The increasing incidence of fungal disease necessitates adequate

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1 Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts Kristie D. Goughenour, a Joan-Miquel Balada-Llasat, b Chad A. Rappleye a Department of Microbiology, Department of Microbial Infection and Immunity, Ohio State University, Columbus, Ohio, USA a ; Department of Clinical Pathology, Ohio State University, Columbus, Ohio, USA b Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 3-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease. The increasing incidence of fungal disease necessitates adequate and timely assessment of antifungal susceptibility to guide the selection and implementation of antifungal therapies. Consequently, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established reference test methods for susceptibility to the major classes of antifungals, which include the polyenes, azoles, and the recently developed echinocandins (1, 2). These standards utilize broth macrodilution or microdilution assays for testing yeasts (M2-A3 [3] and E.DEF.1 [4]) and filamentous fungi (M38-A2 [5] and E.DEF 9.1 [6]). Procedures, including inoculum preparation, media, assay duration, and endpoint definitions, have now been established for testing some of the more commonly encountered yeast-form pathogenic fungi (i.e., Candida species and Cryptococcus). However, notably missing from the M2-A3 and E.DEF.1 standards for testing of yeasts are methods for testing the yeast forms of the dimorphic fungi. The dimorphic fungi that cause systemic disease are characterized by distinct yeast and filamentous morphological states (, 8). Temperature is the principal morphology-determining factor, with yeast forms characterizing infections in mammals (9). Histoplasma capsulatum is the most common clinically encountered dimorphic fungal pathogen in the United States, with an estimated 1, to 2, hospitalizations annually (1). Histoplasma is acquired by inhalation of mycelia-produced conidia from the environment. In the lung, mammalian body temperature triggers differentiation of the conidia into yeasts instead of filamentous hyphae (9). Histoplasma yeasts parasitize phagocytic cells, and the yeast-infected phagocytes can facilitate extrapulmonary dissemination of the fungus to cause life-threatening disseminated histoplasmosis, particularly in immunocompromised individuals. In the majority of otherwise healthy individuals, low-dose inoculation results in a self-limiting disease with the onset of cellmediated immunity. With higher doses, Histoplasma causes acute disease, even in immunocompetent individuals (1, 11). Despite the fact that the yeast form is the state most relevant to disease, some in vitro antifungal testing for Histoplasma has been performed only on the filamentous form (12 14). Both yeast- and filamentous-form Histoplasma cells are generally susceptible to polyenes and triazole-class antifungals in vitro (15 1). Early studies on the echinocandins suggested that this antifungal class, with very low host toxicity, could be effective against Histoplasma infections (13, 18, 19). However, some of these early reports were based on tests with the filamentous form, and subsequent in vitro susceptibility tests employing the yeast form demonstrated that the Histoplasma yeasts are comparatively resistant to the echinocandins (16, 1, 2). This morphology-based discrepancy in antifungal susceptibility is not limited to echinocandins and highlights the need to use the most appropriate cell type during antifungal testing (21). With the increasing importance of monitoring antifungal drug resistance and the acceleration of new an- Received 25 March 215 Returned for modification 29 April 215 Accepted 29 July 215 Accepted manuscript posted online 5 August 215 Citation Goughenour KD, Balada-Llasat J-M, Rappleye CA Quantitative microplate-based growth assay for determination of antifungal susceptibility of Histoplasma capsulatum yeasts. J Clin Microbiol 53: doi:1.1128/jcm Editor: D. W. Warnock Address correspondence to Chad A. Rappleye, rappleye.1@osu.edu. Supplemental material for this article may be found at /JCM Copyright 215, American Society for Microbiology. All Rights Reserved. doi:1.1128/jcm jcm.asm.org Journal of Clinical Microbiology October 215 Volume 53 Number 1

2 Histoplasma Microplate Assay tifungal drug discovery efforts, optimized methods for highthroughput antifungal tests on Histoplasma yeasts are essential. In this study, we standardized a broth microdilution method for assaying Histoplasma yeast growth. This study was motivated by the inadequacy of CLSI-defined yeast culture procedures for the growth of Histoplasma yeasts, the clinical form of Histoplasma. In some previous studies, the CLSI M2-A2 and -A3 standards have been adapted for Histoplasma yeast (22 25), but no consensus methods or optimization of assay parameters have been detailed. Here, we optimize the inoculum size and assay duration to maximize the dynamic range of a microdilution assay for Histoplasma. The assay quantifies yeast growth through spectrophotometric measurement of culture turbidity to provide a more objective conclusion of endpoints than visual inspection allows. To complement culture density measurements, we also determined optimal parameters for the use of colorimetric and fluorescent indicators of