Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
letter
. 2019 May 24;57(6):e00404-19. doi: 10.1128/JCM.00404-19

Triazole Antifungal Susceptibility Patterns among Aspergillus Species in Québec, Canada

Matthew P Cheng a,b,c,, Alexander Lawandi d, Todd C Lee d,e, Philippe J Dufresne f, Danila Seidel g,h, Paul E Verweij i, Oliver A Cornely g,h,j, Donald C Sheppard d,e,k
Editor: Geoffrey A Landl
PMCID: PMC6535597  PMID: 30918044

LETTER

Invasive aspergillosis (IA) is an important cause of morbidity and mortality among immunocompromised patients (13), with over 500 patients developing IA in Canada each year (4). The overall mortality rate of IA is approximately 25% (5, 6) but increases to over 60% in the setting of infection with azole-resistant Aspergillus fumigatus (ARAF) (79). Given the spread of ARAF to countries in Europe (10), Asia (11, 12), Africa, the Middle East, and the Americas (13), we sought to determine the prevalence of azole resistance among Aspergillus isolates in the province of Québec, Canada.

We performed antimicrobial susceptibility testing on all Aspergillus species isolated from sterile sites and respiratory specimens that were processed at the McGill University Health Centre (MUHC) between March 2018 and December 2018. The MUHC is a university-affiliated tertiary-care hospital in Montreal that serves as the central mycology laboratory for 10 hospitals and a population of 1.8 million in the province of Québec.

Clinical isolates of Aspergillus were identified to the species level by conventional microscopy techniques. For a given isolate, sporulating colonies were picked and subcultured on VIPcheck plates (Mediaproducts BV, the Netherlands) and incubated per the manufacturer’s instructions. The VIPcheck plate has a sensitivity of 98% and a specificity of 93% for detecting azole resistance in Aspergillus species (14). Briefly, a moist, sterile swab collected conidia from the Aspergillus colony. A suspension with a 0.5 to 2 McFarland standard was then created in sterile water. All 4 wells of the VIPcheck plate were inoculated with a single drop of the suspension. Finally, the plate was incubated and read after 24 and 48 h. Growth in any well except the control well indicated the possibility of an azole-resistant isolate. Isolates with positive screening results were sent to the Laboratoire de Santé Publique du Québec (LSPQ; the provincial reference center) for molecular identification (by sequencing of internal transcribed spacer [ITS] and BenA regions) and antifungal susceptibility confirmatory testing by broth microdilution per the Clinical and Laboratory Standards Institute method (15, 16). Epidemiological cutoff values (ECV) were used to infer antifungal susceptibility (17).

In total, 113 isolates were received from 5 different hospital centers across the province of Québec. The complete list of species and the results of antifungal susceptibility testing are presented in Table 1. The most common species identified were Aspergillus fumigatus (n = 86; 76.1%) followed by Aspergillus flavus (n = 12; 10.6%), Aspergillus niger (n = 5; 4.4%), and Aspergillus versicolor (n = 4; 3.5%). Only a single isolate (0.8% of total tested) of Aspergillus glaucus screened positive for azole nonsusceptibility in the VIPcheck plate. This isolate had MICs to itraconazole, voriconazole, and posaconazole of 0.25 mg/liter, 0.5 mg/liter, and 0.12 mg/liter, respectively. While there are no defined ECV for A. glaucus, the values are below the ECV for other Aspergillus species (17) and the isolate was thus presumed susceptible. The prevalence of azole resistance in our population is estimated at 0% (95% confidence interval [CI], 0 to 3.2%).

TABLE 1.

Antifungal susceptibility testing results with VIPcheck plates

Antifungal agent A. fumigatus
(n = 86)
A. flavus
(n = 12)
A. niger
(n = 5)
A. versicolor
(n = 4)
A. glaucus
(n = 2)
Aspergillus calidoustus
(n = 2)
Other species
(n = 2)a
Itraconazole (no. [%])b 0 (0) 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 0 (0)
Voriconazole (no. [%]) 0 (0) 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 0 (0)
Posaconazole (no. [%]) 0 (0) 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 0 (0)
Any azole (no. [%]) 0 (0) 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 0 (0)
a

The other species included Aspergillus terreus and Aspergillus nidulans.

b

Values refer to the number and percentage of isolates that had a positive screening test with VIPcheck plates.

