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J Med Microbiol 54 (2005), 981-985; DOI: 10.1099/jmm.0.45942-0
© 2005 Society for General Microbiology
ISSN 0022-2615

Use of chromogenic medium in the isolation of yeasts from clinical specimens

C K Murray1, M L Beckius2{dagger}, J A Green2 {ddagger} and D R Hospenthal1

Department of Medicine, Infectious Disease Service1 and Department of Pathology and Area Laboratory Support2, Brooke Army Medical Center, Fort Sam Houston, TX, USA

Correspondence D. R. Hospenthal Duane.Hospenthal{at}amedd.army.mil

Received October 29, 2004
Accepted June 27, 2005

Over a 1 year period 3296 specimens submitted for fungal culture were plated onto routine mycological media (RM) and CHROMagar Candida (CaC) to evaluate the capability of CaC to improve on RM. With RM, cultures producing single yeast isolates were identified from 802 specimens. CaC produced similar results, with 76 % agreement. Of 761 specimens that yielded a single Candida species by RM, 615 (81 %) produced one or more yeast isolates using CaC. Of concern, 132 negative CaC cultures corresponded to specimens that yielded C. albicans alone on RM. When yeasts were recovered, CaC correctly identified 98 % of C. albicans, 93 % of Candida tropicalis, 96 % of Candida glabrata and 100 % of Candida krusei based on typical colours. CaC did potentially improve on RM by detecting yeasts in 91 specimens that yielded none by routine methods. CaC was noted to recover more yeast isolates than RM when mixed cultures were detected. Overall, the role of CaC in improving RM appears limited.

{dagger}Present address: Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, TX, USA.

{ddagger}Present address: Epidemiological Surveillance Laboratory, Brooks City Base, San Antonio, TX, USA.

Abbreviations: CaC, CHROMagar Candida; CSF, cerebrospinal fluid; RM, routine mycological media.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Yeasts are now the fourth most common organisms recovered from blood culture in hospitals (Edmond et al., 1999; Rangel-Frausto et al., 1999). In the past, Candida albicans was the aetiological agent in approximately 80 % of nosocomial yeast infections (Beck-Sague & Jarvis, 1993). More recently, C. albicans has accounted for less than 50 % of blood stream infections, with increases in the frequencies of Candida glabrata, Candida parapsilosis, Candida tropicalis and other non-albicans species (Rangel-Frausto et al., 1999). This transition has had a significant clinical impact due to the decreased susceptibility of several non-albicans candidal yeasts to antifungal agents, specifically that of C. glabrata and Candida krusei to fluconazole.

Changing candidal epidemiology and the availability of newer antifungal drugs with different antifungal spectrums means that physicians can no longer make therapeutic decisions based on the broad identification of fungi as either yeasts or moulds (Hospenthal et al., 2004); identification of candidal yeasts to the species level is now required. Identifying yeast and yeast-like organisms requires evaluation of microscopic morphology and biochemical studies. Some unusual yeasts may require unique morphological and biochemical studies for identification, occasionally requiring up to 21 days of incubation.

In order to facilitate rapid identification, alternative techniques to standard media such as chromogenic media have been developed. These special media yield microbial colonies with varying pigmentation secondary to chromogenic substrates that react with enzymes secreted by the micro-organisms. CHROMagar Candida (CaC) (www.chromagar.com) employs this methodology to allow the differentiation of several candidal yeasts by colour and morphology; it identifies C. albicans by growth as green colonies, C. tropicalis by growth as steel blue colonies and C. krusei as rough, matted, rose-coloured colonies. Other species such as C. glabrata and Candida dubliniensis may also be reliably identified (Hospenthal et al., 2002; Kirkpatrick et al., 1998; Odds & Bernaerts, 1994; Odds & Davidson, 2000; Pfaller et al., 1996). Another potential advantage of chromogenic media is the straightforward identification of mixed yeast infections, which can have a significant clinical bearing (Hospenthal et al., 2002; Lopez-Ribot et al., 1999; Redding et al., 1999; Willinger et al., 2001; Willinger & Manafi, 1999). Studies assessing the utility of chromogenic media have been performed, but a long-term, large-scale evaluation using one of these media routinely for all samples from a clinical mycology laboratory has not been previously undertaken. We studied the use of CaC in improving routine mycological media (RM) for all specimens referred to the clinical mycology laboratory.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Specimens.

