Medical Mycology, 2022, 60, myac062 https://doi.org/10.1093/mmy/myac062 Original Article Advance access publication date: 0 2022 Candida auris and some Candida parapsilosis strains exhibit similar characteristics on CHROMagarTM Candida Plus Natalia Sasoni1 , Melani Maidana1 , María Gabriela Latorre-Rapela1 , Soraya Morales-Lopez2 , Indira Berrio3 , Soledad Gamarra1 and Guillermo Garcia-Effron 1 ,4 ,∗ 1 Laboratorio de Micología y Diagnóstico Molecular, Cátedra de Parasitología y Micología, Facultad de Bioquímica, Universidad Nacional del Litoral, Ciudad Universitaria, CP 3000 Santa Fe, Argentina 2 Grupo CINBIOS, Programa de Microbiología, Universidad Popular del Cesar, 200002 Valledupar, Colombia. 3 Hospital general de Medellin ’Luz Castro de Gutiérrez’ ESE, 050015 Medellín, Colombia 4 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), CP 3000 Santa Fe, Argentina. ∗ To whom correspondence should be addressed. Guillermo Garcia-Effron, PhD, Laboratorio de Micología y Diagnóstico Molecular, Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Universidad Nacional del Litoral, CP 3000 Santa Fe, Argentina. Tel: +54-342-4575209 ext. 135 (laboratory); E-mail: [email protected]. Abstract Candida auris is considered a public health problem because of its resistance and its tendency to cause nosocomial outbreaks. CHROMagarTM Candida Plus has recently been marketed as capable of presumptively identifying C. auris. The objective of this work was to analyze the ability of this new chromogenic medium to differentiate C. auris from other members of the C. haemulonii complex and from other yeasts commonly isolated in clinical practice. A collection of 220 strains including species of the C. haemulonii (n = 83) and C. parapsilosis (n = 80) complexes was studied. The strains were identified by molecular methods and cultured as individual or as mixed aqueous inoculum on CHROMagarTM Candida Plus plates. Colony morphotypes were evaluated at five time points. CHROMagarTM Candida Plus was a helpful tool for presumptive identification for C. auris. Better reading results were obtained after 48 h of incubation at 35°C. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possible misidentification of C. parapsilosis as C. auris (44.3% showed similar morphotypes). To reduce false positives when it is used in a context of a C. auris outbreak, we propose to supplement the chromogenic medium with 8 μg/ml fluconazole. This modified medium was tested, and it clearly differentiate C. parapsilosis from C. auris. Lay summary CHROMagarTM Candida Plus is able to differentiate Candida auris from other Candida spp., including other species of the C. haemulonii complex. However, 44.3% of the tested C. parapsilosis strains would be misidentified as C. auris. We propose the addition of 8 μg/ml fluconazole to solve this issue. Keywords: CHROMagarTM Candida Plus, Candida auris, Candida parapsilosis sensu lato, phenotypic identification Introduction Candida spp. are a common cause of hospital-acquired infections, and its prevalence is steadily increasing.1–3 Candida albicans is considered the major cause of Candidemia. However, its prevalence is declining at the expense of Candida nonalbicans species, including multidrug-resistant species such as C. auris and other species from C. haemulonii complex.4–8 The identification of these emerging resistant species is challenging.9 All patients colonized or infected by C. auris should be isolated due to the ease of propagation of these yeasts, while the identification of other members of the C. haemulonii complex does not have this implication.10 ,11 To comply with these recommendations, a screening of hospitalized patients and a correct identification of C. auris is mandatory.12 ,13 Phenotypic-based identification methods are not capable of accurately identifying C. auris14 ,15 and rDNA sequencing, rDNA-based PCRs (classical, real-time, isothermal, etc.) and MALDI-TOF MS are the only available tools.14–17 Conversely, these methods are generally beyond the expertise and budget ranges of most clinical laboratories. Recently, a chromogenic isolation medium able to presumptively