Characterization of Two Chromogenic Media of Candida ID and for Preliminary Identification of Yeasts Chien-Fang Peng,, Kun-Mu Lee and Shuan-Hui Lee Laboratory of Microbiology and Clinical Microbiology, Faculty of Biomedical Laboratory Science, College of Health Sciences; Department of Clinical Microbiology, Kaoshuing Medical University Hospital, Kaohsiung Medical University, No. 00, Shih-Chuan st Road, Kaohsiung 807, Taiwan Chromogenic media are frequently used in direct and rapid identification of yeasts because different organisms could produce unique colors and specific colony morphology on these media. A total of 73 clinical isolates and 8 reference strains of yeasts were used to evaluate the performance of two chromogenic media, Candida ID and, on the identification of various yeasts. The fertility of both media was 00%, but selectivity was 86.5% and 96.%, respectively. Sensitivities and specificities of the 5 most clinically important Candida species on Candida ID were as follows: 00% and 95.3% for Candida albicans, 00% and 93.8% for C. glabrata, 90.0% and 8.7% for C. parapsilosis, 95.% and 79.6% for C. tropicalis, and 97.% and 84.5% for C. intermedia. For, the figures were 00% and 94.6% for C. albicans, 90.% and 95.4% for C. glabrata, 90% and 8.3% for C. parapsilosis, 00% and 78.8% for C. tropicalis, and 00% and 83.7% for C. intermedia. In conclusion, Candida ID appears to have a better sensitivity than in the detection and identification of yeasts. Key words: Candida ID,, Rapid identification Introduction Although the frequency of isolation of non-albicans Candida (NAC) species is increasing gradually, Candida albicans is the most common cause of candidiasis [,]. Therefore, rapid identification of C. albicans and other pathogenic yeasts in clinical microbiology laboratories is becoming essential. Several brands of chromogenic media have been shown to have effects of direct and rapid yeast identification [3,4]. These media contain chromogenic substrates that react with enzymes secreted by the microorganisms. These enzymes are species-specific, allowing organisms to be identified to the species level by their color development and colonial features. Routine use of chromogenic media carries the potential efficiency in the clinical microbiology laboratory. Candida ID (biomérieux, Marcy ľetoile, France) [5] is a new version of chromogenic medium derived from a serial evolution of the Albicans ID medium [6], Albicans ID medium [7], and Candida ID medium (biomérieux, Marcy ľetoile, France) [8,9]. It allows direct inoculation from the clinical specimens, the isolation of yeasts and molds, rapid identification of C. albicans by producing metallic blue colonies, and differentiation between C. tropicalis, C. lusitaniae and C. kefyr after 4 h incubation at 35. (CHROMagar, Paris, France) [0] is another chromogenic medium which, after 48 h incubation at 35 C, is useful for the identification of C. albicans by producing light to medium green colonies, of C. tropicalis by producing steel blue colonies accompanied by purple pigment diffusion into surrounding agar, and of C. krusei by producing large, fuzzy, and rose-colored colonies with white edges. The aim of this study was to compare the performance and colony forming speeds of Candida ID and for the presumptive identification of C. albicans and other NAC species. Received: January 3, 007 Revised: April 5, 007 Accepted: August 7, 007 Address for correspondence: Laboratory of Microbiology and Clinical Microbiology, Faculty of Biomedical Laboratory Science, College of Health Sciences, Kaohsiung Medical University, No. 00, Shih-Chuan st Road, Kaohsiung 807, Taiwan, ROC Tel: +886-7-30 ext. 35 Fax:+886-7-33449 E-mail: chfape@ kmu.edu.tw J Biomed Lab Sci 007 Vol 9 No 63