yeast cell density and vitality. These methodologies were validated for use in a clinical microbiology laboratory setting by establishing antifungal susceptibilities of multiple clinical isolates from both North American phylogenetic groups of Histoplasma and a Latin American isolate. MATERIALS AND METHODS Fungal strains and culture. Histoplasma capsulatum strains used were the wild-type North American type 2 (NAm2) strain G21B (ATCC 2632), the wild-type Panama strain G186A (ATCC 2629), clinical sample isolates from the Ohio State Medical Center, and 3 clinical isolates from MiraVista Labs (24, 26, 2). Isolates were assigned to the North America NAm1 and NAm2 phylogenetic groups (28) based on PCR-restriction fragment length polymorphism markers (29). Histoplasma cells were maintained as yeasts by growth at 3 C on a medium optimized for Histoplasma yeast growth (Histoplasma macrophage medium [HMM]) supplemented with 25 M FeSO 4 and solidified with.6% agarose (3)oron a brain heart infusion (BHI) medium (Becton, Dickenson and Co.) solidified with.6% agarose instead of agar. For liquid culture, basal cell culture media were used as the rich growth media. F-12 (Gibco) and RPMI 164 (Gibco) media were compared, as F-12 is the base composition of HMM medium, and RPMI is more commonly used in clinical laboratories for microdilution assays. Both broth media were buffered to a ph of. with 25 mm HEPES and were optionally supplemented with 1.5% glucose and/or. mm cystine for nutritional tests. For growth in microtiter plates (see below), yeasts were added to wells in a total volume of 1 l. For quantitative platings, serial dilutions of yeast suspensions were plated on solid HMM medium, as this medium supports the robust growth of individual colonies, and incubated at 3 C until colonies developed (8 to 1 days). Preparation of Histoplasma inocula. Microtiter plate inocula of Histoplasma yeasts were prepared either by suspending yeast colonies in F-12 medium or from broth cultures of Histoplasma yeasts that had been pregrown in culture tubes for 48 h. For pregrowth of Histoplasma yeasts before dilution into microtiter plates, yeast colonies were inoculated into 2.5 ml liquid medium in 16 mm 1 mm test tubes. Tubes were placed at a 3-degree angle and incubated for 2 days at 3 C with shaking (2 rpm) in a Multitron orbital shaker (Infors, Inc.). Yeast density was determined directly using hemacytometer counts, estimated by optical density at 595 nm (OD 595 ), or by a turbidity comparison to McFarland standards. For estimation of yeast density by OD 595, a standard curve of OD 595 versus the yeast cell number was used. For comparison to McFarland standards, McFarland standards (of.5, 1., or 1.5) were prepared by mixing defined volumes of 1% BaCl 2 (1.15% BaCl 2 dihydrate) with 1% H 2 SO 4 (5 l, 1 l, and 2 l of BaCl 2 in a final volume of 1 ml). Yeast suspensions were adjusted with growth medium to a density equivalent to 1. McFarland standard (representing approximately yeasts/ml) and further diluted to the desired inoculum. Microtiter plate growth. A total of 5 l of yeast culture at twice the desired final yeast density was added to wells of a 96-well flat-bottomed microtiter plate. Subsequently, 5 l of growth medium or medium containing antifungal drugs at twice the desired concentration (typically 2-fold dilutions from 32 g/ml to.3 g/ml) was added to each well for a final volume of 1 l. Plates were incubated at 3 C for 5 days with twice-daily agitation (3 s at 1, rpm) on a microplate mixer (Eppendorf) to improve aeration. Culture turbidity was determined by measurement of the absorbance of each well at 595 nm with a Synergy 2 microplate reader (Biotek). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide based yeast vitality assay. Histoplasma yeasts in 1 l of growth medium were added to 96-well plates and grown as described above for approximately 96 h. A 5 solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was prepared in growth medium containing.5 mm menadione as an electron shuttle. Then, 25 lofthe MTT solution was added to each well that contained Histoplasma yeasts as well as to wells that contained growth medium without Histoplasma yeasts. Plates were incubated at 3 C to allow for reduction of MTT and sufficient color development. The assay was terminated by the addition to each well of 5 l of 2% SDS with 1 mm HCl. After at least 2hof incubation at ambient temperature for reduced MTT extraction, the microtiter plates were centrifuged for 5 min at 2, g to collect yeast cell debris, and 5 l of the supernatant was transferred to a microtiter plate containing 5 l of distilled H 2 O. The extracted MTT was quantified by absorbance readings at 5 nm after correction with absorbance at 69 nm using a Synergy 2 plate reader (BioTek). Resazurin-based yeast vitality assay. Histoplasma yeasts in 1 lof growth medium were added to 96-well plates and grown as described above for approximately 96 h. 1 l ofa1 concentrated solution of resazurin (-hydroxy-1-oxidophenoxazin-1-ium-3-one) were added to each well, including control wells containing growth media without yeast cells and the microtiter plates incubated at 3 C. Kinetics of resorufin (reduced resazurin; -hydroxyphenoxazin-3-one) production was quantified by measurement of fluorescence (53/25 excitation, 59/35 nm emission wavelengths) every 2 min using a Synergy2 