identify C. auris was developed (CHROMagarTM Candida Plus) and reports of its performance were published by different European labs.18–20 In these reports, CHROMagarTM Candida Plus was considered a good tool to differentiate C. auris from several clinically relevant Candida spp. and non-Candida species. However, in one of these reports no C. haemulonii complex species beside C. auris were studied (no C. haemulonii, C. duobushemulonii, C. vulturna or C. pseudohaemulonii strains were included)18 while in another no C. vulturna or C. pseuduohaemulonii were included (19). Moreover, discrepancies on C. parapsilosis sensu lato morphotypes Received: May 16, 2022. Revised: July 8, 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: [email protected] 2 (some strains showed bluish color halo as C. auris) was reported in one of these works18 and in the others only one C. parapsilosis strain19 or none20 were included. Considering that C. parapsilosis sensu lato is highly prevalent in South America2 and that the identification of C. auris implies the isolation of colonized and/or infected patients12 ,14 , we decided to evaluate the performance of CHROMagarTM Candida Plus using a collection of 220 strains with a significant number of isolates of the C. haemulonii and C. parapsilosis species complexes (n = 163) in order to establish whether there is any possibility of misidentifications between these yeast species. Material and methods Fungal strains and identification The yeasts used through this work (n = 220) are conserved in the strain collection of the Laboratorio de Micología y Diagnóstico Molecular (CONICET-UNL). It includes Candida spp. and non-Candida (formerly named as Candida) species usually isolated from clinical samples as etiological agents or contaminants. Within the included strains there were control strains (e.g., ATCC) and strains isolated from clinical samples including (in alphabetic order): C. albicans (n = 10, including ATCC 90028 and ATCC 36082); C. auris (n = 53, all clinical strains isolated in different Colombian cities, Clade IV); C. dubliniensis (n = 5, including NCPF3949); C. duobushaemulonii (n = 5); C. haemulonii (n = 15); C. metapsilosis (n = 5); C. orthopsilosis (n = 5); C. parapsilosis sensu stricto (n = 70, including ATCC 22019); C. pseudohaemulonii (n = 5); C. tropicalis (n = 10, including ATCC 750); C. vulturna (n = 5); Clavispora (Candida) lusitaniae (n = 10, including ATCC 200950); Nakaseomyces (Candida) glabrata (n = 11, including ATCC 90 030); Nakaseomyces (Candida) bracarensis (n = 2); Nakaseomyces (Candida) nivariensis (n = 4); Pichia kudriavzevii (Candida krusei) (n = 5, including ATCC 6258). Strains were identified by molecular-based procedures17 and confirmed by rDNA sequencing (ITS regions).21–23 CHROMagarTM Candida Plus performance was evaluated by inoculating plates with aqueous suspensions of all the 220 strains as described before.18–20 Briefly, each strain was inoculated in YPD slants (1% yeast extract, 2% Bacto peptone, 2% dextrose, 1.5% agar) and incubated at 35°C for 24 h. Yeast cells were suspended in sterile physiological saline to obtain a suspension corresponding to a 0.5 McFarland standard (1–5 × 106 cell/ml) that was subsequently diluted 1:100 (final concentration of ∼104 CFU/ml). Successively, CHROMagarTM Candida Plus and YPD agar plates were spotted with 3 μl of the suspensions in parallel in a grid fashion (16 strains per plate). Both plates were incubated for 72 h at 35°C. The chromogenic media was used to record the colony´s morphotype, front and back color and growth speed. YPD agar plates served as growth control. The same aqueous inoculum was used to evaluate the capacity of the chromogenic media to differentiate C. auris from other commonly found yeast in mixed cultures (in an attempt to mimic a plate taken to assess colonization). Inoculum of three Colombian C. auris clinical strains with different antifungal susceptibility profiles (LMDM-1162, LMDM-1205 and LMDM-1234)24 were mixed (50:50) with C. haemulonii, C. albicans, C. tropicalis and two different strains of C. parapsilosis sensu stricto (ATCC 22019: white colonies Sasoni