in cyp51A overexpression (4). This molecular mechanism was initially found in clinical and environmental isolates from the Netherlands (5). Some studies suggest that the widespread use of triazole fungicides in agriculture might have selected strains carrying this substitution (6, 7). The aims of the present study were to investigate the prevalence of azole resistance among A. fumigatus clinical isolates from a large Italian historical culture collection and to identify the year in which the TR34/L98H substitution appeared in Italy.
A total of 533 A. fumigatus clinical isolates, from 441 patients, that had been collected between 1995 and 2006 and were stored in the Laboratory of Medical Mycology of the Università degli Studi di Milano were screened on Sabouraud dextrose agar (SDA) plates with azoles added, to detect azole resistance. Briefly, fresh conidia from 3-day-old cultures on SDA were suspended in sterile distilled water with 0.05% Tween 20 added (Sigma, St. Louis, MO, USA), to reach turbidity of about a 0.5 McFarland standard. The suspension was inoculated on three plates containing SDA supplemented with azoles and on one azole-free plate, as a control. The plates were prepared by adding azoles dissolved in dimethyl sulfoxide (Sigma) to reach final concentrations of 4 mg/liter, 1 mg/liter, and 0.5 mg/liter for itraconazole (Sigma), voriconazole (Sigma), and posaconazole (Sigma), respectively. The method was preliminarily validated using five azole-susceptible A. fumigatus isolates and five azole-resistant A. fumigatus isolates with different mechanisms of resistance. A resistant A. fumigatus strain (IUM 11-0396; MICs of >16, 2, and 2 mg/liter for itraconazole, voriconazole, and posaconazole, respectively) was used to validate each test. Plates were incubated at 37°C and examined after 48 h (Fig. 1).
FIG 1.
Screening for azole resistance. Isolates were subcultured on SDA plates with added itraconazole (4 mg/liter), voriconazole (1 mg/liter), or posaconazole (0.5 mg/liter).
Isolates grown on azole-containing agar plates were tested with the broth microdilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol (8). Tests were performed in duplicate. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality controls. The isolates were considered resistant, intermediate, or susceptible according to the EUCAST breakpoints (9, 10), i.e., isolates with itraconazole and voriconazole MICs of ≥4 mg/liter and those with posaconazole MICs of ≥0.5 mg/liter were considered resistant; those with itraconazole and voriconazole MICs of ≤1 mg/liter and those with posaconazole MICs of ≤0.12 mg/liter were considered susceptible. Genomic DNA was extracted from the azole-resistant isolates using PrepMan Ultra sample preparation reagents (Applied Biosystems, Foster City, CA).
A. fumigatus sensu strictu isolates were identified by amplification and sequencing of a portion of the β-tubulin gene, as described previously (4). In addition, the entire cyp51A gene and its promoter were sequenced, as described previously (11), to detect the presence of point mutations. The cyp51A sequence from A. fumigatus strain 237 (GenBank accession no. AF338659) was used as a wild-type reference.
Azole-resistant isolates were genotyped by microsatellite analysis using the primers STRAf3A, STRAf3B, STRAf3C, STRAf4A, STRAf4B, and STRAf4C, as described previously (12). The results were compared with those obtained previously for a set of resistant environmental isolates from Italy (11).
A total of 78 isolates showed positive or weak growth on at least one azole-containing plate; 31 isolates were confirmed to be resistant by using the broth microdilution method. Table 1 shows the number of itraconazole-resistant isolates according to the year of isolation. No resistance was detected in isolates collected between 1995 and 1997 (85 isolates from 62 patients). Beginning in 1998, the resistance rate was 6.9%, i.e., 31 resistant isolates of 448 screened isolates. A total of 24 patients (6.25%) harbored a resistant isolate. This resistance rate is consistent with data from other European countries (13).
TABLE 1.
