Comparison of four commercially available chromogenic media to identify Candida albicans and other medically relevant Candida species

The number of invasive Candida infections has significantly increased in recent decades. For the successful treatment of fungal infections, rapid identification at the species level, particularly in polyfungal infections, is a key factor.


| INTRODUC TI ON
The number of invasive infections due to Candida spp. has increased in recent years, including an increase in the percentage of infections caused by non-C albicans Candida species (NCAC). [1][2][3] The shift of species from mainly C albicans towards NCAC in invasive Candida infections may be due to the increased application of accurate diagnostic methods and/or an increased in the use of azole therapy. 4 Thus, the overall Candida species shift from antifungal susceptible C albicans to potentially more resistant NCAC isolates, makes the rapid identification of Candida isolates at the species level essential. 4 Routine diagnostic methods for Candida identification are commonly culture-based, in which micro-and macromorphology are analysed.
Identification through physical characteristics is frequently completed with an array of biochemical tests as part of standard procedures for identifying yeast. 5 For proper detection of yeast in polymicrobial samples, selective media are crucial, for example Sabouraud dextrose agar (SDA) or malt extract agar. Nevertheless, neither the detection of polyfungal infections, nor the differentiation of species is possible by the sole use of these conventional media. The first report of chromogenic agar for identification and differentiation of Candida species was in 1994. 6 The commercially available chromogenic media for Candida identification are hexosaminidase-based, enabling the identification of C albicans.
However, further discrimination between NCAC species differs in the various media by the use of numerous diverse substrates or by detection of either hexosaminidase activity or alkaline phosphatase activity.
In this study, four different commercially available chromogenic media were compared for their performance in C albicans identification in clinical specimens, including primary sterile materials and respiratory samples. Additionally, performance of the media for NCAC identification was also evaluated.

| Strains and specimens
In the first part of the study, a total of 13 control strains were used for evaluation of growth, colour and texture on chromogenic media:

| Data analysis
Statistical analysis was performed to determine sensitivity and specificity for the identification of C albicans, for the prospective part of the study. Due to three independent investigators and the resulting variations in reading, results were depicted as the mode value.
Samples were assigned a true positive result only after macroscopic identification was confirmed by MALDI-TOF MS. A false positive result was assigned to cultures incorrectly identified macroscopically, due to a deviation in colony colour from that described by the manufacturer, when compared to MALDI-TOF MS. A false negative reading was assigned if no growth on the medium could be detected but culturing on alternate media was successful. True negative status was assigned in the absence of growth on chromogenic media and on blood agar or SDA.

| Ethics
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. All clinical samples were used after performing a conventional microbiological analysis. The study did not include patients` details and did not result in additional constraints for the patients. All procedures and methods were carried out in accordance with approved guidelines.

| RE SULTS
MALDI-TOF MS analysis results were compared with culture-based identification of Candida spp., using the four included chromogenic media. The modal observation of the obtained results from three independent investigators was further used in this study.
In the first instance, the four tested chromogenic media were examined based on their capacity to induce growth. All reference strains grew on each of the tested chromogenic medium with visible colonies after 24 hours. The media differ in their complexity.  (Table S1) Species-specific sensitivity and specificity were calculated for C albicans (Table 4). Sensitivity varied between 32% (BCA) and 69% (CS4 and CCA) after 24 hours and 68% (BBL) and 82% (BCA) after 48 hours, while specificity ranged between 62% (BBL) and 81% (CCA) after 24 hours and 82% (BBL) and 85% (CS4) after 48 hours. Best specificity (85% and 83%) and sensitivity (81% and 82%) was exhibited by CS4 and BCA after 48 hours. Because of the low number of identified NCAC, no sensitivity and specificity were defined for NCAC identification. to identify Candida species and to detect polyfungal infections. 6,[11][12][13][14][15][16][17][18][19][20][21][22] To our knowledge, this is the first study evaluating and comparing the performance of the currently commercially available CS4, CCA, BBL and BCA for accurate and rapid identification and differentiation of Candida species from clinical specimens.

| D ISCUSS I ON
Identification of C albicans was reliable using each of the included media as previously reported. 12

TA B L E 3 Results of identification of
Candida on four chromogenic media after 24 and 48 h incubation of 181 respiratory materials (100%) when using BBL to identify C albicans, 11 this study detected C albicans with only 68% sensitivity and 82% specificity using BBL.
Reasons for the disparity in performance of BBL in the aforementioned studies may be due to variations in the types of samples tested. 12 Baixench et al compared BCA and CHROMagar Candida and determined a sensitivity of 96.6% and a specificity of 100% for identifying C albicans after 48 hours. 19 In contrast, after 24 hours incubation, only 19 of 89 colonies showed a characteristic colour, 19 which was also observed in the current study. Here, CS4 and BCA showed the most robust positive results and the highest sensitivity for C albicans identification, whereas the highest specificity was obtained using CS4, CCA and BCA, when identifying colonies macroscopically after 48 hours of incubation.
We were able to identify C krusei on all included agar media. This was only possible when morphological characteristics of the colonies were analysed in concert with pigmentation assessment. Similar observations have been reported in other studies. [11][12][13][14]17,18 We could observe that, on BBL agar, non-krusei Candida species are almost undiscernible from C krusei after the first 24 hours. Each of the false positive C krusei isolates was identified as C glabrata. Additionally, nearly all of these C glabrata isolates have been shown to originate from polymicrobial samples also containing C albicans. Only one misidentified C krusei has been confirmed to be a pure culture of C glabrata. However, those false positive results did not occur when identification was performed after 48 hours of incubation.
A relatively low identification rate was reached for C tropicalis on the chromogenic media with the exception of BBL, which enabled the true positive identification of 83% of C tropicalis. On BBL, C tropicalis develops a unique and easy-to identify colour. This has similarly been observed for BCA with a 75% identification rate. Vecchione et al were able to detect C tropicalis properly after 24 hours on CS4, BBL and BCA, but this study only included yeast isolates from a strain collection. 14 Another study group reported 95% sensitivity and 79% specificity when 21 C tropicalis isolates were investigated on CCA. 15