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Different detection capabilities by mycological media for Candida isolates from mono- or dual-species cultures

Different detection capabilities by mycological media for Candida isolates from mono- or... OPENACCESS The aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) Citation: De Angelis G, Menchinelli G, Torelli R, De Carolis E, Posteraro P, Sanguinetti M, et al. (2020) media in regard to their capability of detecting Candida isolates from mono- or dual-species Different detection capabilities by mycological cultures. We prepared Candida isolates’ suspensions to obtain mono-species (n = 18) or media for Candida isolates from mono- or dual- dual-species (n = 153) culture plates per each medium, and three readers independently species cultures. PLoS ONE 15(3): e0226467. observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as https://doi.org/10.1371/journal.pone.0226467 correct, over or under detection compared to the expected species number(s). BCG showed Editor: Alex Friedrich, University Medical Center Groningen, NETHERLANDS significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had cor- Received: November 27, 2019 rect (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had cor- Accepted: March 4, 2020 rect (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for Published: March 23, 2020 an accurate laboratory diagnosis of Candida infections. Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: Introduction https://doi.org/10.1371/journal.pone.0226467 Almost concurrent with the enormous advances in medical diagnosis and treatment, a grow- Copyright:© 2020 De Angelis et al. This is an open ing number of individuals have become susceptible to acquiring fungal infections [1], and the access article distributed under the terms of the majority of these infections is lethal for more than 1.5 million people [2]. Fungal infections Creative Commons Attribution License, which such as mucosal/skin infections, though non-lethal, can reduce the quality of life for >1 billion permits unrestricted use, distribution, and affected people [2]. As opportunistic fungi, Candida species are the prevalent causes of invasive reproduction in any medium, provided the original author and source are credited. (e.g. candidaemia) and non-invasive (e.g. vulvovaginal candidiasis) fungal diseases, with an estimated ~700,000 invasive candidiasis cases occurring annually [2]. Data Availability Statement: All relevant data are Early diagnosis and, consequently, prompt treatment of invasive Candida infections is within the manuscript and its Supporting Information files. crucial to prevent mortality [3]. Five Candida species, Candida albicans, Candida glabrata, PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 1 / 9 PLOS ONE Mycological media for Candida isolation Funding: This study was supported by Università Candida tropicalis, Candida parapsilosis and Candida krusei, are responsible for 92% of cases Cattolica del Sacro Cuore (Fondi Ateneo, linea D1- of candidaemia globally [4]. However, the gamut of clinically relevant Candida species is 2018). expanding [5] and, notably, less common species (i.e. Candida guilliermondii complex) [6], Competing interests: The authors have declared rare species (e.g. Candida inconspicua [Torulopsis inconspicua], Candida pararugosa and that no competing interests exist. Pichia norvegensis [Candida norvegensis]) [7], or emerging species (i.e. Candida auris) [8] may exhibit high antifungal resistance levels, thereby compromising infection outcomes. Further- more, mixed bloodstream infections with Candida species in single patient-episodes are not uncommon [6, 9], consequently leading to misdiagnoses because of apparently pure isolates in mycological cultures. Implementation of matrix-assisted laser desorption ionization-time-of-flight (MALDI-- TOF) mass spectrometry (MS) in clinical mycology diagnostics has greatly shortened the time for fungal species identification [10]. However, the accuracy of identification still relies on the precision of picking fungal colonies from primary culture plates (i.e. directly derived from clinical samples) and on the media used to enable fungal colony growth. Chromogenic media (e.g. Brilliance Candida agar; Oxoid, Thermo Scientific, Basingstoke, UK) are currently used for both isolating and/or presumptively identifying Candida species from primary cultures in clinical microbiology laboratories [11, 12]. By contrast, the traditional Sabouraud dextrose agar (SDA) medium (Vacutest Kima S.r.l.) allows to isolate from and differentiate Candida species in primary cultures based on macromorphology features. Since several years, our labo- ratory adopted the Candida bromcresol green (BCG) medium (Vacutest Kima S.r.l., Padua, Italy) [13] as an alternative to the SDA [9]. The BCG had been introduced from Difco Labora- tories (Detroit, MI, USA) as a differential and selective medium for primary isolation and detection of Candida species from clinical samples. However, to the best of our knowledge, no study published did include the BCG medium in their mycological media evaluation. We compared the performance of BCG medium with those of Brilliance Candida agar medium (hereafter referred to as CHROM medium) and SDA medium, using Candida species allowed to grow in pure (mono-species) or mixed (dual-species) cultures, respectively. In addi- tion to the species claimed by the CHROM medium’s manufacturer as presumptively identifi- able (i.e. C. albicans, C. krusei and C. tropicalis) [12], we tested other species (including C. auris) to comprise five common and 13 uncommon species of Candida in total. Materials and methods We used 18 selected Candida species that belonged to the clinical isolate collection hold at the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome (Italy). Only C. auris was obtained from the Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen (The Netherlands). The species (isolate) included in the study were, in alphabetic order, C. albicans (UCSC34/23), C. auris (CWZ-1), C. dubliniensis (UCSC35/12), C. glabrata (UCSC61/2), C. guilliermondii (UCSC36/14), C. incospicua (UCSC72/2), Candida kefyr (UCSC51/14), C. krusei (UCSC59/12), Candida lusitaniae (UCSC59/18), Candida nivariensis (UCSC11/3), C. norve- gensis (UCSC64/13), C. parapsilosis (UCSC30/27), C. pararugosa (UCSC35/20), Candida pelliculosa (UCSC72/2), Candida robusta (UCSC54/2), Candida sorbosa (UCSC28/45), C. tropicalis (UCSC49/29) and Candida utilis (UCSC36/21). All the isolates were from patient bloodstream infections. Before testing, we recovered isolates from frozen stocks by culture on SDA plates at 30˚C ensuring vitality and/or pure growth. To confirm their identity, isolates were re-identified using the MALDI-TOF MS based method, as previously described [14]. We used each isolate to prepare a 0.5 McFarland suspension (~10 CFU/ml) in phosphate- buffered saline [12]. Using a checkerboard-like dilution scheme (S1 Fig), we mixed (ratio 1:1) each isolate’s suspension with the suspension of itself or with the suspension of each other PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 2 / 9 PLOS ONE Mycological media for Candida isolation isolate, respectively, in Axygen 96-Deep Well polypropylene plates to reach a 1-ml final vol- ume for a concentration of ~500 CFU per well. To obtain mono-species (n = 18) or dual-spe- cies (n = 153) cultures per medium, we used a spatula to spread a 100-μl (~50 CFU) aliquot from each well on the surfaces of BCG, CHROM or SDA plates. We incubated 513 plates at 30˚C, according to the media manufacturers’ instructions. Preliminarily, we performed con- trols with the isolates’ suspensions to verify growth, number of CFU and identity of Candida species (single or multiple) expected to grow on the plates. Although the common Candida species grew well at 37˚C, at least on the BCG or SDA media, we chose the 30˚C incubation to favour the uncommon Candida species (the majority in the study) growing slowly at 37˚C (e.g. C. guilliermondii). Three authors independently read the plates after 24, 48 and 72 h of incubation, in a blinded manner regarding type(s) and number(s) of Candida species growing on plates. These conditions are those universally accepted for the isolation of medically impor- tant yeasts from clinical specimens [15], but simultaneous incubation at 30˚C and 37˚C may be useful [16]. We scored individual reader results daily regarding how many different colonies, in terms of morphologic appearance (including texture and/or colour), he/she was able to observe. For the BCG, CHROM or SDA plate series (i.e. three plates for each species or combination of spe- cies tested per medium), we recorded the number of colonies observed by the readers on each plate, and we compared the numbers obtained with those expected for each plate of the three series (S1 Table). Thus, we reported reading results as correct detections (when the number of observed species equalled the number of expected species), over detections (when the number of observed species exceeded the number of expected species) or under detections (when the number of observed species was inferior to the number of expected species). Although more colony morphotypes do not necessarily correspond to different species, we considered the term morphotype as the equivalent of species when recorded our reading results. If necessary, we stratified the reading results by all the species (n = 18), common species (n = 5) or uncom- mon species (n = 13) obtained with the Candida isolates grown in either mono-species or dual-species culture plates. We compared detection rates on the BCG versus CHROM or SDA plates using chi-square test. We considered a p value of <0.05 statistically significant. We used the weighted kappa coefficient to assess the inter-reader agreement, with ranges described in literature [17]. Results We obtained 54 mono-species and 459 dual-species Candida cultures (171 on BCG, 171 on CHROM and 171 on SDA plates), and the results of detecting Candida species are shown in Fig 1 (all species), S2 Fig (common species) and S3 Fig (uncommon species). The percentages of correct detections of the three Candida species groups, compared to expected results, ranged with the BCG medium from 73.1 (24 h) to 90.1 (72 h), 73.3 (48/72 h) to 93.3 (72 h), and 64.8 (24 h) to 91.2 (72 h); with the CHROM medium from 44.4 (24 h) to 90.6 (72 h), 53.3 (24 h) to 93.3 (48/72 h), and 40.7 (24 h) to 87.9 (72 h); and with the SDA medium from 60.8 (24 h) to 92.4 (72 h), 86.7 (24/48/72 h) to 93.3 (24/48/72 h), and 52.7 (24 h) to 91.2 (72 h). The percent- ages of over detections ranged with the BCG medium from 0.6 (24 h) to 9.4 (48 h), 6.7 (24/48/ 72 h) to 20.0 (24/48/72 h), and 0.0 (24 h) to 7.7 (72 h); with the CHROM medium from 0.6 (24/48/72 h) to 4.7 (48 h), 0.0 (48/72 h) to 13.3 (48/72 h), and 0.0 (24 h) to 4.4 (24/72 h); and with the SDA medium from 0.0 (24 h) to 4.7 (48/72 h), 0.0 (24 h) to 13.3 (48/72 h), and 0.0 (24/48/72 h) to 6.6 (72 h). The percentages of under detections ranged with the BCG medium from 2.9 (72 h) to 26.3 (24 h), 0.0 (24/48/72 h) to 6.7 (24/48/72 h), and 1.1 (72 h) to 35.2 (24 h); with the CHROM medium from 5.8 (72 h) to 55.0 (24 h), 6.7 (48/72 h) to 40.0 (24 h), and 7.7 PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 3 / 9 PLOS ONE Mycological media for Candida isolation Fig 1. Rates of correct, over or under detections by three readers for the overall Candida species grown as mono- (n = 18) or dual- species cultures (n = 153) on the BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. https://doi.org/10.1371/journal.pone.0226467.g001 (72 h) to 59.3 (24 h); with the SDA medium from 2.9 (72 h) to 39.2 (24 h), 0.0 (24/48/72 h) to 6.7 (24 h), and 2.2 (72 h) to 47.3 (24 h). As for all the Candida species observed by at least one reader, statistically significant differ- ences in the rates of correct or under detections did favour the BCG medium over the CHROM medium (24/48/72 h) and the SDA medium (24 h and 24/48/72 h, respectively) (Fig 1). As for the common Candida species observed by at least one reader, statistically significant differences in the rates of correct or under detections did favour the BCG medium over the CHROM medium (24 h) (S2 Fig). As for the uncommon Candida species observed by at least one reader, statistically significant differences did favour the BCG medium over the CHROM medium in the rates of correct detections (24/48/72 h) and over both the CHROM and SDA media in the rates of under detections (24/48/72 h) (S3 Fig). As shown in Table 1, we analysed detection results regarding inter-reader agreement. The levels of agreement for the 24-h readings of BCG, CHROM and SDA plates, for all or uncom- mon species, were at least moderate (kappa coefficient values, 0.41–0.60), whereas the levels of agreement for the 48-h and 72-h readings of BCG, CHROM and SDA plates were at least fair (kappa coefficient values, 0.21–0.40). Conversely, for common species, the levels of agreement for the 24-h/48-h/72-h readings of CHROM plates and for the 48-h/72-h readings of SDA plates were at least substantial (kappa coefficient values, 0.61–0.80). However, comparing the three readers with respect to the percentages of incorrect (over/under) detections for overall PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 4 / 9 PLOS ONE Mycological media for Candida isolation Table 1. Agreement by readers on the detection results of Candida species cultured on three media plates that were obtained at 24, 48 or 72 h of incubation of the plates. Candida organisms grown in mono- or dual- Culture media N. of overall N. of detections found Kappa coefficient Level of inter-reader species cultures plates read at detections to be in agreement and (95% CI) agreement the indicated scored as times Correct Over Under All species (n = 18) BCG 24 171 108 1 18 0.53 (0.41–0.64) Moderate CHROM 171 59 0 52 0.55 (0.47–0.64) Moderate SDA 171 87 0 26 0.47 (0.37–0.58) Moderate BCG 48 171 120 1 4 0.29 (0.15–0.43) Fair CHROM 171 99 1 19 0.47 (0.36–0.59) Moderate SDA 171 113 1 7 0.34 (0.22–0.48) Fair BCG 72 171 126 2 0 0.25 (0.13–0.38) Fair CHROM 171 109 1 7 0.30 (0.18–0.44) Fair SDA 171 119 1 3 0.31 (0.19–0.43) Fair Common species only (n = 5) BCG 24 15 12 1 0 0.59 (-0.04–1.30) Moderate CHROM 15 7 0 5 0.75 (0.48–1.04) Substantial SDA 15 12 0 0 0.20 (-0.29–0.86) Fair BCG 48 15 9 1 0 0.28 (-0.43–1.16) Fair CHROM 15 12 0 1 0.68 (0.18–1.27) Substantial SDA 15 13 1 0 0.72 (-0.41–2.76) Substantial BCG 72 15 10 1 0 0.36 (-0.41–1.37) Fair CHROM 15 12 0 1 0.63 (-0.03–1.52) Substantial SDA 15 13 1 0 0.72 (-0.41–2.76) Substantial Uncommon species only (n = 13) BCG 24 91 53 0 15 0.59 (0.45–0.74) Moderate CHROM 91 26 0 28 0.49 (0.37–0.61) Moderate SDA 91 40 0 20 0.52 (0.39–0.66) Moderate BCG 48 91 67 0 4 0.39 (0.18–0.62) Fair CHROM 91 47 1 15 0.54 (0.40–0.68) Substantial SDA 91 56 0 4 0.28 (0.11–0.46) Fair BCG 72 91 71 1 0 0.33 (0.17–0.51) Fair CHROM 91 54 1 5 0.35 (0.19–0.53) Fair SDA 91 57 0 2 0.23 (0.08–0.38) Fair Mycological media used for mono- or dual-species cultures of all, common or uncommon species of Candida were BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar). We calculated the kappa coefficient with 95% confidence interval (95% CI) for the comparison among the rates of correct, over or under detections (according to the definitions specified in the text). With regard to the agreement by readers, we used values greater than zero to indicate none to slight (0.01–0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80) or almost perfect (0.81–1.00) levels of agreement, and values lower than/equal to zero to indicate the absence of agreement https://doi.org/10.1371/journal.pone.0226467.t001 readings of BCG, CHROM and SDA plates revealed statistically significant differences between the readers only for CHROM and SDA plates (S2 Table). The BCG medium is similar to other selective and differential media for the primary iso- lation of Candida species. It consists of peptone agar base supplemented with yeast extract (which is absent in the SDA) and dextrose to support growth [13]. However, bromcresol green helps to differentiate and identify Candida species, because a change in the pH causes the medium to take on a yellow colour around the Candida colonies that ferment dextrose [13]. Fig 2 depicts the appearance on the BCG medium for the five common species of Can- dida tested by us. As it can see, C. albicans formed smooth, regular, matte, and white to dark-green colonies, C. glabrata smooth, circular, brilliant and white to pale-green colonies, PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 5 / 9 PLOS ONE Mycological media for Candida isolation Fig 2. Appearance on the Candida bromcresol green (BCG) medium of five Candida isolates included in the study that belong to (A) C. albicans, (B) C. glabrata, (C) C. krusei, (D) C. parapsilosis and (E) C. tropicalis. The isolates were seeded on the BCG plates and incubated at 30˚C before the plates were imaged. (See the text for the detailed description of the isolates’ features). https://doi.org/10.1371/journal.pone.0226467.g002 C. krusei rough, irregular, matte and green and white-edged colonies, C. parapsilosis rough, irregular, small and white colonies, and C. tropicalis smooth, regular, matte and white colonies. Discussion Apart from the overall slight superiority shown by the BCG medium, we noticed that 12 (66.7%) of 18 mono-species cultures at the 24-h readings had correct or over detections (i.e. cultures with�1 colony morphologies observed per plate) in the BCG plates as well in both the CHROM and SDA plates (S1 Table). The only exceptions were C. albicans, C. incospicua, C. lusitaniae, C. pararugosa, C. pelliculosa and C. sorbosa. Interestingly, while detection of C. albicans in the CHROM plates occurred not prior to 48 h of incubation, over detection of C. glabrata occurred always in 100% of BCG plate readings, 55.6% of CHROM plate readings, and 88.9% of SDA plate readings. These results are consistent with those from some previous studies [12, 18]. In one study, 132 (23%) of the 564 C. albicans isolates recovered by routinely used media, did not grow on a chromogenic medium [18]. Another study showed the pre- sumptive