plate reader (BioTek). Antifungal drug susceptibility tests. Antifungals used in clinical practice were applied to Histoplasma yeast and included amphotericin B, fluconazole, and caspofungin. Antifungals were prepared according to the CLSI reference standard M2-A3 (3). For the 5% inhibitory concentration (IC 5 ) and MIC determinations for Histoplasma yeasts, a 2-fold dilution series of antifungal drugs was prepared, and 5 l of drug-containing solution was added to the wells of a 96-well microtiter plate containing 5 l ofhistoplasma yeasts at a density of to yeasts/ml (producing a final yeast density of to yeasts/ml). Yeast growth was assessed by measurement of turbidity at 595 nm after 96 to 144 h and the growth was normalized to wells containing no antifungal drug. Endpoint resazurin reduction assays were subsequently employed to confirm OD 595 readings. Relative growth at each antifungal drug concentration was used to generate dose-response curves, and the IC 5 was determined by nonlinear regression. MIC values were based on the minimum concentration of antifungal drug that prevented development of visual turbidity or lack of resazurin fluorescence compared to growth in the absence of antifungal drug. The morphology of Histoplasma cells following drug treatment was evaluated by phase-contrast microscopy at 1 magnification. RESULTS Microtiter-plate based growth of Histoplasma yeasts. To determine the maximal yeast cell density that could be achieved in microtiter plates, replicate plates (n 3) were inoculated with 1 1,1 1 6,1 1 5, and yeasts/ml of the G21B strain, October 215 Volume 53 Number 1 Journal of Clinical Microbiology jcm.asm.org 328

3 Goughenour et al. A cfu / ml B OD 595 nm time (h) time (h) C.2.15 F-12 RPMI.1.5. media media+glc media+cys media+glc+cys FIG 1 Growth of Histoplasma yeasts in microtiter plates. Histoplasma yeasts were grown in 96-well microtiter plates, and the yeast cell density was measured by CFU (A) or optical density at 595 nm (OD 595 ) (B). Growth media were inoculated with 1 1 (circles), (squares), (diamonds), and (triangles) yeasts/ml, and microtiter plates were incubated at 3 C. Plates were shaken (6 s at 1, rpm) twice daily to improve aeration. Yeast growth was assessed at 24-hour intervals. (A) For quantitative platings, dilutions of yeast cultures from 96-well microtiter plates were plated on solid HMM medium to enumerate viable CFU. (B) Culture turbidity was measured by OD 595 in a plate reader. (C) Effect of supplementation of growth media with glucose (Glc) or cysteine (Cys) on yeast cell density (OD 595 ). Data shown represent growth at 5 days after inoculation with yeasts/ml in Ham s F-12 (black bars) or RPMI (gray bars) buffered to a ph of.. Data points represent the average standard deviation of biological replicate cultures (n 3). a clinical strain representative of North American isolates. Plates were incubated at 3 C for 5 days. Yeast growth and viability were monitored daily by measurement of optical density at 595 nm (OD 595 ) and by plating serial dilutions of the yeast suspension to enumerate CFU, respectively. Maximal growth in 96-well microtiter plates saturated at approximately CFU/ml (Fig. 1A). Yeast growth reached saturation at 2 to 3 days, 4 days, and 5 days for 1 1,1 1 6, and yeasts/ml of starting culture density, respectively. Yeast cultures started at yeasts/ml significantly lagged in growth and reached saturation only after 6 to days of incubation (data not shown). Yeast viability as determined by CFU closely paralleled the optical density of each well at 595 nm (Fig. 1B), indicating that optical density was a reliable indicator of yeast cell growth. Importantly, Histoplasma microdilution assays started at both the high and low inoculum levels recommended for yeasts by the CLSI procedures ( and yeasts/ml, respectively [3]) resulted in a complete failure of Histoplasma yeast growth (data not shown). As maximal growth obtained in microtiter plates was less dense than when Histoplasma yeasts were grown in culture tubes, we investigated whether glucose or cysteine was limiting in the RPMI-based medium. CLSI procedures for growth of yeast-form organisms (i.e., Candida and Cryptococcus) typically utilize a defined rich medium (e.g., RPMI) buffered to a ph of. (3). HMM, a medium optimized for growth of Histoplasma and macrophages, is based on Ham s F-12 cell culture medium and is supplemented with 1.8% glucose and. mm cystine (3). The increased cystine provides additional organic sulfur, since Histoplasma yeast cells and mycelia differ in their requirements for organic sulfur (31 35). Although both F-12 and RPMI have cysteine (.2 mm and.4 mm, respectively), we tested if supplementation of F-12 or RPMI with cystine increased the yield of Histoplasma yeasts in microtiter plates. Neither added cystine nor added glucose significantly improved the overall rate or total growth of Histoplasma yeasts; no significant differences in yield were detected by one-way analysis of variance (ANOVA) (n 3; Fig. 1C). With some strains of Histoplasma, F-12 supported 2% to 8% higher yeast cell densities than RPMI (data not shown). We suspect that trace metals (i.e., Fe 2,Cu 2,Zn 2 ) present in F-12 but absent from the RPMI formulation may account for the improved growth in the F-12 medium. Thus, F-12 (lacking any supplements) buffered to a ph of. was selected as the growth medium for microtiter platebased growth of Histoplasma. Enumeration of yeasts by hemacytometer counts was used to establish a standard curve of optical density versus yeast cell density and to correlate Histoplasma yeast cell number to McFarland standards. Since spectrophotometers vary in their readings for light scattering due to sample turbidity, the McFarland standards provide a defined optical density reference. McFarland standards of.5 to 4. were prepared in triplicate, and their optical densities at 595 nm were determined using a plate reader (Fig. 2A). In parallel, different yeast culture densities (determined by hema jcm.asm.org Journal of Clinical Microbiology October 215 Volume 53 Number 1