We prospectively studied all clinical specimens and cultures referred to the clinical mycology laboratory (CML) at Brooke Army Medical Center (BAMC), Fort Sam Houston, Texas, between January 2002 and January 2003. BAMC is a tertiary-care medical facility that does not perform bone marrow or solid organ transplants. The CML assesses clinical samples that are direct referrals, referrals from the bacteria section of the clinical microbiology laboratory and outside referrals from smaller, regional hospitals. Referrals from the bacteria section include fungal colonies on solid media and liquid media from Gram-stain-positive blood culture bottles.

Cultures.

The media used routinely (RM) in the CML for primary isolation of fungi differed for typically sterile and non-sterile site specimens. Referred fungi growing on or in bacterial media were plated directly as sterile site specimens. Sterile site specimens were plated onto brain heart infusion medium with 10 % sheep blood, chloramphenicol and gentamicin (BHI) (Becton Dickinson) and potato flake agar (Becton Dickinson). Non-sterile site specimens were plated onto Mycosel agar (Sabouraud dextrose agar containing chloramphenicol and cycloheximide) (Becton Dickinson) and BHI.

Identification of yeast and yeast-like organisms was done by germ-tube testing, observation of morphology on corn meal agar (Becton Dickinson), Vitek Yeast Biochemical Cards (bioMérieux), and fermentation of glucose, sucrose and cellobiose. Further studies used to identify unusual yeast included API 20C (bioMérieux), growth at 37 °C and 42 °C, ascospore formation and other biochemical tests.

CaC (CHROMagar Microbiology) was purchased from the distributor (Hardy Diagnostics) and stored in an unlighted laboratory refrigerator. All plates were used within 30 days of arrival at BAMC.

Inoculation of CaC and RM was randomly performed as specimens were received in an attempt to avoid potential inoculum bias. Inoculated plates were incubated at 30 °C and read for up to 7 days. Plates were observed for fungal growth using morphology and colour to determine the presence of yeasts or moulds. As per the manufacturer, C. albicans, C. tropicalis and C. krusei were identified by the production of green, steel blue and rough, matted, rose-coloured colonies, respectively (www.chromagar.com). We have previously described the ability to identify C. glabrata by a dark-violet appearance (Hospenthal et al., 2002). CaC were read independently of standard media results. Fungi noted on CaC were not further identified, and results from these tests were not released to health care providers or used in medical decision making.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Specimens

From January 2002 to January 2003, 3554 clinical specimens were evaluated by the CML. Of these samples, 258 were duplicates or rejected for other reasons. Consequently 3296 specimens were plated onto media and had results available for analysis. Specimens from sterile sites included 359 cerebrospinal fluid, 137 blood and 18 catheter tip samples. Predominately non-sterile sites from which samples were submitted included respiratory (467), urine (204), nail (720), scalp/hair (221) and skin (319). Respiratory samples included specimens labelled sputum, bronchoalveolar lavage, tracheal aspirate, pleural fluid and lung. The remaining 851 samples consisted of specimens from other sites including the oral cavity, genital tract, soft tissues, bone and those listed as ‘unknow’ or ‘other'.