et al. no halo and LMDM-1222: white colonies with blue halo). These mixed inocula were plated onto CHROMagarTM Candida Plus and CHROMagarTM Candida Plus supplemented with 8 μg/ml of fluconazole (see below for a more detailed explanation) and incubated for 72 h at 35°C. Colony morphotypes and color were recorded at 24, 36 (as recommended by the manufacturer), 48, 60 and 72 h. Modified CHROMagar TM Candida Plus In order to better differentiate C. auris from others fluconazole susceptible Candida spp. with similar morphotypes (specially strains identified as C. parapsilosis), we supplemented the commercially available CHROMagar TM Candida Plus with fluconazole (8 μg/ml). The plates with this modified media were used to strike the described mixed inocula. Results The morphotype of 220 unrelated isolates was examined at five time points. At 24 h of inoculation, most strains showed poor growth and were colorless, making it difficult to correctly differentiate among species. On the contrary, at 36 h of incubation most species showed the characteristics described by the manufacturer in terms of color and/or morphology. Only 2 C. tropicalis (10%) and 10 C. auris (18.9%) strains displayed the morphotypes describe by manufacturer (metallic blue w/slight pink halo and white/pale with blue halo, respectively) after 48 h of incubation. These observed morphotypes did not vary at 48 h except by colony size. After 60 and 72 h of incubation, color and other colony characteristics start to vary. It was clearly seen that C. auris grew better (faster and as bigger colonies) than other species of the C. haemulonii complex on CHROMagarTM Candida Plus. The same happens with C. lusitaniae and C. krusei when compared with others Candida spp. All 53 studied C. auris strains grew on CHROMagarTM Candida Plus as white to pale cream colonies with a blue halo (diffusible color) in the adjacent agar (at 35°C for 36– 48 h). In contrast, all the tested members of the C. haemulonii complex (n = 30) grew as white to pale pink colonies with no colored halo. The absence of the diffusible blue halo in these species makes it easy to distinguish C. auris from other species of the C. haemulonii complex. It should be noted that 63.3% (19/30) of the non-auris Candida tested species of the complex had difficulties growing in CHROMagarTM Candida Plus after 48 h of incubation (3/5 C. duobushaemulonii, 12/15 C. haemulonii, 3/5 C. pseudohaemulonii and 1/5 C. vulturna showed poor growth on the tested chromogenic media) (Fig. 1). Turning to other organisms, most prevalent tested species showed the characteristics described by manufacturer (C. albicans/C. dubliniensis: turquoise blue/green, C. krusei: pink to purple with white edges, C. glabrata: pink to purple, C. tropicalis: metallic blue w/slight pink halo and C. lusitaniae: pink to purple) (Table 1, Figs 1 and 2). Conversely, 44.3% (31/70) of the tested C. parapsilosis species complex clinical strains grew on CHROMagarTM Candida Plus as white colonies surrounded by a blue halo (incubated for 36 and 48 h) (Fig. 2). These characteristics make these colonies difficult to differentiate from C. auris. On the other hand, the rest (n = 39) grew as white to light blue colonies with no 3 Medical Mycology, 2022, Vol. 60, No. 00 Figure 1. Representative morphotypes (obverse (A) and reverse (B) sides) of tested yeasts on CHROMagarTM Candida Plus incubated at 35°C for 48 h. Plates were spotted with 3 μl of suspensions of each yeast. 1: C. albicans (ATCC 90028); 2: C. tropicalis (ATCC 750); 3: C. glabrata sensu stricto (ATCC 90 030); 4: C. krusei (ATCC 6258); 5: C. parapsilosis sensu stricto (ATCC 22019); 6–9 and 14: C. auris; 10 and 19: C. pseudohaemulonii; 11, 12, 15–17 and 20: C. haemulonii. 