Yeasts identification of by Candida ID and Materials and Methods Organisms for Fertility Testing A total of 8 isolates, including 73 clinical isolates and 8 reference strains, were studied (Table ). The 73 yeast isolates were identified by ATB ID3C. All isolates tested were subcultured twice on Brain Heart Infusion Agar (BBL, Becton Dickinson Microbiology Systems, Becton Dickinson and Company, Sparks, Md. USA) at 35 for 4 h. One colony was then transferred onto Candida ID and, respectively, and incubated again at 35. Results of the cultures were recorded at 6-8 h, 4 h, and 48 h, respectively, after incubation. In addition, C. albicans ATCC 03 and C. tropicalis ATCC 9968 were used as quality control strains. Typical appearances of the two strains were blue and pink colonies on Candida ID and green and metallic blue colonies on, respectively. Organisms for Selectivity Testing To examine the selectivity of the two media, a total of 5 identified bacterial isolates, including Escherichia coli, Enterococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Proteus-Morganella-Providencia group, and Klebsiella- Enterobacter-Serratia group, were used. They were isolated from various clinical specimens, including sputum, throat swab, and blood. One colony of each isolate was Table. Colony appearance of Candida species and some yeasts grown on Candida ID and CHROMagar Candida after 48 h incubation at 35 Yeast C. albicans C. albicans ATCC 4053* No. of Colony appearances, no. observed strain tested Candida ID 3 Blue 33 Dark green Bright yellow green C. catenulate ATCC 88* Light violet Light purple or lilac C. colliculosa White pink C. dubliniensis 3 Dark blue 3 Dark green 3 C. famata 4 pink 4 Dark green C. glabrata 4 White 4 pink Lightly purple C. guilliermondii ATCC 9058* pink-white Light purple or lilac C. intermedia 36 pink 35 36 Bright yellow green C. krusei White Rose-colored C. lambica ATCC 4750* White Light purple or lilac C. lipolytica ATCC 030* White Light purple or lilac C. lusitaniae 3 pink 3 Olive yellow 3 C. parapsilosis 0 White Light purple or lilac C. sake 5 Deep blue Light purple C. tropicalis C. tropicalis ATCC 4563* 0 Light-pale pink Bright blue with purple 9 3 0 pink Lightly-bright purple Dark green 06 7 37 4 4 5 3 C. zeylanoides ATCC 735* White Lightly-bright purple Cryptococcus humicolus C. humicolus ATCC 4438* 5 Light purple Most dark blue Light purple 4 Dull red purple pink Trichosporon asahi Light greenish blue, fuzzy Light blue *:reference strains 3 64 J Biomed Lab Sci 007 Vol 9 No
picked from blood agar into a sterile plastic tube ( 75 mm) containing ml of normal saline and mixed thoroughly. A loopful of bacterial suspension was then transferred to each of the two tested media and incubated at 35. The plates were examined after 4 h incubation. Comparative Analysis Colony appearances between Candida ID and CHRO- Magar Candida were compared in terms of fertility, selectivity, sensitivity, and specificity. Fertility was defined as the ratio of growing isolates among total isolates in a specific group of bacterial species. Selectivity for organisms that should not grow was calculated as the number of isolates without growth divided by the total number of isolates studied. Sensitivity and specificity were calculated as below. Sensitivity = (true positive) X 00 / (true positive + false negative) Specificity = (true negative) X 00 / (true negative + false positive) Results Comparison of Fertility and Selectivity All of the 8 yeast isolates tested were grown on the two media. The fertility of Candida ID (00%) was identical to that of (00%) for Candida species and some yeasts observed. As to the selectivity, 7 bacterial isolates (3 isolates of Klebsiella pneumoniae, isolates of S. aureus, and isolates of S. epidermidis) were grown on Candida ID, but only isolates of K. pneumoniae were grown on CHROMagar Candida. Therefore, the selectivity of (50 of 5, 96.%) is superior to that of Candida ID (45 of 5, 86.5%). Comparison of Colony Color, Sensitivity, and Specificity All 33 C. albicans isolates were blue on Candida ID. Although 7 isolates of NAC developed blue color on Candida ID, the degree of pigmentation was different in 3 isolates of C. dubliniensis (dark blue), 3 isolates of C. sake (deep blue) and isolate of C. humicolus (most dark blue) (Table ). Candida ID exhibited 00% sensitivity and 95.3% specificity in C. albicans identification. Of the 33 isolates of C. albicans on, 06 were dark green and 7 were bright yellow green; both colors were acceptable as presumptive