4 Histoplasma Microplate Assay A.2.15 OD 595 nm.1.5 B C 4x1 4x1 3x1 3x1 yeasts/ml yeasts/ml 2x1 2x1 1x1 1x McFarland Standard.1.2 OD 595 nm McFarland Standard FIG 2 Correlation of OD 595 with McFarland turbidity standards and yeast culture density. Optical density readings (OD 595 ) were correlated to McFarland turbidity standards (A) or yeast cell density (B). (A) McFarland standards were constructed by mixing BaCl 2 with 1% H 2 SO 4 and then measuring the optical density. Data points represent individual readings for replicate cultures (n 3), and the dashed line indicates the computed linear relationship (B) For determination of yeast cell density, dilutions of Histoplasma yeast cultures were counted by hemacytometer to enumerate yeast cells. Data points represent individual Histoplasma cultures. Dashed line indicates the relationship between OD 595 and yeast cell density by linear regression. (C) The corresponding OD 595 for each McFarland standard was matched to the OD 595 standard curve for Histoplasma yeast culture density to yield a linear relationship for McFarland standards and the titer of the yeast culture. cytometer counts) were correlated to optical density readings (Fig. 2B). Both linear relationships were subsequently combined to produce a standard curve of the turbidity reference (McFarland standards) and the Histoplasma yeast cell density (Fig. 2C). A Mc- Farland standard of 1. was equivalent to Histoplasma yeasts/ml. To optimize the dynamic range of the growth of Histoplasma yeasts in 96-well microtiter plates, we investigated the parameters of starting yeast cell density and incubation time using cultures of G21B in triplicate. Starting inocula of less than yeasts/ml displayed significant lags in growth (Fig. 1A) or no growth at all. While inocula of around 1 1 yeasts/ml produced maximal growth in a short time (2 to 3 days), these inocula already showed significant optical density in the absence of any growth, thereby decreasing the dynamic range (Fig. 3A). Starting concentrations of around yeasts/ml reached maximal growth at 4 days. A OD 595 nm time (h) 3.2x1 /ml 6 8.x1 /ml 6 2.x1 /ml 5 5.x1 /ml B Dynamic range (OD units) inoculum (yeasts/ml) 96 h 8 h 2 h C Z value h 8 h 96 h. 5.x1 5 2.x1 6 8.x x1 1.x1 6 4.x x1 inoculum (yeasts/ml) FIG 3 Optimization of inocula sizes for maximization of the dynamic range of the microplate-based assay. (A) Representative growth curves of Histoplasma yeasts initiated at starting densities of (circles), (squares), (diamonds), or (triangles) yeast cells/ml. Data points represent average optical density readings standard deviation of replicate cultures (n 3). (B) Determination of the dynamic range of OD 595 readings for different inocula after growth at 3 C. The dynamic range was determined as the difference between the initial OD 595 reading at the time of h and then at the time of 2 h (diamonds), 8 h (squares), or 96 h (circles). (C) Z=-factor statistic indicating the discriminatory power of the microtiter plate-based assay of Histoplasma yeast growth. Cultures were initiated at yeasts/ml to yeasts/ml. For computation of the Z=-factor, wells containing cultures grown in the absence of antifungal drug were compared to wells cultured in the presence of 5 g/ml of amphotericin B to suppress growth. Data points represent the average standard deviation of replicate cultures (n 3). October 215 Volume 53 Number 1 Journal of Clinical Microbiology jcm.asm.org 3289

5 Goughenour et al. A.8 8 B.8 Abs 5 nm x1 /ml 4x1 /ml Abs 5 nm no Flc +Flc [MTT] (mg/ml) time (min) C 6 8 D 6 Fluorescence 4 2 x1 /ml 4x1 /ml Fluorescence 4 2 no Flc +Flc [Resazurin] (µm) time (min) FIG 4 Optimization of parameters for metabolic reduction of MTT (A, B) or resazurin (C, D) for relative quantitation of Histoplasma yeast cell density. For optimization of substrate concentration (A, C) Histoplasma yeast suspensions at (circles), 1 (squares), 4 1 (diamonds), and 1 1 (triangles) yeasts/ml were incubated with various amounts of substrate. For optimization of the timing of the development reactions (B, D), Histoplasma yeasts in wells of a 96-well microtiter plate were grown for 96 h in the absence (circles) or presence (squares) of 4 g/ml of fluconazole (Flc) to suppress yeast growth and then incubated with MTT or resazurin substrates. MTT reduction was monitored by formazan product formation, which was extracted from cells with SDS and quantified by absorbance at 5 nm. Resazurin reduction was monitored by fluorescence of the resorufin product. Histoplasma yeasts were incubated with MTT at final concentrations ranging from to 1. mg/ml (A), with.5 mg/ml MTT and the formazan