Itraconazole resistance according to year of isolation
| Year | No. of tested isolates | No. of patients | No. (%) of itraconazole-resistant isolatesa | No. (%) of patients harboring resistant isolates |
|---|---|---|---|---|
| 1995 | 33 | 24 | 0 | 0 |
| 1996 | 21 | 15 | 0 | 0 |
| 1997 | 31 | 23 | 0 | 0 |
| 1998 | 41 | 26 | 5 (12.2) | 5 (19.2) |
| 1999 | 37 | 32 | 4 (10.8) | 2 (6.3) |
| 2000 | 49 | 48 | 3 (6.1) | 3 (6.3) |
| 2001 | 57 | 57 | 4 (7.0) | 4 (7.0) |
| 2002 | 60 | 51 | 4 (6.7) | 3 (5.9) |
| 2003 | 81 | 58 | 8 (9.9) | 4 (6.9) |
| 2004 | 62 | 54 | 1 (1.6) | 1 (1.9) |
| 2005 | 37 | 36 | 0 | 0 |
| 2006 | 24 | 17 | 2 (8.3) | 2 (11.8) |
MIC of >2 mg/liter.
The emergence of azole-resistant strains in 1998 is supported by the network analysis of microsatellite genotyping results (Fig. 2), showing all 1998 clinical isolates located near the core region of the network, where the hypothetical ancestral resistant strain originated. In contrast, most of the environmental isolates collected recently (2011 to 2014) were divergent from the ancestral genotype, suggesting the emergence of new genotypes harboring the azole-resistant mutations.
FIG 2.
Network analysis of the microsatellite genotyping results as drawn by Network v4.6 (Fluxus Technology Ltd.), using the median joining algorithm. Gray circles, clinical isolates (a single isolate from each patient); black circles, environmental isolates.
The characteristics of the resistant isolates, the MIC values, and the types of substitutions are reported in Table 2. All of the isolates were resistant or intermediate to itraconazole and posaconazole and 63% to all three azoles. The TR34/L98H substitution was found in isolates from 17 patients, with a prevalence of 3.8%, as high as that found in the Netherlands (13). Four cystic fibrosis patients undergoing azole therapy harbored isolates with the G54E substitution. Two isolates did not show mutations in the cyp51A gene. All of the resistant isolates were identified as A. fumigatus sensu strictu.
TABLE 2.
Characteristics of resistant isolates
| Isolate no. | Biological sample typea | Date of isolation (day/mo/yr) | Underlying disease/predisposing factor | MIC (mg/liter) |
cyp51A alteration | ||
|---|---|---|---|---|---|---|---|
| Itraconazole | Posaconazole | Voriconazole | |||||
| 98-0264 | Sputum | 23/1/1998 | Bone marrow transplantation | 4 | 0.25 | 2 | L98H |
| 98-0398 | Bronchial secretions | 4/2/1998 | Unknown | 4 | 0.25 | 1 | L98H |
| 98-0420/2 | Bronchial secretions | 5/2/1998 | Intensive care | 2 | 1 | 1 | L98H |
| 98-0834 | Bronchial secretions | 3/3/1998 | Chronic lymphatic leukemia | 16 | 1 | 2 | L98H |
| 98-0873 | BAL fluid | 3/3/1998 | Solid cancer | 2 | 0.5 | 2 | L98H |
| 99-3091 | Bronchial secretions | 9/7/1999 | Surgery | 2 | 1 | 1 | L98H |