While patients with IA treated with an azole may require changes in therapy due to a lack of clinical efficacy, these cases can often be successfully treated with a different azole antifungal (18). Our data suggest that microbiologically confirmed azole resistance is rare in the province of Québec. Therefore, antifungal resistance to triazole antifungals is unlikely a significant contributor to clinical failure in patients with IA in our geographic area, and other factors, including host immune status, antifungal pharmacokinetics, and incorrect diagnosis, should be considered first.

Our results must be interpreted in the context of the study characteristics. Our epidemiologic study did not examine patient outcomes and, therefore, only indirectly addresses the potential causes of treatment failure with azole antifungals. Although the VIPcheck is not 100% sensitive, assuming even a 5% pretest probability of azole resistance, the negative predictive value of the VIPcheck remains excellent at 99.9%, which gives us confidence in our estimate. Our results are thus informative regarding the risk of antifungal resistance across a large geographical area and suggest that the rate of azole antifungal resistance among Aspergillus species is low in the province of Québec.

ACKNOWLEDGMENTS

M.P.C. was supported by a postdoctoral training grant from Fonds de Recherche Québec Santé (FRQS). D.C.S. was supported by a Research Chair from the FRQS. VIPcheck plates were provided through a research grant by Gilead to O.A.C.

M.P.C., A.L., T.C.L., P.J.D., D.S., and D.C.S. have no conflicts of interest to disclose. P.E.V. reports research grants from Merck/MSD and F2G; is a consultant to Amplyx, Cidara, and F2G; and received lecture honoraria from Pfizer, Gilead Sciences, F2G and Merck/MSD. O.A.C. reports research grants from Actelion, Amplyx, Astellas, Basilea, Cidara, Da Volterra, F2G, Gilead, Janssen Pharmaceuticals, Medicines Company, Medpace, Melinta Therapeutics, Merck/MSD, Pfizer, and Scynexis; is a consultant to Actelion, Allecra Therapeutics, Amplyx, Astellas, Basilea, Biosys UK Limited, Cidara, Da Volterra, F2G, Gilead, IQVIA, Matinas, Medpace, Menarini Ricerche, Merck/MSD, Octapharma, Paratek Pharmaceuticals, Pfizer, PSI, Rempex, Scynexis, Seres Therapeutics, Tetraphase, and Vical; and received lecture honoraria from Astellas, Basilea, Gilead, Merck/MSD and Pfizer.