identification of five Candida species (C. albicans, C. dubliniensis, C. krusei, C. tropi- calis and C. parapsilosis) on the CHROM medium (i.e. two additional species besides those identifiable by the medium) [12]. However, discrimination for several Candida species, including C. glabrata, may be diffi- cult. Conversely, the bromcresol green, a non-toxic indicator contained in the BCG medium (i.e. a modified SDA), seems to aid primary isolation and detection of Candida species from clinical samples based on dextrose fermentation [13]. While the medium colour around the colonies becomes yellow (usually within 72 h of incubation), the Candida species grown on the BCG medium produce convex to cone-shaped, smooth to rough colonies. Thus, the BCG medium would allow to easily revealing differences in colour (i.e. tonalities of yellow) as well in morphology (i.e. extents of roughness) (Fig 2). We noted that one reader differed from the two other readers with respect to the over detection at 24, 48 and 72 h mainly for the uncom- mon Candida species (S3 Fig). Although we chose the three readers to represent a medium-to- high extent of expertise in medical mycology, it is plausible that subtle differences in their mycological skills may explain for the moderate or fair levels of agreement found across read- ers (Table 1). Among mixed candidaemia episodes, C. albicans plus C. glabrata is usually the most fre- quent combination [5], but other combinations of Candida species may be of great impor- tance. We noticed that 106 (69.3%), 60 (39.2%) and 78 (51.0%) of 153 dual-species cultures at PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 6 / 9 PLOS ONE Mycological media for Candida isolation the 24-h readings had correct or over detections (i.e. cultures with�2 colony morphologies observed per plate) in the BCG, CHROM or SDA plates, respectively (S1 Table). More interest- ingly, six cultures (C. albicans/C. parapsilosis, C. incospicua/C. pelliculosa, C. incospicua/C. sor- bosa, C. nivariensis/C. pelliculosa, C. pararugosa/C. sorbosa and C. sorbosa/C. tropicalis) in the CHROM medium and two cultures (C. auris/C. guilliermondii and C. auris/C. lusitaniae) in the SDA medium were always under detected with respect to one of the two species grown together. Although a mix of these species seems to be very uncommon, their incomplete detec- tion in candidaemia cases may have clinical repercussions, especially because of different anti- fungal susceptibility profiles exhibited by these species [5–8]. In conclusion, reading primary culture plates from patient samples in the clinical mycology laboratory remains somewhat subjective. For Candida species, the existence of different mor- photypes, which underpins transitions between commensal and pathogenic cell types in the same species [19–21], complicates the situation. However, distinguishing as many as possible Candida colonies, which will likely correspond to different Candida species, in clinical sam- ples, is crucial in order to exploit the established, powerful MALDI-TOF MS capability of iden- tifying any Candida organism to the species level (or even beyond). Therefore, using the BCG medium may represent an essential prerequisite for a specific and accurate diagnosis of the causative infection agent(s), especially in patients suffering from life-threatening candidiasis. Supporting information S1 Table. Results of the detections by three independent readers for mono-species or dual- species Candida cultures recorded at 24, 48 or 72 h of incubation of the BCG, CHROM or SDA culture plates. (DOC) S2 Table. Rates of incorrect (over/under) detections by three readers for the overall BCG, CHROM or SDA cultures of Candida species. Bold indicates statistically significant differ- ences in the comparisons between reader #1 and reader #2, reader #2 and reader #3, or reader #1 and reader #3. (DOC) S1 Fig. Scheme of the checkerboard-like dilution method used to obtain mono-species or dual-species Candida suspensions before spreading them on the mycological media under evaluation. (DOC) S2 Fig. Rates of correct, over or under detections by three readers for the common Candida species grown as mono- (n = 5) or dual-species (n = 10) on the BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabour- aud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. (JPG) S3 Fig. Rates of correct, over or under detections by three readers for the uncommon Can- dida species grown as mono- (n = 13) or dual-species (n = 78) on the BCG (Candida brom- cresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. (JPG) PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 7 / 9 PLOS ONE Mycological media for Candida isolation Acknowledgments We wish to thank Franziska Lohmeyer for her English language assistance. Author Contributions Conceptualization: Giulia De Angelis, Brunella Posteraro. Data curation: Giulia De Angelis, Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro, Brunella Posteraro. Formal analysis: Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro. Investigation: Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro. Methodology: Giulia Menchinelli, Brunella Posteraro. Supervision: Maurizio Sanguinetti. Validation: Maurizio Sanguinetti. Writing – original draft: Giulia De Angelis, Brunella Posteraro. Writing – review & editing: Brunella Posteraro. References 1. Clark C, Drummond RA. 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PLoS Pathog 2017; 13:e1006290. https://doi.org/10.1371/ journal.ppat.1006290 PMID: 28542486 PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 9 / 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Public Library of Science (PLoS) Journal

Different detection capabilities by mycological media for Candida isolates from mono- or dual-species cultures

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Copyright: © 2020 De Angelis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was supported by Università Cattolica del Sacro Cuore (Fondi Ateneo, linea D1-2018). Competing interests: The authors have declared that no competing interests exist.