Culture results

RM identified a single yeast isolate from 802 (24 %) of the specimens and more than one yeast from 51 (2 %) of the specimens. No yeasts were recovered from 2443 (74 %) of the specimens. When compared only by the number of yeasts recovered on each plate (not matching colour or colony characteristics), CaC produced similar results from 2983 of the tested specimens (91 % agreement). Agreement was highest for specimens without growth on RM (96 %, 2352/2443), with lower agreement when RM recovered a single yeast (76 %, 606/802) or multiple yeasts (49 %, 25/51). CaC recovered yeasts from a total of 91 specimens that yielded no growth by RM, but failed to detect yeast growth from 164 of 853 (19 %) specimens that did produce yeast growth using RM. Of the mixed cultures, RM revealed more than one yeast isolate from 51 specimens, whereas CaC recovered more than one isolate from 83 specimens. In total, multiple isolates were recovered from RM, CaC or both for 104 patient specimens. Agreement between the methods was seen with 23 (22 %) of these, with RM recovering more isolates in 26 and CaC recovering more isolates in 55.

Of the 802 specimens that yielded single isolates of yeasts by RM, 761 recovered yeasts of the genus Candida (Table 1). Among these specimens, when yeasts were recovered on CaC, identification was possible with 98 % of C. albicans, 93 % of C. tropicalis, 96 % of C. glabrata and 100 % of C. krusei based on expected colour and morphology. Of the remaining five species not typically identifiable on CaC (Candida parapsilosis, Candida guilliermondii, Candida famata, Candida lusitaniae and Candida lipolytica), colony colours were noted, as described previously, in varying shades of white, pink and lavender. Unfortunately, 146 of the 761 single Candida isolates recovered by RM did not grow candidal yeasts at all on CaC, including 132 (23 %) of 564 C. albicans isolates.


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  		Table 1. Morphology and recovery of Candida by CHROMagar Candida compared with routine media Data are presented for all RM cultures that yielded only one Candida species.
 

The performance of CaC with sterile site specimens was generally better than that seen with non-sterile site specimens, especially dermatological samples (scalp/hair, skin and nail) (Table 2). Agreement was seen between CaC and RM results in 88 % of blood (121/137), 78 % of catheter tip (14/18) and 99 % of cerebrospinal fluid (CSF) (357/359) cultures. Discordance seen with sterile site specimens (blood, catheter tips ans CSF) was most commonly due to lack of any growth on CaC. For blood cultures, 13 samples were negative by CaC but positive for a Candida species by RM. One CaC culture recovered a green yeast colony that corresponded to a RM culture that was negative. The patient from whom this blood sample was obtained had no other positive fungal cultures during their hospitalization and thus the significance of this result is unknown. Two CaC blood specimens each grew two differing colony types that did not correlate with the matched single isolates recovered by RM. For one of these patients, a second blood sample and catheter tip removed the same day produced two different yeasts on both RM and CaC, supporting the CaC results. Discordance between catheter tip cultures resulted from the recovery of no yeasts by CaC from three specimens and the recovery of only one of two isolates from another sample. CSF specimens produced discordant yeast growth on CaC in two cases (out of 359 total submitted specimens), in each of which a review of the clinical data found that the most likely cause was culture or specimen contamination.


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  		Table 2. Performance of CHROMagar Candida by site of specimen collection
 


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Previous evaluation of chromogenic media in the recovery of fungi directly from clinical specimens has been limited by small sample sizes, inclusion of only selected clinical samples or lack of comparison to routine media. We prospectively evaluated the utility of chromogenic media to improve on media routinely used by our hospital mycology laboratory in all clinical specimens submitted during a 12 month period. We expected the most promising application of chromogenic media in this clinical setting to be the ability to identify mixed infections and to improve the detection of yeast. However, CaC did not clearly enhance traditional techniques of fungal identification.

