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

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) media in regard to their capability of detecting Candida isolates from mono- or dual-species cultures. We prepared Candida isolates' suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s). BCG showed 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 correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for an accurate laboratory diagnosis of Candida infections.

Introduction

Almost concurrent with the enormous advances in medical diagnosis and treatment, a growing number of individuals have become susceptible to acquiring fungal infections [1], and the majority of these infections is lethal for more than 1.5 million people [2]. Fungal infections such as mucosal/skin infections, though non-lethal, can reduce the quality of life for >1 billion affected people [2]. As opportunistic fungi, Candida species are the prevalent causes of invasive (e.g. candidaemia) and non-invasive (e.g. vulvovaginal candidiasis) fungal diseases, with an estimated ~700,000 invasive candidiasis cases occurring annually [2].

Early diagnosis and, consequently, prompt treatment of invasive Candida infections is crucial to prevent mortality [3]. Five Candida species, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei, are responsible for 92% of cases of candidaemia globally [4]. However, the gamut of clinically relevant Candida species is expanding [5] and, notably, less common species (i.e. Candida guilliermondii complex) [6], rare species (e.g. Candida inconspicua [Torulopsis inconspicua], Candida pararugosa and Pichia norvegensis [Candida norvegensis]) [7], or emerging species (i.e. Candida auris) [8] may exhibit high antifungal resistance levels, thereby compromising infection outcomes. Furthermore, 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 laboratory 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 Laboratories (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 addition to the species claimed by the CHROM medium’s manufacturer as presumptively identifiable (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. norvegensis (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 (~106 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 isolate, respectively, in Axygen® 96-Deep Well polypropylene plates to reach a 1-ml final volume for a concentration of ~500 CFU per well. To obtain mono-species (n = 18) or dual-species (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 controls 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 important 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 species 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 uncommon 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 percentages 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 (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).

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.

As for all the 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 (24/48/72 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 uncommon 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 readings of BCG, CHROM and SDA plates revealed statistically significant differences between the readers only for CHROM and SDA plates (S2 Table).

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-species cultures	Culture media plates read at the indicated times		N. of overall detections	N. of detections found to be in agreement and scored as			Kappa coefficient (95% CI)	Level of inter-reader agreement
				Correct	Over	Under
All species (n = 18)	BCG	24 h	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 h	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 h	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 h	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 h	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 h	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 h	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 h	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 h	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

The BCG medium is similar to other selective and differential media for the primary isolation 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 Candida 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, 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.

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).

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 presumptive identification of five Candida species (C. albicans, C. dubliniensis, C. krusei, C. tropicalis 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 difficult. 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 uncommon 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 readers (Table 1).

Among mixed candidaemia episodes, C. albicans plus C. glabrata is usually the most frequent combination [5], but other combinations of Candida species may be of great importance. We noticed that 106 (69.3%), 60 (39.2%) and 78 (51.0%) of 153 dual-species cultures at 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 interestingly, six cultures (C. albicans/C. parapsilosis, C. incospicua/C. pelliculosa, C. incospicua/C. sorbosa, 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 detection in candidaemia cases may have clinical repercussions, especially because of different antifungal 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 morphotypes, 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 samples, is crucial in order to exploit the established, powerful MALDI-TOF MS capability of identifying 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.

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)

Click here for additional data file.

Rates of incorrect (over/under) detections by three readers for the overall BCG, CHROM or SDA cultures of Candida species.

Bold indicates statistically significant differences in the comparisons between reader #1 and reader #2, reader #2 and reader #3, or reader #1 and reader #3.

(DOC)

Click here for additional data file.

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)

Click here for additional data file.

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 (Sabouraud dextrose agar) media.

(JPG)

Click here for additional data file.

Rates of correct, over or under detections by three readers for the uncommon Candida species grown as mono- (n = 13) or dual-species (n = 78) on the BCG (Candida bromcresol green), CHROM (chromogenic medium, i.e. Brilliance Candida agar) and SDA (Sabouraud dextrose agar) media.

(JPG)

Click here for additional data file.