| 99-4535 | Sputum | 19/10/1999 | Cystic fibrosis | >16 | 1 | 0.06 | G54E |
| 99-5383b | Sputum | 13/12/1999 | Cystic fibrosis | >16 | 1 | 0.12 | G54E |
| 99-5384b | Sputum | 13/12/1999 | Cystic fibrosis | >16 | 1 | 2 | G54E |
| 00-0080 | Nasal secretions | 4/1/2000 | Otitis | 8 | 0.5 | 2 | L98H |
| 00-0574 | CSF | 4/2/2000 | Intensive care | >16 | 1 | 4 | L98H |
| 00-0542 | Sputum | 2/2/2000 | Unknown | >16 | 0.5 | 4 | L98H |
| 01-4707 | Sputum | 29/12/2001 | Liver transplantation | >16 | 0.25 | 2 | Unknown |
| 01-3192 | Sputum | 1/9/2001 | Bone marrow transplantation | 4 | 0.5 | 2 | L98H |
| 01-2312 | Ear secretions | 8/6/2001 | Otitis | >16 | 1 | 2 | Unknown |
| 01-0368 | Sputum | 24/1/2001 | Unknown | >16 | 1 | 4 | L98H |
| 02-2693/2 | BAL fluid | 8/8/2002 | Hematological malignancy | >16 | 0.25 | 2 | L98H |
| 02-0819/1 | CSF | 26/2/2002 | Intensive care | >16 | 0.25 | >16 | L98H |
| 02-2959 | Sputum | 10/9/2002 | Cystic fibrosis | >16 | 0.5 | 0.12 | G54E |
| 02-3417c | Sputum | 23/10/2002 | Cystic fibrosis | >16 | 1 | 0.25 | G54E |
| 03-202800 | Nasal secretions | 24/6/2003 | Chronic lymphatic leukemia | 8 | 2 | 4 | L98H |
| 03-203060d | Nasal secretions | 10/7/2003 | Chronic lymphatic leukemia | 2 | 0.5 | 1 | L98H |
| 03-203243/1d | Nasal secretions | 23/7/2003 | Chronic lymphatic leukemia | 2 | 1 | 1 | L98H |
| 03-202815d | Nasal secretions | 27/6/2003 | Chronic lymphatic leukemia | 8 | 1 | 1 | L98H |
| 03-203905/1 | BAL fluid | 12/9/2003 | Surgery | 4 | 2 | 2 | L98H |
| 03-200789/1 | Sputum | 3/2/2003 | Non-Hodgkin lymphoma | 2 | 0.5 | 2 | L98H |
| 03-200779e | BAL fluid | 4/2/2003 | Non-Hodgkin lymphoma | 8 | 2 | 4 | L98H |
| 03-203093 | Biopsy | 10/7/2003 | Intensive care | 2 | 2 | 2 | L98H |
| 04-52006446 | Sputum | 11/2/2004 | Cystic fibrosis | >16 | 1 | 0.25 | G54E |
| 06-0135/2 | Sputum | 18/5/2006 | Cystic fibrosis | >16 | 1 | 0.25 | G54E |
| 06-0170 | Sputum | 10/8/2006 | Cystic fibrosis | 8 | ND | 0.25 | ND |
BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; ND, not determined.
Same patient as for 99-4535.
Same patient as for 02-2959.
Same patient as for 03-202800.
Same patient as for 03-200789/1.
The limits of this study were the poor clinical data available, mainly regarding the role of A. fumigatus and the administered antifungal treatments, and the inability of 14% of the isolates to grow after a long period of storage. In conclusion, the present study shows a 6.25% resistance rate, as high as rates in other European countries, and frequent multiazole resistance associated with the TR34/L98H substitution, which has been present in our country since 1998.
ACKNOWLEDGMENT
We have no conflicts of interest to declare.
Funding Statement
This study was partially supported by the Gilead Fellowship Program 2015.