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Abstract

OPENACCESS The aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) Citation: De Angelis G, Menchinelli G, Torelli R, De Carolis E, Posteraro P, Sanguinetti M, et al. (2020) media in regard to their capability of detecting Candida isolates from mono- or dual-species Different detection capabilities by mycological cultures. We prepared Candida isolates’ suspensions to obtain mono-species (n = 18) or media for Candida isolates from mono- or dual- dual-species (n = 153) culture plates per each medium, and three readers independently species cultures. PLoS ONE 15(3): e0226467. observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as https://doi.org/10.1371/journal.pone.0226467 correct, over or under detection compared to the expected species number(s). BCG showed Editor: Alex Friedrich, University Medical Center Groningen, NETHERLANDS significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had cor- Received: November 27, 2019 rect (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had cor- Accepted: March 4, 2020 rect (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for Published: March 23, 2020 an accurate laboratory diagnosis of Candida infections. Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: Introduction https://doi.org/10.1371/journal.pone.0226467 Almost concurrent with the enormous advances in medical diagnosis and treatment, a grow- Copyright:© 2020 De Angelis et al. This is an open ing number of individuals have become susceptible to acquiring fungal infections [1], and the access article distributed under the terms of the majority of these infections is lethal for more than 1.5 million people [2]. Fungal infections Creative Commons Attribution License, which such as mucosal/skin infections, though non-lethal, can reduce the quality of life for >1 billion permits unrestricted use, distribution, and affected people [2]. As opportunistic fungi, Candida species are the prevalent causes of invasive reproduction in any medium, provided the original author and source are credited. (e.g. candidaemia) and non-invasive (e.g. vulvovaginal candidiasis) fungal diseases, with an estimated ~700,000 invasive candidiasis cases occurring annually [2]. Data Availability Statement: All relevant data are Early diagnosis and, consequently, prompt treatment of invasive Candida infections is within the manuscript and its Supporting Information files. crucial to prevent mortality [3]. Five Candida species, Candida albicans, Candida glabrata, PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 1 / 9 PLOS ONE Mycological media for Candida isolation Funding: This study was supported by Università Candida tropicalis, Candida parapsilosis and Candida krusei, are responsible for 92% of cases Cattolica del Sacro Cuore (Fondi Ateneo, linea D1- of candidaemia globally [4]. However, the gamut of clinically relevant Candida species is 2018). expanding [5] and, notably, less common species (i.e. Candida guilliermondii complex) [6], Competing interests: The authors have declared rare species (e.g. Candida inconspicua [Torulopsis inconspicua], Candida pararugosa and that no competing interests exist. Pichia norvegensis [Candida norvegensis]) [7], or emerging species (i.e. Candida auris) [8] may exhibit high antifungal resistance levels, thereby compromising infection outcomes. Further- more, mixed bloodstream infections with Candida species in single patient-episodes are not uncommon [6, 9], consequently leading to misdiagnoses because of apparently pure isolates in mycological cultures. Implementation of matrix-assisted laser desorption ionization-time-of-flight (MALDI-- TOF) mass spectrometry (MS) in clinical mycology diagnostics has greatly shortened the time for fungal species identification [10]. However, the accuracy of identification still relies on the precision of picking fungal colonies from primary culture plates (i.e. directly derived from clinical samples) and on the media used to enable fungal colony growth. Chromogenic media (e.g. Brilliance Candida agar; Oxoid, Thermo Scientific, Basingstoke, UK) are currently used for both isolating and/or presumptively identifying Candida species from primary cultures in clinical microbiology laboratories [11, 12]. By contrast, the traditional Sabouraud dextrose agar (SDA) medium (Vacutest Kima S.r.l.) allows to isolate from and differentiate Candida species in primary cultures based on macromorphology features. Since several years, our labo- ratory adopted the Candida bromcresol green (BCG) medium (Vacutest Kima S.r.l., Padua, Italy) [13] as an alternative to the SDA [9]. The BCG had been introduced from Difco Labora- tories (Detroit, MI, USA) as a differential and selective medium for primary isolation and detection of Candida species from clinical samples. However, to the best of our knowledge, no study published did include the BCG medium in their mycological media evaluation. We compared the performance of BCG medium with those of Brilliance Candida agar medium (hereafter referred to as CHROM medium) and SDA medium, using Candida species allowed to grow in pure (mono-species) or mixed (dual-species) cultures, respectively. In addi- tion to the species claimed by the CHROM medium’s manufacturer as presumptively identifi- able (i.e. C. albicans, C. krusei and C. tropicalis) [12], we tested other species (including C. auris) to comprise five common and 13 uncommon species of Candida in total. Materials and methods We used 18 selected Candida species that belonged to the clinical isolate collection hold at the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome (Italy). Only C. auris was obtained from the Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen (The Netherlands). The species (isolate) included in the study were, in alphabetic order, C. albicans (UCSC34/23), C. auris (CWZ-1), C. dubliniensis (UCSC35/12), C. glabrata (UCSC61/2), C. guilliermondii (UCSC36/14), C. incospicua (UCSC72/2), Candida kefyr (UCSC51/14), C. krusei (UCSC59/12), Candida lusitaniae (UCSC59/18), Candida nivariensis (UCSC11/3), C. norve- gensis (UCSC64/13), C. parapsilosis (UCSC30/27), C. pararugosa (UCSC35/20), Candida pelliculosa (UCSC72/2), Candida robusta (UCSC54/2), Candida sorbosa (UCSC28/45), C. tropicalis (UCSC49/29) and Candida utilis (UCSC36/21). All the isolates were from patient bloodstream infections. Before testing, we recovered isolates from frozen stocks by culture on SDA plates at 30˚C ensuring vitality and/or pure growth. To confirm their identity, isolates were re-identified using the MALDI-TOF MS based method, as previously described [14]. We used each isolate to prepare a 0.5 McFarland suspension (~10 CFU/ml) in phosphate- buffered saline [12]. Using a checkerboard-like dilution scheme (S1 Fig), we mixed (ratio 1:1) each isolate’s suspension with the suspension of itself or with the suspension of each other PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 2 / 9 PLOS ONE Mycological media for Candida isolation isolate, respectively, in Axygen 96-Deep Well polypropylene plates to reach a 1-ml final vol- ume for a concentration of ~500 CFU per well. To obtain mono-species (n = 18) or dual-spe- cies (n = 153) cultures per medium, we used a spatula to spread a 100-μl (~50 CFU) aliquot from each well on the surfaces of BCG, CHROM or SDA plates. We incubated 513 plates at 30˚C, according to the media manufacturers’ instructions. Preliminarily, we performed con- trols with the isolates’ suspensions to verify growth, number of CFU and identity of Candida species (single or multiple) expected to grow on the plates. Although the common Candida species grew well at 37˚C, at least on the BCG or SDA media, we chose the 30˚C incubation to favour the uncommon Candida species (the majority in the study) growing slowly at 37˚C (e.g. C. guilliermondii). Three authors independently read the plates after 24, 48 and 72 h of incubation, in a blinded manner regarding type(s) and number(s) of Candida species growing on plates. These conditions are those universally accepted for the isolation of medically impor- tant yeasts from clinical specimens [15], but simultaneous incubation at 30˚C and 37˚C may be useful [16]. We scored individual reader results daily regarding how many different colonies, in terms of morphologic appearance (including texture and/or colour), he/she was able to observe. For the BCG, CHROM or SDA plate series (i.e. three plates for each species or combination of spe- cies tested per medium), we recorded the number of colonies observed by the readers on each plate, and we compared the numbers obtained with those expected for each plate of the three series (S1 Table). Thus, we reported reading results as correct detections (when the number of observed species equalled the number of expected species), over detections (when the number of observed species exceeded the number of expected species) or under detections (when the number of observed species was inferior to the number of expected species). Although more colony morphotypes do not necessarily correspond to different species, we considered the term morphotype as the equivalent of species when recorded our reading results. If necessary, we stratified the reading results by all the species (n = 18), common species (n = 5) or uncom- mon species (n = 13) obtained with the Candida isolates grown in either mono-species or dual-species culture plates. We compared detection rates on the BCG versus CHROM or SDA plates using chi-square test. We considered a p value of <0.05 statistically significant. We used the weighted kappa coefficient to assess the inter-reader agreement, with ranges described in literature [17]. Results We obtained 54 mono-species and 459 dual-species Candida cultures (171 on BCG, 171 on CHROM and 171 on SDA plates), and the results of detecting Candida species are shown in Fig 1 (all species), S2 Fig (common species) and S3 Fig (uncommon species). The percentages of correct detections of the three Candida species groups, compared to expected results, ranged with the BCG medium from 73.1 (24 h) to 90.1 (72 h), 73.3 (48/72 h) to 93.3 (72 h), and 64.8 (24 h) to 91.2 (72 h); with the CHROM medium from 44.4 (24 h) to 90.6 (72 h), 53.3 (24 h) to 93.3 (48/72 h), and 40.7 (24 h) to 87.9 (72 h); and with the SDA medium from 60.8 (24 h) to 92.4 (72 h), 86.7 (24/48/72 h) to 93.3 (24/48/72 h), and 52.7 (24 h) to 91.2 (72 h). The percent- ages of over detections ranged with the BCG medium from 0.6 (24 h) to 9.4 (48 h), 6.7 (24/48/ 72 h) to 20.0 (24/48/72 h), and 0.0 (24 h) to 7.7 (72 h); with the CHROM medium from 0.6 (24/48/72 h) to 4.7 (48 h), 0.0 (48/72 h) to 13.3 (48/72 h), and 0.0 (24 h) to 4.4 (24/72 h); and with the SDA medium from 0.0 (24 h) to 4.7 (48/72 h), 0.0 (24 h) to 13.3 (48/72 h), and 0.0 (24/48/72 h) to 6.6 (72 h). The percentages of under detections ranged with the BCG medium from 2.9 (72 h) to 26.3 (24 h), 0.0 (24/48/72 h) to 6.7 (24/48/72 h), and 1.1 (72 h) to 35.2 (24 h); with the CHROM medium from 5.8 (72 h) to 55.0 (24 h), 6.7 (48/72 h) to 40.0 (24 h), and 7.7 PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 3 / 9 PLOS ONE Mycological media for Candida isolation Fig 1. Rates of correct, over or under detections by three readers for the overall Candida species grown as mono- (n = 18) or dual- species cultures (n = 153) on the BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. https://doi.org/10.1371/journal.pone.0226467.g001 (72 h) to 59.3 (24 h); with the SDA medium from 2.9 (72 h) to 39.2 (24 h), 0.0 (24/48/72 h) to 6.7 (24 h), and 2.2 (72 h) to 47.3 (24 h). As for all the Candida species observed by at least one reader, statistically significant differ- ences in the rates of correct or under detections did favour the BCG medium over the CHROM medium (24/48/72 h) and the SDA medium (24 h and 