Our study examined the culture results of 3296 clinical isolates, 853 of which produced yeast growth on RM. The largest study reported in the literature examined 6150 clinical specimens (Bouchara et al., 1996). Most of these specimens were non-sterile site surveillance samples and only 366 were also plated on Sabouraud medium for comparison. The three largest studies to compare CaC to a routine medium, Sabouraud medium either with or without chloramphenicol, examined 951–1784 specimens. These included 1784 vaginal swabs in the largest comparative study (Houang et al., 1997), 951 mostly genital and stool specimens in another (Baumgartner et al., 1996) and 1150 unspecified clinical samples in the third (Willinger & Manafi, 1999). Smaller comparative studies have also been reported (Odds & Bernaerts, 1994; Pfaller et al., 1996; Willinger et al., 2001; Ainscough & Kibbler, 1998; Bernal et al., 1996; Freydiere et al., 1997; Momani, 2000), most of which examined non-sterile site specimens, commonly surveillance cultures. None of these other studies performed long-term parallel comparative examination of CaC versus RM with all samples submitted to their hospital mycology laboratories.

In this study the chromogenic medium CHROMagar Candida failed to detect yeast growth from 19 % of specimens that did produce yeast growth using routine laboratory culture, including 132 specimen cultures that produced C. albicans on RM. In the other studies referenced above, CaC generally recovered at least 90 % of those yeasts recovered by RM, and often more yeasts were recovered by CaC than RM. However, as also stated previously, the clinical specimens examined in these other studies generally were from surveillance sites. The sites included were those that typically yield high concentrations of yeasts.

CaC did potentially improve on RM by detecting yeasts in 91 specimens that produced no yeast growth by routine methods and detecting more yeasts than RM in 38 additional specimens. Chromogenic media have been reported to enhance the identification of mixed cultures to varying degrees over traditional media (Pfaller et al., 1996; Willinger & Manafi, 1999; Baumgartner et al., 1996; Beighton et al., 1995).

Overall, 97 % (464/477) of the C. albicans, C. tropicalis and C. krusei isolates recovered were noted to produce the colours and morphology described by the manufacturer of CaC. We also described alternative colours of some yeasts on CaC that are not consistent with the traditionally identifiable colours. This may represent recovery of yeasts of different species on some plates or colour variability among strains of these species, as has been reported by others (Cooke et al., 2002). This reveals a limitation of our study in that we did not identify to the species level all yeasts detected on CaC in parallel with RM.

Chromogenic agar carries the potential of improving identification of yeast, especially in mixed cultures. Our evaluation of CaC as a routine fungal medium revealed disappointing results. Its role as a primary medium to improve the recovery of yeasts or the detection of mixed cultures appears limited.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
The views expressed herein are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defence or the US Government. The authors are employees of the US government. This work was prepared as part of their official duties and, as such, there is no copyright to be transferred.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

  • Ainscough, S. & Kibbler, C. C. (1998). An evaluation of the cost-effectiveness of using CHROMagar for yeast identification in a routine microbiology laboratory. J Med Microbiol 47, 623–628.[Abstract]

  • Baumgartner, C., Freydiere, A. M. & Gille, Y. (1996). Direct identification and recognition of yeast species from clinical material by using Albicans ID and CHROMagar Candida plates. J Clin Microbiol 34, 454–456.[Abstract]

  • Beck-Sague, C. & Jarvis, W. R. (1993). National Nosocomial Infections Surveillance System: secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J Infect Dis 167, 1247–1251.[Medline]

  • Beighton, D., Ludford, R., Clark, D. T. & 9 other authors (1995). Use of CHROMagar Candida medium for isolation of yeasts from dental samples. J Clin Microbiol 33, 3025–3027.[Abstract]

  • Bernal, S., Mazuelos, E. M., Garcia, M., Aller, A. I., Martinez, M. A. & Gutierrez, M. J. (1996). Evaluation of CHROMagar Candida medium for the isolation and presumptive identification of species of Candida of clinical importance. Diagn Microbiol Infect Dis 24, 201–204.[CrossRef][Medline]

  • Bouchara, J. P., Declerck, P., Cimon, B., Planchenault, C., de Gentile, L. & Chabasse, D. (1996). Routine use of CHROMagar Candida medium for presumptive identification of Candida yeast species and detection of mixed fungal populations. Clin Microbiol Infect 2, 202–208.[Medline]