product extracted at 3 min intervals for quantification (B), with resazurin at final concentrations ranging from to 2 M (C), or with 1 M resazurin and the fluorescence of the resorufin product measured at 2 min intervals (D). Data points represent the average standard deviation of replicate cultures (n 3). Comparison of the OD 595 readings at 3 to 4 days (2 to 96 h) with the OD 595 readings at the start of the assay showed that initial inocula at to yeasts/ml had the largest dynamic range (Fig. 3B) and still produced near-maximal growth by 4 days of incubation. A total of 96 h of incubation provided the maximum difference, with an overall dynamic range of 35-fold and -fold for and yeasts/ml inocula, respectively. As a further test for optimal starting yeast cell density, different inocula of Histoplasma yeasts were grown in microtiter plates in the presence or absence of the antifungal drug amphotericin B. Calculation of the Z=-factor, a statistical measure of the discriminatory power of an assay for comparing growth in the presence or absence of drug (36), showed that, while all starting inocula could discriminate between growth and no growth (Z=.5), an inoculum of to yeasts/ml was optimal. From these results, we established yeasts/ml as the starting yeast cell density and 96 h for the growth time for the microtiter plate-based OD 595 growth assay. Greater consistency in growth kinetics was found when the initial inoculum was prepared from a liquid culture of Histoplasma yeast pregrown in culture tubes for 2 days than when yeast suspensions were made from colonies collected directly from solid medium. This was not due to transfer of any spent medium, since washing the pregrown cells before inoculation of microtiter plates yielded identical results as simply diluting pregrown cells (data not shown). The initial inoculum for the microdilution assay in 96-well plates may be determined by hemacytometer counts or by the adjustment of a yeast suspension to a McFarland standard of 1. followed by a 2-fold dilution of the adjusted suspension. Optimization of microtiter-based assays with endpoint indicators of yeast metabolism. Measurement of optical density provides an easy and efficient way to quantify yeast growth over time. Metabolic indicators of cell density, based on metabolism-dependent reduction of colorimetric and fluorescent dyes, provide an alternative measure of yeast cell density as well as provide an indicator of yeast vitality. Two accepted endpoint assays are cellular reduction of the tetrazolium dye MTT to its formazan, which can be monitored by absorption at 59 nm, and reduction of resazurin to fluorescent resorufin. The colorimetric change in both reactions can also be used as a more objective qualitative indicator than visual determination of turbidity. To determine the suitability of these assays for measuring Histoplasma yeast cell growth, we tested the ability of Histoplasma yeasts to reduce these dyes and optimized the dye concentration used and the timing of maximal color/fluorescence development. To determine the optimal concentration of MTT and resazurin substrates to be used, MTT was added to suspensions of Histoplasma yeasts at final concentrations of to 1. mg/ml, and resazurin, at final concentrations of to 2 M(n 3 for each). We used yeast cell densities at or above the maximum titer obtained in 96-well microtiter plate growth to ensure that the substrates would not be limiting for yeast grown to saturation density in microtiter plates. A dose-response relationship between the yeast cell number and formazan formation was observed (Fig. 4A). Sat- 329 jcm.asm.org Journal of Clinical Microbiology October 215 Volume 53 Number 1

6 Histoplasma Microplate Assay A 1. B 1. C 1. Relative growth Relative growth Relative growth [AmB] (μg/ml). μ [Fluconazole] (μg/ml) [Caspofungin] (μg/ml) yeasts IC 5 = 1. μg/ml MIC = 2. μg/ml yeasts IC 5 =.2 μg/ml MIC =.5 μg/ml yeasts IC 5 = 1.4 μg/ml MIC = 32. μg/ml mycelia IC 5 =.8 μg/ml MIC = 2.5 μg/ml mycelia IC 5 = 2. μg/ml MIC = 5. μg/ml mycelia IC 5 =.4 μg/ml MIC = 1.6 μg/ml FIG 5 Antifungal dose-response curves for Histoplasma cells tested with 96-well microtiter plate microdilution assays. Charts depict representative doseresponse curves of yeast to the antifungal compounds amphotericin B (A), fluconazole (B), and caspofungin (C) as determined by quantitative growth assay (turbidity at 595 nm). Histoplasma yeasts were inoculated into buffered F-12 media at a density of yeasts/ml and grown for 96 hours, at which point the OD 595 was measured. Yeast cell density was normalized to wells with no antifungal drugs added, and the relative growth was plotted. Data points represent the average standard deviation of replicate cultures (n 3) for each antifungal drug concentration tested. Trend lines indicate the regression line for the yeast-phase dose response which was used to derive the IC 5 for each antifungal drug. MICs were determined by visual inspection of wells and identification of wells without turbidity. urating levels of the MTT substrate for the highest yeast cell densities occurred around.4 mg/ml. Thus,.5 mg/ml MTT was chosen as the substrate concentration that would not be limiting for the assay with Histoplasma yeasts. To determine the timing for maximal formazan formation, we grew Histoplasma yeasts in microtiter plates for 96 h, added.5 