24/48/72 h, respectively) (Fig 1). As for the common Candida species observed by at least one reader, statistically significant differences in the rates of correct or under detections did favour the BCG medium over the CHROM medium (24 h) (S2 Fig). As for the uncommon Candida species observed by at least one reader, statistically significant differences did favour the BCG medium over the CHROM medium in the rates of correct detections (24/48/72 h) and over both the CHROM and SDA media in the rates of under detections (24/48/72 h) (S3 Fig). As shown in Table 1, we analysed detection results regarding inter-reader agreement. The levels of agreement for the 24-h readings of BCG, CHROM and SDA plates, for all or uncom- mon species, were at least moderate (kappa coefficient values, 0.41–0.60), whereas the levels of agreement for the 48-h and 72-h readings of BCG, CHROM and SDA plates were at least fair (kappa coefficient values, 0.21–0.40). Conversely, for common species, the levels of agreement for the 24-h/48-h/72-h readings of CHROM plates and for the 48-h/72-h readings of SDA plates were at least substantial (kappa coefficient values, 0.61–0.80). However, comparing the three readers with respect to the percentages of incorrect (over/under) detections for overall PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 4 / 9 PLOS ONE Mycological media for Candida isolation Table 1. Agreement by readers on the detection results of Candida species cultured on three media plates that were obtained at 24, 48 or 72 h of incubation of the plates. Candida organisms grown in mono- or dual- Culture media N. of overall N. of detections found Kappa coefficient Level of inter-reader species cultures plates read at detections to be in agreement and (95% CI) agreement the indicated scored as times Correct Over Under All species (n = 18) BCG 24 171 108 1 18 0.53 (0.41–0.64) Moderate CHROM 171 59 0 52 0.55 (0.47–0.64) Moderate SDA 171 87 0 26 0.47 (0.37–0.58) Moderate BCG 48 171 120 1 4 0.29 (0.15–0.43) Fair CHROM 171 99 1 19 0.47 (0.36–0.59) Moderate SDA 171 113 1 7 0.34 (0.22–0.48) Fair BCG 72 171 126 2 0 0.25 (0.13–0.38) Fair CHROM 171 109 1 7 0.30 (0.18–0.44) Fair SDA 171 119 1 3 0.31 (0.19–0.43) Fair Common species only (n = 5) BCG 24 15 12 1 0 0.59 (-0.04–1.30) Moderate CHROM 15 7 0 5 0.75 (0.48–1.04) Substantial SDA 15 12 0 0 0.20 (-0.29–0.86) Fair BCG 48 15 9 1 0 0.28 (-0.43–1.16) Fair CHROM 15 12 0 1 0.68 (0.18–1.27) Substantial SDA 15 13 1 0 0.72 (-0.41–2.76) Substantial BCG 72 15 10 1 0 0.36 (-0.41–1.37) Fair CHROM 15 12 0 1 0.63 (-0.03–1.52) Substantial SDA 15 13 1 0 0.72 (-0.41–2.76) Substantial Uncommon species only (n = 13) BCG 24 91 53 0 15 0.59 (0.45–0.74) Moderate CHROM 91 26 0 28 0.49 (0.37–0.61) Moderate SDA 91 40 0 20 0.52 (0.39–0.66) Moderate BCG 48 91 67 0 4 0.39 (0.18–0.62) Fair CHROM 91 47 1 15 0.54 (0.40–0.68) Substantial SDA 91 56 0 4 0.28 (0.11–0.46) Fair BCG 72 91 71 1 0 0.33 (0.17–0.51) Fair CHROM 91 54 1 5 0.35 (0.19–0.53) Fair SDA 91 57 0 2 0.23 (0.08–0.38) Fair Mycological media used for mono- or dual-species cultures of all, common or uncommon species of Candida were BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar). We calculated the kappa coefficient with 95% confidence interval (95% CI) for the comparison among the rates of correct, over or under detections (according to the definitions specified in the text). With regard to the agreement by readers, we used values greater than zero to indicate none to slight (0.01–0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80) or almost perfect (0.81–1.00) levels of agreement, and values lower than/equal to zero to indicate the absence of agreement https://doi.org/10.1371/journal.pone.0226467.t001 readings of BCG, CHROM and SDA plates revealed statistically significant differences between the readers only for CHROM and SDA plates (S2 Table). The BCG medium is similar to other selective and differential media for the primary iso- lation of Candida species. It consists of peptone agar base supplemented with yeast extract (which is absent in the SDA) and dextrose to support growth [13]. However, bromcresol green helps to differentiate and identify Candida species, because a change in the pH causes the medium to take on a yellow colour around the Candida colonies that ferment dextrose [13]. Fig 2 depicts the appearance on the BCG medium for the five common species of Can- dida tested by us. As it can see, C. albicans formed smooth, regular, matte, and white to dark-green colonies, C. glabrata smooth, circular, brilliant and white to pale-green colonies, PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 5 / 9 PLOS ONE Mycological media for Candida isolation Fig 2. Appearance on the Candida bromcresol green (BCG) medium of five Candida isolates included in the study that belong to (A) C. albicans, (B) C. glabrata, (C) C. krusei, (D) C. parapsilosis and (E) C. tropicalis. The isolates were seeded on the BCG plates and incubated at 30˚C before the plates were imaged. (See the text for the detailed description of the isolates’ features). https://doi.org/10.1371/journal.pone.0226467.g002 C. krusei rough, irregular, matte and green and white-edged colonies, C. parapsilosis rough, irregular, small and white colonies, and C. tropicalis smooth, regular, matte and white colonies. Discussion Apart from the overall slight superiority shown by the BCG medium, we noticed that 12 (66.7%) of 18 mono-species cultures at the 24-h readings had correct or over detections (i.e. cultures with�1 colony morphologies observed per plate) in the BCG plates as well in both the CHROM and SDA plates (S1 Table). The only exceptions were C. albicans, C. incospicua, C. lusitaniae, C. pararugosa, C. pelliculosa and C. sorbosa. Interestingly, while detection of C. albicans in the CHROM plates occurred not prior to 48 h of incubation, over detection of C. glabrata occurred always in 100% of BCG plate readings, 55.6% of CHROM