  • Cooke, V. M., Miles, R. J., Price, R. G., Midgley, G., Khamri, W. & Richardson, A. C. (2002). New chromogenic agar medium for the identification of Candida spp. Appl Environ Microbiol 68, 3622–3627.[Abstract/Free Full Text]

  • Edmond, M. B., Wallace, S. E., McClish, D. K., Pfaller, M. A., Jones, R. N. & Wenzel, R. P. (1999). Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin Infect Dis 29, 239–244.[Medline]

  • Freydiere, A. M., Buchaille, L. & Gille, Y. (1997). Comparison of three commercial media for direct identification and discrimination of Candida species in clinical specimens. Eur J Clin Microbiol Infect Dis 16, 464–467.[CrossRef][Medline]

  • Hospenthal, D. R., Murray, C. K., Beckius, M. L., Green, J. A. & Dooley, D. P. (2002). Persistence of pigment production by yeast isolates grown on CHROMagar Candida media. J Clin Microbiol 40, 4768–4770.[Abstract/Free Full Text]

  • Hospenthal, D. R., Murray, C. K. & Rinaldi, M. G. (2004). The role of antifungal susceptibility testing in the therapy of candidiasis. Diagn Microbiol Infect Dis 48, 153–160.[CrossRef][Medline]

  • Houang, E. T., Chu, K. C., Koehler, A. P. & Cheng, A. F. (1997). Use of CHROMagar Candida for genital specimens in the diagnostic laboratory. J Clin Pathol 50, 563–565.[Abstract/Free Full Text]

  • Kirkpatrick, W. R., Revankar, S. G., McAtee, R. K. & 7 other authors (1998). Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar Candida screening and susceptibility testing of isolates. J Clin Microbiol 36, 3007–3012.[Abstract/Free Full Text]

  • Lopez-Ribot, J. L., McAtee, R. K., Perea, S., Kirkpatrick, W. R., Rinaldi, M. G. & Patterson, T. F. (1999). Multiple resistant phenotypes of Candida albicans coexist during episodes of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 43, 1621–1630.[Abstract/Free Full Text]

  • Momani, O. M. (2000). Cost-effectiveness and efficacy of CHROMagar Candida medium in clinical specimens. East Mediterr Health J 6, 968–978.[Medline]

  • Odds, F. C. & Bernaerts, R. (1994). CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 32, 1923–1929.[Abstract/Free Full Text]

  • Odds, F. C. & Davidson, A. (2000). ‘Room temperature’ use of CHROMagar Candida. Diagn Microbiol Infect Dis 38, 147–150.[CrossRef][Medline]

  • Pfaller, M. A., Houston, A. & Coffmann, S. (1996). Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata. J Clin Microbiol 34, 58–61.[Abstract]

  • Rangel-Frausto, M. S., Wiblin, T., Blumberg, H. M. & 8 other authors (1999). National Epidemiology of Mycoses Survey (NEMIS): variations in rates of bloodstream infections due to Candida species in seven surgical intensive care units and six neonatal intensive care units. Clin Infect Dis 29, 253–258.[Medline]

  • Redding, S. W., Zellars, R. C., Kirkpatrick, W. R. & 7 other authors (1999). Epidemiology of oropharyngeal Candida colonization and infection in patients receiving radiation for head and neck cancer. J Clin Microbiol 37, 3896–3900.[Abstract/Free Full Text]

  • Willinger, B. & Manafi, M. (1999). Evaluation of CHROMagar Candida for rapid screening of clinical specimens for Candida species. Mycoses 42, 61–65.[CrossRef][Medline]

  • Willinger, B., Hillowoth, C., Selitsch, B. & Manafi, M. (2001). Performance of Candida ID, a new chromogenic medium for presumptive identification of Candida species, in comparison to CHROMagar Candida. J Clin Microbiol 39, 3793–3795.[Abstract/Free Full Text]





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