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

Abstract

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.

Keywords: Talaromyces marneffei; Talaromycosis; antifungal susceptibility testing; dimorphic fungi.

Figure 1.

AlamarBlue antifungal susceptibility method for Tm. Tm inoculum was prepared and diluted according to CLSI standard broth microdilution method (top left). Twofold dilutions of seven antifungal drugs were added to 96-well plates in columns 1–10, with columns 11 and 12 used as positive control (Tm inoculum without drug) and negative control (media only), respectively (top right). AlamarBlue is a resazurin dye that changes from blue to fluorescent pink in proportion to cellular metabolism (bottom left). AlamarBlue was added to each well after 24 h. After an additional 48 h (total 72 h) of incubation time, the MIC was determined visually by a change of color from pink to blue, by OD or by FI.

Figure 2.

The growth curves of six Tm isolates tested in two different inoculum sizes over 8 days. Growth of six Tm isolates was assessed by OD daily over 8 days in the CLSI inoculum of 1–5 x 103 CFUs/ml (solid blue) and in the EUCAST inoculum of 1–5 x 105 CFUs/ml (solid red). The broken blue and red lines represent the average OD600 values over time for the six Tm isolates by each inoculum. More robust growth was observed with the lower CLSI inoculum compared to the EUCAST inoculum. Growth reached the stationary phase between day 3 and day 4 for most isolates in both inoculums. Day 3 was determined the most optimal time to read the MIC for the alamarBlue assay.

Figure 3.

Effect of adding alamarBlue on the growth phases of two representative isolates of Tm. The effect of alamarBlue addition at 0 h (gray), 24 h (red), and 48 h (green) following Tm inoculation on Tm growth as assessed by fluorescent intensity reading every 24 h over 96 h. All samples were run in triplicate. Addition of alamarBlue at 0 h resulted in delay and reduction of growth of Tm northern strain (TmN) and delay of growth by 24 h of Tm southern strain (TmS). Addition of alamarBlue at 24 h was determined to be the most optimal time, as it allows ample time (another 48 h) for alamarBlue to fully incorporate into the exponential phase and reach the stationary phase before reading the MIC at 72 h.

Figure 4.

Distribution of MICs of seven antifungal drugs against 32 individual Tm isolates. MIC, minimum inhibitory concentration; CLSI, Clinical and Laboratory Standards Institute; V, visual; OD, optical density; aB, alamarBlue; FI, fluorescence intensity; error bars represent GM and 95% confidence interval. MIC data for aB-OD method were assessed for 16 strains only.