REFERENCES
- 1.Verweij PE, Chowdhary A, Melchers WJG, Meis JF. 2015. Azole resistance in Aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles. Clin Infect Dis doi: 10.1093/cid/civ885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.van der Linden JWM, Arendrup MC, Warris A, Lagrou K, Pelloux H, Hauser PM, Chryssanthou E, Mellado E, Kidd SE, Tortorano AM, Dannaoui E, Gaustad P, Baddley JW, Uekötter A, Lass-Flörl C, Klimko N, Moore CB, Denning DW, Pasqualotto AC, Kibbler C, Arikan-Akdagli S, Andes D, Meletiadis J, Naumiuk L, Nucci M, Melchers WJG, Verweij PE. 2015. Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus. Emerg Infect Dis 21:1041–1044. doi: 10.3201/eid2106.140717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Denning DW, Bowyer P. 2013. Voriconazole resistance in Aspergillus fumigatus: should we be concerned? Clin Infect Dis 57:521–523. doi: 10.1093/cid/cit321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Mellado E, Garcia-Effron G, Alcázar-Fuoli L, Melchers WJ, Verweij PE, Cuenca-Estrella M, Rodríguez-Tudela JL. 2007. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother 51:1897–1904. doi: 10.1128/AAC.01092-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Verweij PE, Te Dorsthorst DT, Rijs AJ, De Vries-Hospers HG, Meis JF. 2002. Nationwide survey of in vitro activities of itraconazole and voriconazole against clinical Aspergillus fumigatus isolates cultured between 1945 and 1998. J Clin Microbiol 40:2648–2650. doi: 10.1128/JCM.40.7.2648-2650.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Snelders E, Camps SM, Karawajczyk A, Schaftenaar G, Kema GH, van der Lee HA, Klaassen CH, Melchers WJ, Verweij PE. 2012. Triazole fungicides can induce cross-resistance to medical triazoles in Aspergillus fumigatus. PLoS One 7:e31801. doi: 10.1371/journal.pone.0031801. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. 2009. Azole resistance in A. fumigatus: a side effect of environmental fungicide use? Lancet Infect Dis 9:789–795. doi: 10.1016/S1473-3099(09)70265-8. [DOI] [PubMed] [Google Scholar]
- 8.Rodriguez-Tudela JL, Donnelly JP, Arendrup MC, Arikan S, Barchiesi F, Bille J, Chryssanthou E, Cuenca-Estrella M, Dannaoui E, Denning DW, Fegeler W, Gaustad P, Lass-Flörl C, Moore C, Richardson M, Schmalreck A, Velegraki A, Verweij P. 2008. EUCAST technical note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming moulds. Clin Microbiol Infect 14:982–984. doi: 10.1111/j.1469-0691.2008.02086.x. [DOI] [PubMed] [Google Scholar]
- 9.Arendrup MC, Cuenca-Estrella M, Lass-Florl C, Hope WW, European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing. 2012. EUCAST technical note on Aspergillus and amphotericin B, itraconazole, and posaconazole. Clin Microbiol Infect 18:E248–E250. doi: 10.1111/j.1469-0691.2012.03890.x. [DOI] [PubMed] [Google Scholar]
- 10.Hope WW, Cuenca-Estrella M, Lass-Florl C, Arendrup MC. 2013. EUCAST technical note on voriconazole and Aspergillus spp. Clin Microbiol Infect 19:E278–E280. doi: 10.1111/1469-0691.12148. [DOI] [PubMed] [Google Scholar]
- 11.Prigitano A, Venier V, Cogliati M, De Lorenzis G, Esposto MC, Tortorano AM. 2014. Azole-resistant Aspergillus fumigatus in the environment of northern Italy, May 2011 to June 2012. Euro Surveill 19(12):pii=20747 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20747. [DOI] [PubMed] [Google Scholar]
- 12.de Valk HA, Meis JF, Curfs IM, Muehlethaler K, Mouton JW, Klaassen CH. 2005. Use of a novel panel of nine short tandem repeats for exact and high-resolution fingerprinting of Aspergillus fumigatus isolates. J Clin Microbiol 43:4112–4120. doi: 10.1128/JCM.43.8.4112-4120.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Vermeulen E, Lagrou K, Verweij PE. 2013. Azole resistance in Aspergillus fumigatus: a growing public health concern. Curr Opin Infect Dis 26:493–500. doi: 10.1097/QCO.0000000000000005. [DOI] [PubMed] [Google Scholar]