REFERENCES

  • 1.Pappas PG, Alexander BD, Andes DR, Hadley S, Kauffman CA, Freifeld A, Anaissie EJ, Brumble LM, Herwaldt L, Ito J, Kontoyiannis DP, Lyon GM, Marr KA, Morrison VA, Park BJ, Patterson TF, Perl TM, Oster RA, Schuster MG, Walker R, Walsh TJ, Wannemuehler KA, Chiller TM. 2010. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis 50:1101–1111. doi: 10.1086/651262. [DOI] [PubMed] [Google Scholar]
  • 2.Singh N, Paterson DL. 2005. Aspergillus infections in transplant recipients. Clin Microbiol Rev 18:44–69. doi: 10.1128/CMR.18.1.44-69.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Taccone F, Van den Abeele A-M, Bulpa P, Misset B, Meersseman W, Cardoso T, Paiva J-A, Blasco-Navalpotro M, De Laere E, Dimopoulos G, Rello J, Vogelaers D, Blot SI. 2015. Epidemiology of invasive aspergillosis in critically ill patients: clinical presentation, underlying conditions, and outcomes. Crit Care 19:7. doi: 10.1186/s13054-014-0722-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Dufresne SF, Cole DC, Denning DW, Sheppard DC. 2017. Serious fungal infections in Canada. Eur J Clin Microbiol Infect Dis 36:987–992. doi: 10.1007/s10096-017-2922-y. [DOI] [PubMed] [Google Scholar]
  • 5.Bitar D, Lortholary O, Le Strat Y, Nicolau J, Coignard B, Tattevin P, Che D, Dromer F. 2014. Population-based analysis of invasive fungal infections, France, 2001–2010. Emerg Infect Dis 20:1149–1155. doi: 10.3201/eid2007.140087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Slobbe L, Polinder S, Doorduijn JK, Lugtenburg PJ, el Barzouhi A, Steyerberg EW, Rijnders BJA. 2008. Outcome and medical costs of patients with invasive aspergillosis and acute myelogenous leukemia–myelodysplastic syndrome treated with intensive chemotherapy: an observational study. Clin Infect Dis 47:1507–1512. doi: 10.1086/591531. [DOI] [PubMed] [Google Scholar]
  • 7.Steinmann J, Hamprecht A, Vehreschild MJGT, Cornely OA, Buchheidt D, Spiess B, Koldehoff M, Buer J, Meis JF, Rath P-M. 2015. Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany. J Antimicrob Chemother 70:1522–1526. doi: 10.1093/jac/dku566. [DOI] [PubMed] [Google Scholar]
  • 8.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]
  • 9.Lestrade P, Bentvelsen RG, Schauwvlieghe AFAD, Schalekamp S, van der Velden WJFM, Kuiper EJ, van Paassen J, van der Hoven B, van der Lee HA, Melchers WJG, de Haan AF, van der Hoeven HL, Rijnders BJA, van der Beek MT, Verweij PE. 2018. Voriconazole resistance and mortality in invasive aspergillosis: a multicenter retrospective cohort study. Clin Infect Dis doi: 10.1093/cid/ciy859. [DOI] [PubMed] [Google Scholar]
  • 10.Mortensen KL, Mellado E, Lass-Florl C, Rodriguez-Tudela JL, Johansen HK, Arendrup MC. 2010. Environmental study of azole-resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain. Antimicrob Agents Chemother 54:4545–4549. doi: 10.1128/AAC.00692-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chowdhary A, Kathuria S, Randhawa HS, Gaur SN, Klaassen CH, Meis JF. 2012. Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India. J Antimicrob Chemother 67:362–366. doi: 10.1093/jac/dkr443. [DOI] [PubMed] [Google Scholar]
  • 12.Liu M, Zeng R, Zhang L, Li D, Lv G, Shen Y, Zheng H, Zhang Q, Zhao J, Zheng N, Liu W. 2015. Multiple cyp51A-based mechanisms identified in azole-resistant isolates of Aspergillus fumigatus from China. Antimicrob Agents Chemother 59:4321–4325. doi: 10.1128/AAC.00003-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wiederhold NP, Gil VG, Gutierrez F, Lindner JR, Albataineh MT, McCarthy DI, Sanders C, Fan H, Fothergill AW, Sutton DA. 2016. First detection of TR34 L98H and TR46 Y121F T289A Cyp51 mutations in Aspergillus fumigatus isolates in the United States. J Clin Microbiol 54:168–171. doi: 10.1128/JCM.02478-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Buil JB, van der Lee HAL, Rijs AJMM, Zoll J, Hovestadt JAMF, Melchers WJG, Verweij PE. 2017. Single-center evaluation of an agar-based screening for azole resistance in Aspergillus fumigatus by using VIPcheck. Antimicrob Agents Chemother 61:e01250-17. doi: 10.1128/AAC.01250-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Clinical and Laboratory Standards Institute. 2017. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. CLSI document M38, 3rd ed Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 16.Clinical and Laboratory Standards Institute. 2017. Performance standards for antifungal susceptibility testing of filamentous fungi. CLSI document M61, 1st ed Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 17.Clinical and Laboratory Standards Institute. 2018. Epidemiological cutoff values for antifungal susceptibility testing. CLSI supplement M59, 2nd ed Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 18.Cheng MP, Orejas JL, Arbona-Haddad E, Marty FM, Koo S, Hammond S. 2018. Comparison of voriconazole (VORI), isavuconazole (ISAV), and posaconazole (POSA) in the initial treatment of patients with invasive aspergillosis (IA). Open Forum Infect Dis 5:S140–S141. doi: 10.1093/ofid/ofy210.369. [DOI] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES