Antifungal susceptibility of Aspergillus genus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.
Susceptibilidad a los antifúngicos del género Aspergillus determinada por el método Etest®: once años de experiencia en el Instituto Médico La Floresta. Caracas, Venezuela.
Keywords:
susceptibility, Aspergillus spp, cryptic species, antifungals, Etest diffusion method, minimal inhibitory concentration
Abstract
This research aimed to determine the susceptibility of Aspergillus spp. to four antifungal agents using the Etest® method in several clinical samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among others) from a private health center in Venezuela. Thirty-three strains were evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillus strain was prepared on Potato Dextrose agar and then inoculated on Sabouraud agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspofungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concentrations (MIC) in μg/mL were determined after 24 and 48 hours of incubation at 35 °C and th range (R), geometric mean (GM), MIC 50, and MIC 90 were calculated. The results for the 33 Aspergillus spp. tested after 24 h were the following: VCZ (R = 0.031- 16; GM = 0.145; MIC 50 = 0.125 and MIC 90 = 0.5), AMB (R= 0.031-16; GM = 0.644; MIC 50 = 0.5 and MIC 90 = 8), CAS (R = 0.031-16; GM= 0.1076; MIC 50 = 0.063 and MIC 90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755; MIC 50 = 0.063 and MIC 90 = 0.25). This investigation allowed assessing the antifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples by the Etest® method, which is practical, reproducible and easy to perform in microbiology laboratories.
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References
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Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD, Andes D, Kontoyiannes DP, Perrone G, Peterson S, Brandt ME, Pappas PG, Chiller T. Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J Clin Microbiol 2009; 47:3138 –3141. https://doi. org/10.1128/JCM.01070-09.
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Snelders E, Van Der Lee HAL, Kuijpers J, Rijs AJ M.M, Varga J, Samson RA, Mellado E, Donders ART, Melchers WJG, Verweij PE. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLOS Medicine 2008; 5(11): 1629-1637. [e219]. https://doi.org/10.1371/journal.pmed.0050219.
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Bedin Denardi, L, Hoch Dalla-Lana B, Pantella Kunz de Jesus F, Bittencourt Severo C, Morais Santurio J, Zanette RA, Alves SH. In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil. The Braz J Infect Dis 2018; 22(1), 30–36. https://doi.org/10.1016/j. bjid.2017.10.005.
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Guinea J, Peláez T, Alcalá L, Bouza E. Correlation between the E-Test and the CLSI M38-A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus. Diagn Micr Infec Dis 2007; 57:273-276. https://doi. org/10.1016/j.diagmicrobio.2006.09.003.
Imbert S, Normand AC, Ranque S, Costa JM, Guitard J, Accoceberry I, Bonnal C, Fekkar A, Bourgeois N, Houzé S, Hennequin C, Piarroux R, Dannaoui E, Botterel F. Species identification and in vitro antifunagal susceptibility of Aspergillus terreus species complex clinical isolates from a French Multicenter Study. Antimicrob Agents Chemother 2018; 62: e02315-02317. https//doi.org/10.1128/ AAC.02315-17.
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antifungal Susceptibility Testing of Filamnetous Fungi, Approved Guideline. CLSI document M61. Wayne, PA; Clinical and Laboratory Standards Institute; 2017.
Guinea J. Updated EUCAST clinical breakpoints against Aspergillus, implications for the clinical. J Fungi 2020; 6: 343. https://doi.org/10.3390/jof6040343.
Castanheira M, Deshpande LM, Davis AP, Rhomberg PR, Pfaller MA. Monitoring antifungal resistance in a global collection of invasive yeasts and molds: application of CLSI epidemiological cut-off values and whole-genome sequencing analysis for detection of azole resistance in Candida albicans. Antimicrob Agents Chemother 2017; 22;61(10): e00906-17. https://doi. org/10.1128/AAC.00906-17.
Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 2005; 4:625– 632. https://doi.org/10.1128/EC.4.3.625-632.2005.
Pérez-Cantero A, López-Fernández L, Guarro J, Capilla J. Azole resistance mechanisms in Aspergillus: update and recent advances. Int J Antimicrob Agents 2020;55(1):105807. https://doi.org/10.1016/j.ijantimicag.2019.09.011.