13: C. vulturna; 18: C. duobushaemulonii. The photographs on the backs of the plates are mirror images of the original photographs (Digital Edition). This change was made to facilitate observation of the results. Table 1. CHROMagarTM Candida Plus performance’s summary. Organism (n) Morphotype Candida albicans (10)a Candida tropicalis (10)b Candida glabrata (11)c Candida krusei (5) Candida lusitaniae (10)d Candida auris (53)b Non-C. auris species of C. haemulonii complex (30)e , f Candida parapsilosis (70)e , g Turquoise blue/green Metallic blue w/slight pink halo Pink to purple Pink to purple with white edges Pink to purple White/pale with blue halo White/pink slow growth White/light blue Sensitivity (%) Specificity (%) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 86.04h a Candida dubliniensis was excluded from the analysis. It was not possible to differentiate C. albicans from C. dubliniensis using this media. Some strains displayed all the described morphotype only after 48 h of incubation. This analysis corresponds to C. glabrata sensu stricto. The cryptic species of C. glabrata species complex were indistinguishable from each other. d Same color as C. glabrata but bigger colonies. e Non-blue halo and blue halo were the pondered characteristic to consider a result as true positive and true negative, respectively. f Candida haemulonii (n = 15), C. duobushaemulonii (n = 5), C. pseudohaemulonii (n = 5) and C. vulturna (n = 5) were included. g This analysis corresponds to C. parapsilosis sensu stricto. The cryptic species of C. parapsilosis species complex were indistinguishable from each other. h Blue halo in 31 strains (false positive). b c halo. Similarly, 2/10 strains identified as C. orthopsilosis and C. metapsilosis (cryptic species of the C. parapsilosis species complex) showed morphotypes that may be confused with C. auris (1 strain of each species showed white colonies with blue halo). When mixed inocula (C. auris plus another Candida spp.) were plated onto CHROMagarTM Plus, the morphotypes were maintained. Candida auris was easily differentiated from C. haemulonii, C. albicans, C. tropicalis and C. parapsilosis ATCC 22019. On the other hand, C. parapsilosis LMDM-1234 (isolated from blood) grew as small white colonies with blue halo that may be confused with C. auris (although C. auris showed bigger colonies) (Fig. 3). Knowing that most (>93%) C. auris strains show high fluconazole MICs (≥16 μg/ml)25 we repeated the described experiments using CHROMagarTM Candida Plus with 8 μg/ml fluconazole. We found that in this modified media, C. auris was easily isolated maintaining their morphotypes and no C. parapsilosis colonies were obtained (Fig. 3). Discussion Chromogenic media are well established as a presumptive identification tool in clinical laboratories. Moreover, mixed infections can be uncovered by its use.18 In this work, we evaluate CHROMagarTM Candida Plus and it showed an excellent capability for species differentiation, as previously reported.18–20 Its main advantage is that it is able to differentiate C. auris in pure and in mixed cultures. Moreover, it is able to easily differentiate C. auris from other members of the C. haemulonii complex. This point must be highlighted since several commercially available identification methods confuse C. auris with other members of the complex.9 ,11 What is more important is that the identification of C. auris implies the isolation of the patient, while if a patient is colonized/infected by a non-C. auris species of the complex, this isolation is not necessary.10 ,11 Thus, this medium could be a useful tool to reduce the extension of C. auris outbreaks. One point to consider is that in 18.9% of the studied C. auris strains, the blue halo appeared at 48 h of incubation. Consequently, this reading time point should be used for 4 Sasoni et al. Figure 2. Representative morphotypes (obverse (A) and reverse (B) sides) of tested yeasts on CHROMagarTM Candida Plus incubated at 35°C for 48 h. Plates were spotted with 3 μl of suspensions of each yeast. 1: C. dubliniensis (NCPF3949); 2: C. parapsilosis (ATCC 22019); 3–8, 13–16 and 27: C. parapsilosis sensu stricto; 9–12, 17, 22–26: C. auris; 18 and 19: C. orthopsilosis; 20 and 21: C. metapsilosis; 28: C. haemulonii; 29, 30: C. lusitaniae; 31: C. tropicalis. The photographs on the backs of the plates are mirror images of the original photographs (Digital Edition). This change was made to facilitate observation of the results. Figure 3. Surveillance CHROMagarTM Candida Plus plates (top panels) and CHROMagarTM Candida Plus with 8 μg/ml of fluconazole (bottom panels) plates with mixed inoculum of C. auris with C. parapsilosis ATCC 22019 (left panels) and C. parapsilosis clinical strain LMDM 1234 (right panels) incubated for 48 h at 35°C. Circular photographs are digital close-ups of the colonies growing on CHROMagarTM Candida Plus. optimal performance (the possibility of the existence of C. auris in a sample before 48 h should not be ruled out). Similar observations were done before.18 ,20 One potential disadvantage of the evaluated media is the high percentage (44.3%) of false positives (white colonies with blue halo after 36–48 h of incubation) obtained with our collection of clinical C. parapsilosis strains. Mulet-Bayona et al.18 reported that some of their 10 C. parapsilosis isolates showed light bluish colonies. On the other hand, in other studies where CHROMagarTM Candida Plus was evaluated, only one C. parapsilosis clinical strain was used.19 This strain grew as white colonies with no blue halo. It is difficult to explain the reason of the observed morphotypes in C. parapsilosis (some resemblant to C. auris) since the composition of the chromogenic mixture is not known. However, these media are known to contain chromogenic enzyme substrates that take on different colors depending on the action of specific enzymes that could be present or absent in different Candida spp. In addition, it is estimated that the intensity of these colors may vary depending on the level of expression of these enzymes, which may be dependent on the strain under study. For C. parapsilosis, the manufacturer does not describe what color should be observed. However, in reports where the predecessor medium (CHROMagarTM Candida) was described,26 C. parapsilosis showed white or pale pink colonies. Thus, observed colors for this species are known to be strain dependent. For this reason, we can speculate that the variations in the observed morphotypes (blue halo present or absent) on CHROMagarTM Candida Plus among the evaluated strains may be due to a similar strain-dependent phenomenon. In order to reduce the possible misinterpretation of results, especially when C. auris surveillances studies are performed, we propose the use of a modified CHROMagarTM Candida Plus media with the addition of Fluconazole (8 μg/ml). 5 Medical Mycology, 2022, Vol. 60, No. 00 Using this modified media, tested C. parapsilosis were unable to grow and it could facilitate the presumptive identification of C. auris. If these modified plates with fluconazole are used, the only possible false-negative or false-positive results that would be obtained would be the case of a C. auris with a low fluconazole MIC (<7%, belonging mainly to C. auris clade II)26 ,27 or a C. parapsilosis with a high MIC to fluconazole MIC,28 respectively. From a practical point of view, we might suggest that clinical samples be streaked onto duplicate plates of the chromogenic agar, one with fluconazole and other with no drug. By doing so, users would isolate all Candida spp. (fluconazole susceptible and resistant) on the drug-free plate, whereas on modified CHROMagarTM Candida Plus medium, colonies with a blue halo would be easily identified as C. auris. A second approach would be sequential. Firstly, the clinical sample would be streaked onto a drug-free CHROMagarTM Candida Plus plate. After 48 h of incubation, individual colonies with a blue halo would be spotted onto the chromogenic medium with 8 μg/ml of fluconazole to confirm the identification as a C. auris. The choice of one or the other approach will depend on the budget (the first option is more expensive) and the expected response speed (the second option is slower). As a conclusion, we can state that CHROMagarTM Candida Plus is a helpful tool for presumptive identification for C. auris. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possibility of misidentification of C. parapsilosis as C. auris that would be reduced if fluconazole is added to the chromogenic media. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 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