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Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei

Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a...

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Autores principales: Thu, Nguyen Thi Mai, Borda, Hannah, Vitsupakorn, Shawin, Reddy, Kaushik Sreerama, Kasmani, Navsin, Barwatt, Joseph, Schwartz, Ilan S, Giamberardino, Charles, Perfect, John R, Hoa, Ngo Thi, Le, Thuy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10681740/
https://www.ncbi.nlm.nih.gov/pubmed/37994652
http://dx.doi.org/10.1093/mmy/myad111
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author Thu, Nguyen Thi Mai
Borda, Hannah
Vitsupakorn, Shawin
Reddy, Kaushik Sreerama
Kasmani, Navsin
Barwatt, Joseph
Schwartz, Ilan S
Giamberardino, Charles
Perfect, John R
Hoa, Ngo Thi
Le, Thuy
author_facet Thu, Nguyen Thi Mai
Borda, Hannah
Vitsupakorn, Shawin
Reddy, Kaushik Sreerama
Kasmani, Navsin
Barwatt, Joseph
Schwartz, Ilan S
Giamberardino, Charles
Perfect, John R
Hoa, Ngo Thi
Le, Thuy
author_sort Thu, Nguyen Thi Mai
collection PubMed
description Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008–0.025 μg/ml for itraconazole, 0.004–0.13 μg/ml for voriconazole, 0.03–0.13 μg/ml for posaconazole, 0.06–0.5 µg/ml for flucytosine, 0.5–1 µg/ml for amphotericin B, 0.5–4 µg/ml for caspofungin, and 0.5–16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%–100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.
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spelling pubmed-106817402023-11-22 Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei Thu, Nguyen Thi Mai Borda, Hannah Vitsupakorn, Shawin Reddy, Kaushik Sreerama Kasmani, Navsin Barwatt, Joseph Schwartz, Ilan S Giamberardino, Charles Perfect, John R Hoa, Ngo Thi Le, Thuy Med Mycol Original Article Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008–0.025 μg/ml for itraconazole, 0.004–0.13 μg/ml for voriconazole, 0.03–0.13 μg/ml for posaconazole, 0.06–0.5 µg/ml for flucytosine, 0.5–1 µg/ml for amphotericin B, 0.5–4 µg/ml for caspofungin, and 0.5–16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%–100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology. Oxford University Press 2023-11-22 /pmc/articles/PMC10681740/ /pubmed/37994652 http://dx.doi.org/10.1093/mmy/myad111 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Original Article
Thu, Nguyen Thi Mai
Borda, Hannah
Vitsupakorn, Shawin
Reddy, Kaushik Sreerama
Kasmani, Navsin
Barwatt, Joseph
Schwartz, Ilan S
Giamberardino, Charles
Perfect, John R
Hoa, Ngo Thi
Le, Thuy
Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title_full Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title_fullStr Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title_full_unstemmed Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title_short Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei
title_sort development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus talaromyces marneffei
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10681740/
https://www.ncbi.nlm.nih.gov/pubmed/37994652
http://dx.doi.org/10.1093/mmy/myad111
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