Ruiz-Camps I, Cuenca-Estrella M. Antifúngicos para uso sistémico. Enferm Infecc Microbiol Clin 2009; 27(6):353-362. https:// doi.org/10.1016/j.eimc.2009.04.001.
Cornely OA, Mertens J, Winston DJ, Perfect J, Ullmann AJ, Wals TJ, Helfgott D, Holowiecki J, Stockelberg D, Goh YT, Petrini M, Hardalo C, Suresh R, Angulo- Gonzalez D. Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007; 356: 348-359. https://doi.org/10.1056/NEJMo a061094.
Buil JB, Hagen F, Chowdhary A, Verweij PE, Meis JF. Itraconazole, voriconazole, and posaconazole CLSI MIC distributions for wild-type and azole-resistant Aspergillus fumigatus isolates. J Fungi (Basel, Switzerland). 2018; 4(3): e103. https://doi.org/10.3390/jof4030103.
Fuhren J, Voskuil WS, Boel CH, Haas PJA, Hagen F, Meis JF, Kusters JG. High prevalence of azole resistance in Aspergillus fumigatus isolates from high-risk patients. J Antimicrob Chemother 2015; 70:2894–2898. https://doi.org/ 10.1093/ jac/dkv177.
Pfaller MA, Messer SA, Boyken L, Rice C, Tendolkar S, Hollis R, Diekema DJ. In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species. J Clin Microbiol 2008; 46:2568–2572. https://doi.org/ 10.1128/ JCM.00535-08.
Pham CD, Reiss E, Hagen F, Meis JF, Lockhart SR. Passive surveillance for azole resistant Aspergillus fumigatus, United States, 2011–2013. Emerg Infect Dis 2014; 20:1498–1503. https://doi.org/10.3201/eid2009.140142.
Alcazar-Fuoli L, Mellado E, Alastruey- Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL. Aspergillus Section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 2008;52(4):1244–1251. https://doi.org/10.1128/AAC.00942-07.
San Juan JL, Fernández CM, Almaguer M, Perurena MR, Martínez GF, Velar RE, IIInait MT. Sensibilidad in vitro de cepas cubanas de Aspergillus spp. de origen clínico y ambiental. Biomédica 2017; 37:452-459. https://doi.org/10.7705/biomedica. v34i2.3447.
Cavassin FB, Bau´-Carneiro JL, Vilas-Boas RR, Queiroz-Telles F. Sixty years of amphotericin B: an overview of the main antifungal agent used to treat invasive fungal infections. Infect Dis Ther 2021; 10(1):115-147. https://doi.org/10.1007/ s40121-020-00382-7.
Seo K, Akiyoshi H, Ohnishi Y. Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus. Microbiol Immunol 1999; 43:1017-1025. https://doi.org/10.1111/j.1348-0421.19 99.tb01231. x.
Mayr A, Aigner M, Lass-Flörl C. Caspofungin: ¿When and how? The microbiologist’s view. Mycoses 2011; 55:27-35. https://doi. org/10.1111/j.1439-0507.2011.02039.x.
Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, Diekema DJ. Wild-type minimum effective concentration distributions and epidemiologic cut-off values for caspofungin and Aspergillus spp. as determined by Clinical and Laboratory Standards Institute broth microdilution methods. Diagn Microbiol Infect Dis 2010; 67(1):56-60. https://doi. org/10.1016/j.diagmicrobio.2010.01.001.
Alastruey-Izquierdo A, Mellado E, Peláez T, Pemán J, Zapico S, Alvarez M, Rodriguez-Tudela JL, Cuenca-Estrella M, FILPOP Study Group. Population- based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study). Antimicrob Agents Chemother 2013; 57(7):3380–3387. https://doi. org/10.1128/AAC.00383-13.
Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD, Andes D, Kontoyiannes DP, Perrone G, Peterson S, Brandt ME, Pappas PG, Chiller T. Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J Clin Microbiol 2009; 47:3138 –3141. https://doi. org/10.1128/JCM.01070-09.