mg/ml MTT, and incubated the plates at 3 C for different time intervals from to 24 min before stopping the reaction. Maximal MTT reduction to its formazan was achieved at 18 min after MTT addition (Fig. 4B). As with the MTT assay, a dose-response relationship between the yeast cell number and resorufin formation from resazurin was observed (Fig. 4C). Levels of the resazurin substrate became saturating at 1 M. Concentrations of resazurin above 1 M partially inhibited the reduction of resazurin to resorufin (Fig. 4C and data not shown). Maximum fluorescence was achieved by 1 to 12 min of incubation with 1 M resazurin (Fig. 4D). At 9 min, the reaction was near but not yet at saturation levels; thus, 9 min was designated the optimal time for the resazurin reduction assay. To determine if the MTT- and resazurin-based assays were suitable to detect inhibition of Histoplasma growth, the assays were applied to Histoplasma yeasts grown in microtiter plates in the presence of the antifungal drug fluconazole (Fig. 4B and D). This test showed that the MTT assay could efficiently discriminate between growth and inhibition of growth of Histoplasma yeasts, with a dynamic range of -fold. No additional formazan was formed by incubation times longer than 18 min; however, the background of nongrowing yeasts continued to increase, thereby falsely reporting the relative difference between inhibited and uninhibited yeasts. Comparison of the resazurin assay between Histoplasma yeasts grown in the presence or absence of the antifungal drug fluconazole showed that the resazurin assay also had excellent discrimination between yeast growth and growth inhibition, with a Z=-factor of.85 and a dynamic range of 12-fold. Application of the microtiter assay for drug sensitivity tests. To validate the optimized 96-well microtiter plate assay for drug sensitivity studies, we determined the concentrations for 5% inhibition (IC 5 ) and the MICs of antifungal drugs against the G21B strain of Histoplasma capsulatum (Fig. 5). Using the OD 595 parameters optimized above, yeast growth was determined over a range of antifungal drug concentrations in triplicate cultures. As optical density can quantify partial yeast growth, dose-response curves could be constructed, and the IC 5 could be determined from linear regression. MICs were determined by visual inspection of the plate for lack of turbidity. Treatment of Histoplasma yeasts with amphotericin B showed an IC 5 of 1..3 g/ml (mean standard deviation [SD]) and an MIC of 2. g/ml (Fig. 5A). Fluconazole had an IC 5 of.24.1 g/ml (mean SD) and an MIC of.5 g/ml (Fig. 5B). For the echinocandin caspofungin, Histoplasma yeasts were characterized by an IC 5 of g/ml (mean SD) and an MIC of 32 g/ml (Fig. 5C). These results are in agreement with established drug sensitivities for Histoplasma by macrodilution assay and modified microdilution assays (2, 24, 3, 38). Examination of Histoplasma cells by microscopy at inhibitory and subinhibitory drug concentrations showed no drug-induced morphological conversion to mycelia from the yeast-form inocula (data not shown). Using the microdilution assay, we confirmed that the antifungal susceptibility profile of yeasts differed from mycelia; 2-fold more caspofungin was required to inhibit yeasts than mycelia (Fig. 5C). In contrast mycelia were more resistant to fluconazole than were the Histoplasma yeasts (Fig. 5B). No significant difference in the susceptibility to amphotericin B was found between the two Histoplasma morphologies (Fig. 5A), consistent with previous observations of amphotericin B effects on yeasts and mycelia (16). The variation in antifungal drug effects on the different morphologies underscores the importance of using the clinically relevant form (i.e., yeasts) for antifungal susceptibility profiling of Histoplasma. We applied the optimized microtiter plate-based assay to test the drug sensitivity profile of clinical isolates of Histoplasma using yeast-phase cells. The isolates represent clinical strains from the North America phylogenetic group 1 (NAm1), the more clinically prevalent North America group 2 (NAm2), and a Latin American October 215 Volume 53 Number 1 Journal of Clinical Microbiology jcm.asm.org 3291

7 Goughenour et al. TABLE 1 Antifungal susceptibility of clinical isolates Susceptibility data b ( g/ml) for: Amphotericin B Fluconazole Caspofungin Isolate Phylogenetic group a IC 5 MIC IC 5 MIC IC 5 MIC HC1 NAm HC1 NAm HC1 NAm HC14 NAm HC16 NAm HC22 NAm HC3 Lam G186A Pan IN2 NAm IN14 NAm NY1 RFLP-VI a Phylogenetic group classification as described by Kasuga et al. (28): NAm1, North American type 1; NAm2, North American type 2; LAm Latin American isolates or, in the case of NY1, based on the RFLP classification scheme of Keath et al. (4). b IC 5 expressed as average standard deviation (n 3); MIC expressed as range (low to high; n 3). isolate (LAm), as well as from a laboratory strain originally from Panama (G186A; Pan). The antifungal susceptibility results of these clinical isolates are presented in Table 1. In general, Histoplasma clinical isolates were similar in their susceptibility to amphotericin B but NAm1, LAm, and the Panama isolates had slightly higher natural resistance to fluconazole (2- to 3-fold higher IC 5 ). All isolates were relatively resistant to caspofungin, with MICs above 8 g/ml. Of note, the NAm1, LAm, and