14 Jan 2020

PONE-D-19-32923

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

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Reviewer #1: In this work the Authors compared the performance of 3 different mycological media, namely BCG, CHROM and the non chromogenic SDA, for the detection of different Candida species cultures, both in mono-species and dual-species culture plates. Plates were read at 24, 48 and 72 hrs from 3 indipendent readers, and results were interpreted as correct detection, over detection, or under detection. Overall, BCG medium showed the best performance with higher rates of correct detection and lower rates of under detection for all Candida species analyzed, but also by splitting Candida spp in common and uncommon species, although with differences in the statistical significance. The Authors conclude BCG medium may represent an accurate tool for the correct identification to the species level and for the laboratory diagnosis of candidemia.

The matter of the manuscript is of interest in clinical diagnostics and provides valuable indications for the microbiology laboratory in the accurate diagnosis of Candida infections.

Some considerations:

- From what stated in the text, it would be interesting to know the opinion for the right time and temperature of incubation of plates for clinical specimens for the isolation of Candida spp, also as indicated by the literature.

- It is not clear if the term “over detection” means culture with more colony morphotypes, but not necessarily different species, or more Candida species than those expected. Please, clarify it.

- It would be interesting to know the true clinical impact of mixed Candida infections, specifically in candidemia (page 8, lines 170-174, the mixed cultures reported seems to be very uncommon, right?)?

- page 5, line 84-88. Please, it is necessary to better specify the final volume (the final concentration) reached in each well. Could the Authors provide some references to support their choice for the methodologies?

- Page 8, lines 161-165. I would add this part also in the Results section along with the reference of Fig 4, with a brief description of the characteristics of the Candida media used.

- The resolution of Figures 1, 2, 3, needs to be much improved, they are blurred. Moreover, it is also difficult to notice what the Authors state for the Figure 4 with regard to the differences in the colony morphologies.

- There is a variability between reader 1 and the others respect to the over detection at 24, 48 and 72 hrs mainly for all Candida species and the uncommon Candida species, as indicated in the Figures 1 to 3 and in Table 1 (moderate or fair levels of agreement).What is the reason of it? Does this could affect the results?

Reviewer #2: The ms by De Angelis et al is a comparison study among different commercially available media (three) in order to understand if these media can be used for detecting different Candida spp from primary cultures.

The AA used 18 strains belonging to different species of Candida, all isolates from BSI. A C.auris strain was also included. Following a CK scheme, cultures were variably combined to obtain mono, dual and triple combination of strains. All these unique combinations were plated on the three media and results were reported in 1 table and many figures.

The ms is clearly written and results obtained , even with limitations described by the AA (reading plates remain very subjective, and the different morphotype of Candida) are in favor of the use of BCG.

In my opinion, two minor points need to be addressed:

1) the measure of the level of inter reader agreement is somewhat vague. Any comment from the AA? Is it possible to measure this agreement in term of errors?

2) Number of figures can be reduced, summarizing the most significative results without losing the meaning of the message (the original figures can be added at the supplemental material section)

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Reviewer #1: Yes: Giovanni Gherardi

Reviewer #2: No

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27 Jan 2020

PONE-D-19-32923

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer: We ensured that our manuscript was in line with the PLOS ONE's style requirements. See the revised manuscript throughout.

2. For reproducibility purposes, please provide the ID # for all clinical isolates used in this analysis.

Answer: We provided the ID # for all the clinical isolates used in our analysis. See the text and the footnote of S1 Fig.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

'This study was supported by Università Cattolica del Sacro Cuore (Fondi Ateneo, linea D1-2018).'

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Answer: We apologize for the incongruence of our statements. You will find the updated statements in the Funding Section.

Reviewer #1: In this work, the Authors compared the performance of 3 different mycological media, namely BCG, CHROM and the non-chromogenic SDA, for the detection of different Candida species cultures, both in mono-species and dual-species culture plates. Plates were read at 24, 48 and 72 hrs from 3 independent readers, and results were interpreted as correct detection, over detection, or under detection. Overall, BCG medium showed the best performance with higher rates of correct detection and lower rates of under detection for all Candida species analyzed, but also by splitting Candida spp. in common and uncommon species, although with differences in the statistical significance. The Authors conclude BCG medium may represent an accurate tool for the correct identification to the species level and for the laboratory diagnosis of candidemia.

The matter of the manuscript is of interest in clinical diagnostics and provides valuable indications for the microbiology laboratory in the accurate diagnosis of Candida infections.

Some considerations:

- From what stated in the text, it would be interesting to know the opinion for the right time and temperature of incubation of plates for clinical specimens for the isolation of Candida spp, also as indicated by the literature.