Sabino R, Gonçalves P, Melo A, Simões D, Oliveira M, Francisco M, Viegas C, Carvalho D, Martins C, Ferreira T, Toscano C, Simões H, Veríssimo C. Trends on Aspergillus epidemiology—Perspectives from a National Reference Laboratory Surveillance Program. J Fungi 2021; 7, 28. https://doi.org/10.3390/jof7010028.
Snelders E, Van Der Lee HAL, Kuijpers J, Rijs AJ M.M, Varga J, Samson RA, Mellado E, Donders ART, Melchers WJG, Verweij PE. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLOS Medicine 2008; 5(11): 1629-1637. [e219]. https://doi.org/10.1371/journal.pmed.0050219.
Cantón E, Córdoba S, Melhem M, Pemán J, Rivas P. Estudio de la sensibilidad a los antifúngicos en los pacientes con Enfermedad Fúngica Invasora. En: Aproximación Clínica Diagnóstica de la Enfermedad Fúngica Invasora. Capítulo 7. Micellium 2017.
Bassetti M, Bouza E. Invasive mould infections in the ICU setting: complexities and solutions. J Antimicrob Chemother 2017;72(1): i39-i47. https://doi. org/10.1093/jac/dkx032.
Cornely OA, Hoenigl M, Lass-Flörl C, Chen SC, Kontoyiannis DP, Morrissey CO, Thompson III GR, for the Mycoses Study Group Education and Research Consortium (MSG -ERC) and the European Con-federation of Medical Mycology (ECMM). Defining breakthrough invasive fungal infection-Position paper of the mycoses study group education and research consortium and the European Confederation of Medical Mycology. Mycoses 2019;62(9):716- 729. https://doi.org/10.1111/myc.12960.
Bedin Denardi, L, Hoch Dalla-Lana B, Pantella Kunz de Jesus F, Bittencourt Severo C, Morais Santurio J, Zanette RA, Alves SH. In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil. The Braz J Infect Dis 2018; 22(1), 30–36. https://doi.org/10.1016/j. bjid.2017.10.005.
De Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of Clinical Fungi. Segunda edición. Central bureau voor Schimmel cultures, Utrecht: Universitat Rovira i Virgili, Reus; 2000. p. 442-519.
Klich MA, Pitt JI. A laboratory guide to the common Aspergillus species and their 19 teleomorphs. Canberra: CSIRO Division of Food Processing. 2da. Edition. 1998. p. 116.
Espinel-Ingroff A, Rezusta A. E-test method for testing susceptibilities of Aspergillus spp. to the new triazoles voriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method. J Clin Microbiol 2002; 40(6):2101-2107. https://doi.org/10.1128/JCM.40.6.2101-2107.2002.
Guinea J, Peláez T, Alcalá L, Bouza E. Correlation between the E-Test and the CLSI M38-A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus. Diagn Micr Infec Dis 2007; 57:273-276. https://doi. org/10.1016/j.diagmicrobio.2006.09.003.
Imbert S, Normand AC, Ranque S, Costa JM, Guitard J, Accoceberry I, Bonnal C, Fekkar A, Bourgeois N, Houzé S, Hennequin C, Piarroux R, Dannaoui E, Botterel F. Species identification and in vitro antifunagal susceptibility of Aspergillus terreus species complex clinical isolates from a French Multicenter Study. Antimicrob Agents Chemother 2018; 62: e02315-02317. https//doi.org/10.1128/ AAC.02315-17.
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antifungal Susceptibility Testing of Filamnetous Fungi, Approved Guideline. CLSI document M61. Wayne, PA; Clinical and Laboratory Standards Institute; 2017.
Guinea J. Updated EUCAST clinical breakpoints against Aspergillus, implications for the clinical. J Fungi 2020; 6: 343. https://doi.org/10.3390/jof6040343.
Castanheira M, Deshpande LM, Davis AP, Rhomberg PR, Pfaller MA. Monitoring antifungal resistance in a global collection of invasive yeasts and molds: application of CLSI epidemiological cut-off values and whole-genome sequencing analysis for detection of azole resistance in Candida albicans. Antimicrob Agents Chemother 2017; 22;61(10): e00906-17. https://doi. org/10.1128/AAC.00906-17.
Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 2005; 4:625– 632. https://doi.org/10.1128/EC.4.3.625-632.2005.