identification of C. albicans. Eight isolates of Candida species, including C. dubliniensis (3 isolates), C. sake (3 isolates) and C. famata ( isolates), also showed a green color on. The sensitivity and specificity for in C. albicans identification were 00% and 94.6%, respectively (Table ). Colony color, specificity and sensitivity of five species of Candida on Candida ID and CHROMagar Candida are listed in Table 3. With the exception of one isolate showing a purple color, the other 0 C. tropicalis were pale pink on Candida ID. However, there were also 53 non-tropicalis Candida isolates grew as pale Table. Comparison of 33 C. albicans isolates grown on Candida ID and after 4 h incubation Features Candida ID Predictive Candida species Predictive Candida species color (No. observed) color (No. observed) True positive Blue C. albicans (33) BG C. albicans (34) False negative non-blue C. albicans (0) BG C. albicans (0) True negative non-blue non-c. albicans (4) BG non-c. albicans (40) False positive Blue non-c. albicans (7): BG non-c. albicans (8): C. dubliniensis (3) C. dubliniensis (3) C. sake (3) C. sake (3) C. humicolus () C. famata () Sensitivity 00 00 Specificity 95.3 94.6 BG: bright green or fresh bamboo color (including dark green and bright yellow green) Sensitivity = (true positive) X 00 / (true positive + false negative) Specificity = (true negative) X 00 / (true negative + false positive) J Biomed Lab Sci 007 Vol 9 No 65
Yeasts identification of by Candida ID and pink colonies. The sensitivity and specificity for Candida ID in C. tropicalis identification were 95.% and 79.6% respectively. A total of isolates of C. tropicalis had a dull greenish blue on, but another 55 isolates of non-tropicalis Candida grew blue, too. Thus the sensitivity and specificity for in C. tropicalis identification were 00% and 78.8%, respectively. A total of 4 isolates of C. glabrata were white on Candida ID (sensitivity 00%), whereas 5 isolates of non-glabrata Candida formed white colonies (specificity 93.8%). On, 37 of the 4 C. glabrata isolates were pale pink, and 4 were lightly purple (sensitivity 90.%); however, isolates of non-glabrata Candida also grew as pale pink colonies (specificity 95.4%). A total of 9 isolates of C. parapsilosis grew as white colonies on Candida ID (sensitivity 90.0%), but 47 isolates of non-parapsilosis Candida also had the same white color (specificity 8.7%) (Table 3). On, except that one isolate had a dull greenish blue color, 9 other isolates of C. parapsilosis were purple, including pale pink and lightly purple (sensitivity 90%); however, 48 isolates of nonparapsilosis Candida were of the same color (specificity 8.3%). With the exception of one isolate developed a bright yellow green color, the other 35 C. intermedia isolates had a pink color (sensitivity 97.%) on Candida ID, but 38 non-intermedia Candida isolates also had the same color (specificity 84.5%). On CHROMagar Candida, all the 36 C. intermedia isolates had a dull greenish blue color (sensitivity 00%), but 40 nonintermedia Candida isolates were also of the same color (specificity 83.7%). Comparison of Colony Sizes After 6-8 h incubation at 35, differentiation of the colony appearance on either Candida ID or CHROMagar Candida were still not easy due to small colony sizes. All 8 yeast isolates grew well after 48 h of incubation on both media. In comparison, the average sizes of the strains tested on Candida ID (3.0±0.87 mm) were bigger than those on (.94±0.55 mm). Colony sizes of the five most common Candida species tested on Candida ID and are summarized in Table 4. The average sizes of colonies of C. albicans grown on both media were nearly identical. However, the average sizes of C. glabrata on were larger than those on Candida ID. Discussion The occurrence rate of C. albicans in human candidiasis is high. In this study, Candida ID and CHROMagar Table 3. Colony color, specificity and sensitivity of five species of Candida grow on Candida ID and Candida species (No. tested) Color expected Candida ID Sensitivity Specificity C. albicans (33) Blue 00.0 95.3 Color expected Bright green Sensitivity Specificity 00.0 94.6 C. glabrata (4) White 00.0 93.8 C. parapsilosis (0) White 90.0 8.7 pink pink or lightly purple 90. 95.4 90.0 8.3 C. tropicalis () pink 95. 79.6 Dull greenish blue 00.0 78.8 C. intermedia (36) pink 97. 84.5 Dull greenish blue 00.0 83.7 66 J Biomed Lab Sci 007 Vol 9 No