Answer: We provided details about the optimal time and temperature of incubation of plates for clinical specimens that are required for the isolation of Candida species. See lines 100–102 of the revised manuscript.

- It is not clear if the term “over detection” means culture with more colony morphotypes, but not necessarily different species, or more Candida species than those expected. Please, clarify it.

Answer: We clarified this issue adding a sentence as appropriate. See lines 111–113 of the revised manuscript.

- It would be interesting to know the true clinical impact of mixed Candida infections, specifically in candidemia (page 8, lines 170-174, the mixed cultures reported seems to be very uncommon, right?).

Answer: We added a comment about this interesting aspect raised by the reviewer. See lines 199–201 of the revised manuscript.

- page 5, line 84-88. Please, it is necessary to better specify the final volume (the final concentration) reached in each well. Could the Authors provide some references to support their choice for the methodologies?

Answer: We specified the final volume reached in each well as well as provided a reference to support our methodological choice. See lines 88–92 of the revised manuscript.

- Page 8, lines 161-165. I would add this part also in the Results section along with the reference of Fig 4, with a brief description of the characteristics of the Candida media used.

Answer: We added the part indicated by the reviewer in the Results section along with the reference of Fig 2 (formerly Fig 4) and a brief description of the media’s characteristics. See lines 155–164 of the revised manuscript.

- The resolution of Figures 1, 2, 3 needs to be much improved, they are blurred. Moreover, it is also difficult to notice what the Authors state for the Figure 4 with regard to the differences in the colony morphologies.

Answer: We improved all the Figures, namely Fig 1, S1 Fig (formerly Fig 2) and S2 Fig (formerly Fig 3) for quality. In particular, we redone the image of Fig 2 (formerly Fig 4) to make more evident the differences in the colony morphologies of the isolates photographed.

- There is a variability between reader 1 and the others respect to the over detection at 24, 48 and 72 hrs mainly for all Candida species and the uncommon Candida species, as indicated in the Figures 1 to 3 and in Table 1 (moderate or fair levels of agreement).What is the reason of it? Does this could affect the results?

Answer: We added a comment about this interesting aspect raised by the reviewer. See lines 185–189 of the revised manuscript.

Reviewer #2: The ms by De Angelis et al is a comparison study among different commercially available media (three) in order to understand if these media can be used for detecting different Candida spp from primary cultures.

The AA used 18 strains belonging to different species of Candida, all isolates from BSI. A C. auris strain was also included. Following a CK scheme, cultures were variably combined to obtain mono, dual and triple combination of strains. All these unique combinations were plated on the three media and results were reported in 1 table and many figures.

The ms is clearly written and results obtained, even with limitations described by the AA (reading plates remain very subjective, and the different morphotype of Candida) are in favor of the use of BCG.

In my opinion, two minor points need to be addressed:

1) The measure of the level of inter reader agreement is somewhat vague. Any comment from the AA?

Is it possible to measure this agreement in term of errors?

Answer: We agree with the reviewer on the vagueness of measuring the level of inter-reader agreement. Unfortunately, we were unable to measure the agreement in terms of errors. However, we added data about the rates of incorrect (over/under) detections of the three readers in order to underline the statistically significant differences between the readers with respect to the incorrect reading results. See lines 151–154 and a new Table in the Supporting information (S2 Table) of the revised manuscript.

2) Number of figures can be reduced, summarizing the most significative results without losing the meaning of the message (the original figures can be added at the supplemental material section).

Answer: As suggested, we move Fig 2 (now S1 Fig) and Fig 3 (now S2 Fig) to Supporting information.

Submitted filename: PONE-D-19-32923 Response to Reviewers.doc

Click here for additional data file.

5 Mar 2020

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

PONE-D-19-32923R1

Dear Dr. Sanguinetti,

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: All comments have been adequately addressed. The revision version of the manuscript is much improved. This is an interesting manuscript and the entire manuscript is clear, concise and well written, the results obtained and discussion are strongly supported by the experimental data, with the overall high quality and appropriateness of the testing performed.

Reviewer #2: The AA addressed all my comments and in my opinion the manuscript is now acceptable for publication.

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Reviewer #1: Yes: Giovanni Gherardi

Reviewer #2: No

9 Mar 2020

PONE-D-19-32923R1

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

Dear Dr. Sanguinetti:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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