Panama isolates were more resistant than NAm2 clinical isolates to the -glucan synthase inhibitor caspofungin. As further confirmation of the parameters for the Histoplasma microdilution assay for clinical and laboratory application, we tested three isolates with established resistance to fluconazole. Clinical isolates IN2, IN14, and NY1 originated from AIDS patients who had failed or relapsed following fluconazole therapy (24, 26, 2). Compared to typical inhibitory levels of fluconazole for NAm2-class Histoplasma yeasts, we observed 22- to 2-fold increases in the IC 5 s and MICs of fluconazole for isolates IN2 and IN14, respectively (Table 1) (see Fig. S1 in the supplemental material). The NY1 strain also had decreased susceptibility to fluconazole (MICs, 128 to 256 g/ml). Interestingly, the IN14 isolate also showed approximately 3-fold higher resistance to caspofungin (see Fig. S1 in the supplemental material), even though this antifungal was not part of the treatment regimen for the patient from whom it was isolated. Further investigation of the very high fluconazole resistance of the IN14 isolate identified a mutation in the ERG11B gene that resulted in substitution at amino acid 165 (L165R; data not shown). DISCUSSION In this study, we optimized parameters for growing and measuring Histoplasma yeast growth in 96-well microtiter plates. Differences in antifungal susceptibilities of Histoplasma yeasts cells compared to mycelia (16, 1, 2) raise the importance of standardizing methodologies for Histoplasma yeasts, the clinically relevant morphological form. Although CLSI has developed procedures for yeast-form fungal pathogens, these methods do not work for the culture of Histoplasma yeasts. Some studies on Histoplasma have adapted the CLSI macrobroth procedures for dimorphic fungi (23, 24, 3, 39), but no optimized methodology for a microdilution assay has been standardized. Our optimized methods are summarized in Fig. 6 and differ from the CLSI procedures for yeast by the following: higher inoculum density (2 1 6 to yeasts/ml compared to 5 to 2,5 yeasts/ml), longer growth time (4 to 6 days compared to 1 to 2 days) due to the much slower growth of Histoplasma, and growth in lower volume of media (1 l compared to 2 l) and in flat-bottomed wells (instead of U-bottom wells) to improve oxygen diffusion. Using these parameters, saturated growth densities of approximately Histoplasma yeasts/ml were achieved. We suspect that reduced oxygen availability caused by growth in microtiter plate wells accounts for the lower densities than are typically achieved in culture tubes. Nonetheless, sufficient growth was present to reliably distinguish growth of Histoplasma over the inoculum density. We observed that improved consistency of growth of Histoplasma yeasts in microtiter plates was achieved if the initial inoculum was prepared from pregrown liquid cultures. Nevertheless, preparation of the initial inoculum by suspension of yeast colonies taken directly from solid BHI medium was sufficient for the microdilution assay if the yeasts were obtained from freshly streaked plates (4 to 8 days old). Measurement of culture turbidity by optical density at 595 nm provides an easy and efficient means of determining the Histoplasma yeast number, which facilitates high-throughput assays and antifungal susceptibility profiling. Daily OD 595 measurements provide a kinetic view of Histoplasma yeast growth. OD 595 readings are less subjective than visual inspection of wells, and, furthermore, spectrophotometric quantitation provides data for partially, but not fully, inhibited growth. Measurement of light scatter due to turbidity differs between spectrophotometers; thus, the creation of standard curves for OD 595 versus turbidity using McFarland standards should be performed for individual plate reader machines to calibrate readings. Starting yeast cell densities of to yeasts/ml provide an optimal balance between length of growth period in microtiter plates and dynamic range for growth measurement. An inoculum of to yeasts/ml yields initial plate readings (or readings of fully inhibited yeast growth) near zero, and the yeast typically reach saturation in 4 to 5 days (Fig. 4), although the growth period is extended to 6 days for some slower growing strains. This results in 3292 jcm.asm.org Journal of Clinical Microbiology October 215 Volume 53 Number 1

8 Histoplasma Microplate Assay Microdilution Assay Parameters Medium: F-12 buffered to ph. 6 Inoculum density*: 2-4x1 yeasts/ml (final) Growth conditions: 1 μl total volume** flat-bottom microtiter plate wells 3 C for 4-6 days * a 2X inoculum can be prepared by adjusting yeast suspensions to a McFarland Standard of 1. equivalent ** 5 μl of 2X inoculum + 5 μl of media ± 2X drug Results Determination Turbidity MTT assay Resazurin assay.5 mg/ml MTT +.1 mm menadione 18 minutes at 3 C 1 μm resazurin 9 minutes at 3 C visual quantitative turbidity OD 595 color change (yellow purple) Absorbance at 5 nm* color change (blue pink) fluorescence (53 ex / 59 em) * A5 read after formazan extraction with 1% SDS + 5 mm HCl FIG 6 Summary of the optimized growth and assay parameters for microdilution testing of Histoplasma yeasts. Inoculum size, growth medium, and growth conditions are listed for initiation of the tests in 96-well microtiter plates. Assays that can be used for determination of