Pérez-Cantero A, López-Fernández L, Guarro J, Capilla J. Azole resistance mechanisms in Aspergillus: update and recent advances. Int J Antimicrob Agents 2020;55(1):105807. https://doi.org/10.1016/j.ijantimicag.2019.09.011.
Ruiz-Camps I, Cuenca-Estrella M. Antifúngicos para uso sistémico. Enferm Infecc Microbiol Clin 2009; 27(6):353-362. https:// doi.org/10.1016/j.eimc.2009.04.001.
Cornely OA, Mertens J, Winston DJ, Perfect J, Ullmann AJ, Wals TJ, Helfgott D, Holowiecki J, Stockelberg D, Goh YT, Petrini M, Hardalo C, Suresh R, Angulo- Gonzalez D. Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007; 356: 348-359. https://doi.org/10.1056/NEJMo a061094.
Buil JB, Hagen F, Chowdhary A, Verweij PE, Meis JF. Itraconazole, voriconazole, and posaconazole CLSI MIC distributions for wild-type and azole-resistant Aspergillus fumigatus isolates. J Fungi (Basel, Switzerland). 2018; 4(3): e103. https://doi.org/10.3390/jof4030103.
Fuhren J, Voskuil WS, Boel CH, Haas PJA, Hagen F, Meis JF, Kusters JG. High prevalence of azole resistance in Aspergillus fumigatus isolates from high-risk patients. J Antimicrob Chemother 2015; 70:2894–2898. https://doi.org/ 10.1093/ jac/dkv177.
Pfaller MA, Messer SA, Boyken L, Rice C, Tendolkar S, Hollis R, Diekema DJ. In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species. J Clin Microbiol 2008; 46:2568–2572. https://doi.org/ 10.1128/ JCM.00535-08.
Pham CD, Reiss E, Hagen F, Meis JF, Lockhart SR. Passive surveillance for azole resistant Aspergillus fumigatus, United States, 2011–2013. Emerg Infect Dis 2014; 20:1498–1503. https://doi.org/10.3201/eid2009.140142.
Alcazar-Fuoli L, Mellado E, Alastruey- Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL. Aspergillus Section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 2008;52(4):1244–1251. https://doi.org/10.1128/AAC.00942-07.
San Juan JL, Fernández CM, Almaguer M, Perurena MR, Martínez GF, Velar RE, IIInait MT. Sensibilidad in vitro de cepas cubanas de Aspergillus spp. de origen clínico y ambiental. Biomédica 2017; 37:452-459. https://doi.org/10.7705/biomedica. v34i2.3447.
Cavassin FB, Bau´-Carneiro JL, Vilas-Boas RR, Queiroz-Telles F. Sixty years of amphotericin B: an overview of the main antifungal agent used to treat invasive fungal infections. Infect Dis Ther 2021; 10(1):115-147. https://doi.org/10.1007/ s40121-020-00382-7.
Seo K, Akiyoshi H, Ohnishi Y. Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus. Microbiol Immunol 1999; 43:1017-1025. https://doi.org/10.1111/j.1348-0421.19 99.tb01231. x.
Mayr A, Aigner M, Lass-Flörl C. Caspofungin: ¿When and how? The microbiologist’s view. Mycoses 2011; 55:27-35. https://doi. org/10.1111/j.1439-0507.2011.02039.x.
Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, Diekema DJ. Wild-type minimum effective concentration distributions and epidemiologic cut-off values for caspofungin and Aspergillus spp. as determined by Clinical and Laboratory Standards Institute broth microdilution methods. Diagn Microbiol Infect Dis 2010; 67(1):56-60. https://doi. org/10.1016/j.diagmicrobio.2010.01.001.
Published
2023-11-20
How to Cite
Moreno Calderón, X., Macero Estévez , C., & Oliveira Oliveira, D. (2023). Antifungal susceptibility of Aspergillus genus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.: Susceptibilidad a los antifúngicos del género Aspergillus determinada por el método Etest®: once años de experiencia en el Instituto Médico La Floresta. Caracas, Venezuela. Investigación Clínica, 64(4), 471-481. https://doi.org/10.54817/IC.v64n4a4
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