Table 4. Comparison of the average sizes of colony for five commonly isolated species of Candida on Candida ID and after 48 h of incubation Candida species (No. tested) Candida ID Size of colonies (mm ± SD) C. albicans (33).80 ± 0.35.7 ± 0.39 C. glabrata (4).67 ± 0.36 3.38 ± 0.55 C. parapsilosis (0) 3.0 ± 0.54.60 ± 0.30 C. tropicalis () 4.04 ± 0.66 3.3 ± 0.3 C. intermedia (36) 4.6 ± 0.5 3.9 ± 0.39 Candida both demonstrated a good sensitivity and specificity for the identification of C. albicans. Some isolates of C. dubliniensis, C. sake and C. humicolus developed similar blue color on Candida ID. To differentiate these isolates, we used C. albicans ATCC 03 with the typical blue color for comparison. Different colony pigmentation from bright, dark, deep to most dark blue color were found in the four Candida species. On CHROMagar Candida, some isolates of C. dubliniensis, C. sake and C. famata also produced similar green color to those shown by C. albicans. These controversial results are in consistent with previous reports by other investigators [5,6, -4]. Among the 8 species of Candida and other yeasts investigated, 7 species of Candida isolates (C. colliculosa, C. glabrata, C. krusei, C. lambica, C. lipolytica, C. parapsilosis, and C. zeylanoides) yielded white-colored colonies, and 5 species of Candida isolates showed a pale pink color (C. famata, C. guilliermondii, C. intermedia, C. lusitaniae, and C. tropicalis) on Candida ID. Blue, white, and pale pink colors were the three predominant colors produced by Candida species on Candida ID. These results provided a useful presumptive method for identification of Candida species. Regarding, 6 species of Candida isolates (C. famata, C. intermedia, C. parapsilosis, C. sake, C. tropicalis, and C. humicolus) formed a dull greenish blue color. Four other species of Candida isolates (C. catenulata, C. guilliermondii, C. lambica, and C. lipolytica) were of a light purple or lilac color, and another 4 species of Candida and other yeast isolates (C. colliculosa, C. glabrata, C. parapsilosis, and C. humicolus) were pale pink. Colonies grown on appeared to demonstrate more color changes, which may facilitate the isolation and identification of the five most common species of Candida isolates. However, its sensitivity and specificity were lower than that demonstrated by Candida ID. According to our serial observations, colony morphology and color appearance were poor within 6-8 h of incubation on both Candida ID and CHROMagar Candida. Therefore, routine application of these media in clinical microbiology laboratories should allow for a sufficient incubation time of at least 4 h. Colony sizes on Candida ID were larger and thus easier to be observed than those on at 4 h incubation. Therefore, identification of Candida species with colony appearance on Candida ID can be accomplished more quickly. Identification of Candida isolates to the species level is now required for the appropriate selection of antifungal agents. Early reporting to physicians can be beneficial for a patient s successful therapy. In this regard, Candida ID appears to show a significant increase in sensitivity for identification of C. albicans by differentiating the organism from other Candida species in mixed cultures. In a mycology laboratory, the role of Candida ID and can be used as either selective isolation media for detection of yeasts from clinical specimens or differential media for direct identification of clinical isolates of Candida species in less than 48 h. With the typical colors shown on the two media, they may well replace Sabouraud dextrose agar and conventional biochemical tests, including assimilation tests, germ tube tests, and fermentation tests, used for direct identification of C. albicans. Therefore, clinical microbiologists will be able to save time and cost for the diagnosis of mycosis from clinical specimens usually encountered in general practices and hospitals through the use of either chromogenic medium. However, discrimination of white colonies of other Candida spp. on Candida ID agar required other complementary tests for further identification. Also, Candida species producing pale pink colonies detected on Candida ID agar necessitate additional supplemental testings for further differentiation in the clinical laboratory. In con- J Biomed Lab Sci 007 Vol 9 No 67