plate readings, and 88.9% of SDA plate readings. These results are consistent with those from some previous studies [12, 18]. In one study, 132 (23%) of the 564 C. albicans isolates recovered by routinely used media, did not grow on a chromogenic medium [18]. Another study showed the pre- sumptive identification of five Candida species (C. albicans, C. dubliniensis, C. krusei, C. tropi- calis and C. parapsilosis) on the CHROM medium (i.e. two additional species besides those identifiable by the medium) [12]. However, discrimination for several Candida species, including C. glabrata, may be diffi- cult. Conversely, the bromcresol green, a non-toxic indicator contained in the BCG medium (i.e. a modified SDA), seems to aid primary isolation and detection of Candida species from clinical samples based on dextrose fermentation [13]. While the medium colour around the colonies becomes yellow (usually within 72 h of incubation), the Candida species grown on the BCG medium produce convex to cone-shaped, smooth to rough colonies. Thus, the BCG medium would allow to easily revealing differences in colour (i.e. tonalities of yellow) as well in morphology (i.e. extents of roughness) (Fig 2). We noted that one reader differed from the two other readers with respect to the over detection at 24, 48 and 72 h mainly for the uncom- mon Candida species (S3 Fig). Although we chose the three readers to represent a medium-to- high extent of expertise in medical mycology, it is plausible that subtle differences in their mycological skills may explain for the moderate or fair levels of agreement found across read- ers (Table 1). Among mixed candidaemia episodes, C. albicans plus C. glabrata is usually the most fre- quent combination [5], but other combinations of Candida species may be of great impor- tance. We noticed that 106 (69.3%), 60 (39.2%) and 78 (51.0%) of 153 dual-species cultures at PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 6 / 9 PLOS ONE Mycological media for Candida isolation the 24-h readings had correct or over detections (i.e. cultures with�2 colony morphologies observed per plate) in the BCG, CHROM or SDA plates, respectively (S1 Table). More interest- ingly, six cultures (C. albicans/C. parapsilosis, C. incospicua/C. pelliculosa, C. incospicua/C. sor- bosa, C. nivariensis/C. pelliculosa, C. pararugosa/C. sorbosa and C. sorbosa/C. tropicalis) in the CHROM medium and two cultures (C. auris/C. guilliermondii and C. auris/C. lusitaniae) in the SDA medium were always under detected with respect to one of the two species grown together. Although a mix of these species seems to be very uncommon, their incomplete detec- tion in candidaemia cases may have clinical repercussions, especially because of different anti- fungal susceptibility profiles exhibited by these species [5–8]. In conclusion, reading primary culture plates from patient samples in the clinical mycology laboratory remains somewhat subjective. For Candida species, the existence of different mor- photypes, which underpins transitions between commensal and pathogenic cell types in the same species [19–21], complicates the situation. However, distinguishing as many as possible Candida colonies, which will likely correspond to different Candida species, in clinical sam- ples, is crucial in order to exploit the established, powerful MALDI-TOF MS capability of iden- tifying any Candida organism to the species level (or even beyond). Therefore, using the BCG medium may represent an essential prerequisite for a specific and accurate diagnosis of the causative infection agent(s), especially in patients suffering from life-threatening candidiasis. Supporting information S1 Table. Results of the detections by three independent readers for mono-species or dual- species Candida cultures recorded at 24, 48 or 72 h of incubation of the BCG, CHROM or SDA culture plates. (DOC) S2 Table. Rates of incorrect (over/under) detections by three readers for the overall BCG, CHROM or SDA cultures of Candida species. Bold indicates statistically significant differ- ences in the comparisons between reader #1 and reader #2, reader #2 and reader #3, or reader #1 and reader #3. (DOC) S1 Fig. Scheme of the checkerboard-like dilution method used to obtain mono-species or dual-species Candida suspensions before spreading them on the mycological media under evaluation. (DOC) S2 Fig. Rates of correct, over or under detections by three readers for the common Candida species grown as mono- (n = 5) or dual-species (n = 10) on the BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabour- aud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. (JPG) S3 Fig. Rates of correct, over or under detections by three readers for the uncommon Can- dida species grown as mono- (n = 13) or dual-species (n = 78) on the BCG (Candida brom- cresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar) media. Asterisks indicate statistically significant differences between the rates of detections obtained with the BCG medium and those of the CHROM or SDA media. (JPG) PLOS ONE | https://doi.org/10.1371/journal.pone.0226467 March 23, 2020 7 / 9 PLOS ONE Mycological media for Candida isolation Acknowledgments We wish to thank Franziska Lohmeyer for her English language assistance. Author Contributions Conceptualization: Giulia De Angelis, Brunella Posteraro. Data curation: Giulia De Angelis, Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro, Brunella Posteraro. Formal analysis: Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro. Investigation: Giulia Menchinelli, Riccardo Torelli, Elena De Carolis, Patrizia Posteraro. Methodology: Giulia Menchinelli, Brunella Posteraro. Supervision: Maurizio Sanguinetti. Validation: Maurizio Sanguinetti. Writing – original draft: Giulia De Angelis, Brunella Posteraro. Writing – review & editing: Brunella Posteraro. References 1. Clark C, Drummond RA. 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