Histoplasma growth (culture turbidity, MTT reduction, and resazurin assays) provide both visual (qualitative) and spectrophotometric (quantitative) results. Further details of the endpoint assay methodologies are found in the text. a dynamic range over 3-fold between measurements of the inoculum and saturated growth. Metabolic activity indicator dyes can also be used to quantify Histoplasma yeasts grown in microtiter plates. We show that both MTT- and resazurin-based endpoint assays are applicable to the Histoplasma microdilution assay. With both assays, color can be used as a qualitative indicator of yeast cell number, which is less subjective than the visual determination of turbidity. Spectrophotometric measurement of formazan formation or fluorescence due to the conversion of resazurin to resorufin also provides more objective, quantitative results. The accurate determination of relative growth requires that the measurements for maximum numbers of Histoplasma yeasts do not saturate the assay color or fluorescence development. For MTT, we determined that.5 mg/ml MTT was not limiting and that a reaction time of 3hat3 C maximized the dynamic range of the assay. For resazurin, 1 M and a time of 9 min at 3 C were the nonlimiting substrate concentration and optimal assay reaction time before saturation, respectively. The MTT and resazurin assays both are particularly useful for measuring the growth of Histoplasma strains which exhibit clumped rather than dispersed growth in a liquid medium (e.g., strains of the NAm1 class or Latin American isolates), since these assays are based on cellular metabolism rather than on yeast particles which, when aggregated, can lead to variable turbidity readings. Testing of multiple clinical Histoplasma isolates from diverse geographic locations demonstrates that the optimized microdilution assay is broadly applicable to Histoplasma isolates. Antifungal susceptibility testing of the isolates showed similar susceptibility profiles for fungicidal amphotericin B. NAm1, LAm, and the Panama isolates had slightly increased natural resistance to fluconazole compared with the NAm2 isolates, but all were still below the fluconazole breakpoints defined for Candida yeasts (4, 41). Yeasts from the LAm and Panama, but not the NAm2, phylogenetic groups are characterized by cell walls that have -glucan polysaccharides in addition to the -glucan polysaccharide (42 46), and these isolates showed increased resistance to caspofungin compared to the resistance of NAm2 isolates. This correlation between -glucan and less sensitivity to -glucan synthase inhibitors is intriguing, yet the mechanism remains undefined at this point. Nonetheless, it indicates that Histoplasma strains should not be considered a homogeneous group with regard to the natural profiles of antifungal susceptibility. In addition, testing of isolates from patients that had failed or relapsed following fluconazole treatment confirms the acquisition of fluconazole resistance in these isolates and shows the applicability of the microdilution assay to provide accurate information on antifungal susceptibilities to improve clinical management of histoplasmosis. In conclusion, the optimized parameters for measurement of Histoplasma yeast growth in 96-well microtiter plates, either through optical density measurement or through endpoint colorimetric/fluorescence assays, fill a gap in the CLSI methodology for October 215 Volume 53 Number 1 Journal of Clinical Microbiology jcm.asm.org 3293

9 Goughenour et al. antifungal susceptibility testing of Histoplasma yeasts. It is hoped that these standardized methods will provide for more reliable profiling of clinical isolates of Histoplasma as well as accelerate high-throughput screening of compounds for new antifungals active against the pathogenic phase of Histoplasma (22). ACKNOWLEDGMENTS We thank Rosely M Zancopé-Oliveira (Fiocruz; Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) for the Latin American Histoplasma isolate and Patty Connolly and L. Joseph Wheat at MiraVista Diagnostics for the fluconazole-resistant Histoplasma strains. We thank Gabriel Silveira d Almeida for initial work on inocula and parameter optimization. This work was supported in part by NIH/NIAID grant R21AI1943. REFERENCES 1. Cuenca-Estrella M, Rodriguez-Tudela JL. 21. The current role of the reference procedures by CLSI and EUCAST in the detection of resistance to antifungal agents in vitro. 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Clin Microbiol Infect 14: Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard 2nd ed. CLSI document M38-A2 28. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Rodriquez-Tudela JL, Donnelly JP, Arendrup MC, Arikan S, Barchiesi F, Bille J, Chryssanthou E, Cuenca-Estrella M, Dannaoui E, Denning D, Fegeler W, Gaustad P, Lass-Flörl C, Moore C, Richardson M, Schmalreck A, Velegraki A, Verweij P, Subcommittee on Antifungal Susceptibility Testing of the ESCMID European Committee for Antimicrobial Susceptibility Testing. 28. EUCAST technical note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming molds. Clin Microbiol Infect 14: Klein BS, Tebbets B. 2. Dimorphism and virulence in fungi. Curr Opin Microbiol 1: Maresca B, Kobayashi GS. 2. Dimorphism in Histoplasma capsulatum and Blastomyces dermatitidis. 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