Yeasts identification of by Candida ID and clusion, these chromogenic media allow the direct identification of Candida species with a relative reading comfort. Acknowledgements This work was supported in part by a grant from biomérieux, Marcy ľetoile, France. References. Abi-Said D, Anaissie E, Uzun O, et al: The epidemiology of hematogenous candidiasis caused by different Candida species. Clin Infect Dis 997; 4: -8.. Rangel-Frausto MS, Wiblin T, Blumberg HM, et al: Variations in rates of bloodstream infections due to Candida species in seven surgical intensive case units and six neonatal intensive care units. Clin Infect Dis 999; 9: 53-8. 3. Ainscough S and Kibbler CC: An evaluation of the costeffectiveness of using CHROMagar for yeast identification in a routine microbiology laboratory. J Med Microbiol 998; 47: 63-8. 4. Koehler AP, Chu K, Houang ETS, et al: Simple, reliable, and cost-effective yeast identification scheme for the clinical laboratory. J Clin Microbiol 999; 37: 4-6. 5. Roche JM, Mula V and Villeval F: Evaluation of Candida ID, a new chromogenic medium for yeasts. Poster No. P075, Glasgow, 003, 3 th ECCMID. 6. Contreras I, San-Millan R, Agustin-Barrasa A, et al: Utility of Albicans ID plate for rapid identification of Candida albicans in clinical samples. Rapid identification of Candida albicans. Mycopathologia 996; 36: 7-0. 7. Cardenes CD, Carrillo AJ, Arias A, et al: Comparison of Albicans ID agar plate with the germ tube for presumptive identification of Candida albicans. Diagn Microbiol Infect Dis 00; 4: 8-5. 8. Valerie LB, Meyer MH, Galoisy AC, et al: Prospective evaluation of the new chromogenic medium Candida ID, in comparison with Candiselect, for isolation of molds and isolation and presumptive identification of yeast species. J Clin Microbiol 00; 40: 508-0. 9. Willinger B, Hillowoth C, Selitsch B, et al: Performance of Candida ID, a new chromogenic medium for presumptive identification of Candida species, in comparison to. J Clin Microbiol 00; 39: 3793-5. 0. Odds FC and Bernaerts R:, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 994; 3: 93-9.. Freydiere AM, Buchaille L and Gille Y: Comparison of three commercial media for direct identification and discrimination of Candida species in clinical specimens. Eur J Clin Microbiol Infect Dis 997; 6: 464-7.. Fricker-Hidalgo H, Orenga S, Lebeau B, et al: Evaluation of Candida ID, a new chromogenic medium for fungal isolation and preliminary identification of some yeast species. J Clin Microbiol 00; 39: 647-9. 3. Hoppe JE and Frey P: Evaluation of six commercial tests and the germ-tube test for presumptive identification of Candida albicans. Eur J Clin Microbiol Infect Dis 999; 8: 88-9. 4. Hospenthal DR, Murray CK, Beckius ML, et al: Persistence of pigment production by yeast isolates grown on medium. J Clin Microbiol 00; 40: 4768-70. 68 J Biomed Lab Sci 007 Vol 9 No
評估兩種顯色培養基 Candida ID 及 對酵母菌初步鑑定的比較 彭健芳, 李昆畝 李宣慧 高雄醫學大學健康科學院生物醫學檢驗學系高雄醫學大學附設中和紀念醫院檢驗部微生物檢驗室 顯色培養基因為能依菌種不同而形成特殊顏色及特有菌落型態, 常應用於直接且快速的酵母菌鑑定 我們共收集了 73 株臨床分離的酵母菌株及 8 株標準酵母菌株, 以比較 Candida ID 與 這兩種顯色培養基對於各種不同酵母菌鑑定的效果 結果顯示, 兩種培養基均可培養出所有試驗菌株, 其菌種的選擇性則分別為 86.5% 及 96.% 依照臨床常見前五種的酵母菌菌株分析敏感性及專一性在 andida ID 的情形如下 :Candida albicans 00% 95.3%,C. glabrata 00% 93.8%,C. parapsilosis 90.0% 8.7%,C. tropicalis 95.% 79.6%,C. intermedia 97.% 84.5% 而 的情形則如下 :C. albicans 00% 94.6%,C. glabrata 90.% 95.4%,C. parapsilosis 90% 8.3%,C. tropicalis 00% 78.8%,C. intermedia 00% 83.7% 從以上結果得知, 在酵母菌的偵測及鑑定方面,Candida ID 似乎比 有較高的敏感性 關鍵詞 :Candida ID 快速鑑定 收稿日期 :96 年 月 3 日修稿日期 :96 年 4 月 5 日接受日期 :96 年 8 月 7 日通訊作者 : 彭健芳高雄醫學大學健康科學院生物醫學檢驗學系 Tel: +886-7-30 ext. 35 Fax: +886-7-33449 E-mail: chfape@kmu.edu.tw J Biomed Lab Sci 007 Vol 9 No 69