CHROMagar Candida as the Sole Primary Medium for Isolation of Yeasts and as a Source Medium for the Rapid-Assimilation-of-Trehalose Test

The chromogenic medium BBL CHROMagar Candida (CAC) was evaluatedas a sole primary medium for the isolation of yeasts from clinicalspecimens in which yeasts are the primary concern. Additionally,the reliability of the rapid-assimilation-of-trehalose (RAT)test in yielding correct results with isolates taken from CACwas assessed. A total of 270 throat, urine, and genital (TUG)specimens were streaked onto CAC, Sabouraud dextrose agar (SDA),inhibitory mold agar (IMA), and Mycosel (MYC). A total of 69blood culture broths that were smear positive for yeast werestreaked onto CAC and SDA. A 1-h RAT test (NCCLS M35-A) wasperformed simultaneously on isolates from CAC and SDA. A totalof 112 TUG specimens yielded yeast colonies (CAC, 111 colonies;IMA, 105; SDA, 103; MYC, 91). The 69 blood culture yeasts grewon both CAC and SDA. Mixed cultures of yeasts were detectedon 11 CAC plates but were unrecognized on other media. Coloniessuspected of being C. glabrata on 32 CAC plates were all RATtest positive and confirmed to be C. glabrata; of 59 colonieswith various characteristics of color and morphology on CAC,none were RAT positive, and all were conventionally identifiedas yeasts other than C. glabrata (sensitivity and specificity,100%). The same isolates from SDA tested for RAT produced sixfalse negatives and no false positives (sensitivity, 81%; specificity,100%). The results show that CAC can be used as the sole primarymedium for recovery of yeasts from clinical specimens. Additionally,isolates grown on CAC yield excellent results with the RAT testutilized in this study.

INTRODUCTION

Top

Introduction

Candida species are now the fourth-most-common cause of hospital-acquiredbloodstream infections (4, 13) and are the organisms commonlysought in fungal cultures of throat, urine, and genital (TUG)specimens. Over the past 4 decades, rates of Candida infectionshave steadily increased, with non-albicans Candida species makingup a greater proportion of nosocomial infections (4, 13, 14).Candida glabrata, C. parapsilosis, C. tropicalis, and C. kruseiconstitute the majority of species other than C. albicans isolatedin most institutions. Of note, C. glabrata and C. krusei havebeen observed to be 4- to 32-fold less susceptible than C. albicansto fluconazole (3, 11, 12, 14). Rapid species-level identificationis therefore an essential task for the clinical mycology laboratory,as it can have a direct bearing on treatment decisions.

The usefulness of a selective and differential medium for theisolation of Candida spp. has long been noted (8, 10). CHROMagarCandida (CAC) is a selective medium for the isolation of fungithat simultaneously provides direct differentiation and identificationof several Candida species (9). The yeasts produce enzymes thatreact with chromogenic substrates in the CAC medium, producingcolonies of different colors. These enzymes are specific, allowingsome yeasts to be identified to the species level by their colorand colony characteristics. Colonies of C. albicans and C. dubliniensisappear lighter and darker green, respectively, C. tropicaliscolonies appear dark blue to metallic blue, and C. krusei coloniesappear light pink and dry with a light border. Other yeastsare noted to appear cream colored or develop a light- to dark-pinktone.

The manufacturer does not presently claim the ability to identifyC. glabrata by color or texture, although several studies havestated that, with experience, the species can be identifiedby colony color and size on CAC (2, 6, 12). In our laboratory,it has been found to be a very subjective call that requiresconfirmation. Classically, C. glabrata identification has beenbased on growth characteristics and biochemical tests (5) thatcan take 2 to 3 days to complete. In contrast, a rapid-assimilation-of-trehalose(RAT) test (method of Stockman and Roberts) (1, 7) that requiresonly 1 h of incubation has been developed specifically for itsidentification. Because of the increased prevalence of C. glabratain bloodstream and other infections and its decreasing susceptibilityto fluconazole, it is imperative that a clinical mycology laboratorybe capable of identifying it with certainty as rapidly as possible.

Currently, most clinical laboratories utilize a battery of 2to 3 primary media for each specimen submitted for fungus culture.For identification to the species level of a yeast other thanC. albicans, the biochemical testing of the isolate typicallytakes 1 to 3 days.

The purpose of our study was (i) to evaluate the performanceof CAC for use as the sole primary medium for yeasts from selectedclinical specimens and (ii) to verify that the RAT test willyield correct results when the isolate is taken from CAC, enablingidentification of C. glabrata on the same day as colony growth.

MATERIALS AND METHODS

Top

Materials and Methods

A total of 270 clinical specimens in which yeasts were the anticipatedfungus, i.e., TUG specimens, were streaked onto CAC, Sabourauddextrose agar (SDA), inhibitory mold agar (IMA), and Mycosel(MYC) (all media from Becton Dickinson/BBL, Sparks, Md.). Atotal of 69 blood cultures yielding a positive signal on a continuousmonitoring system (BacT/Alert; bioMerieux, Durham, N.C.) andfound to be smear positive for yeast were streaked onto CACand SDA. The CAC plates were incubated in atmospheric air at37°C, as recommended by the manufacturer. The other mediawere incubated at 30°C, the temperature routinely used forfungal cultures in our clinical mycology laboratory. All cultureswere read at 48 h and again on days 4 and 7 if no growth wasobserved on the first reading. If a sparse amount of yeast grewon one agar only, it was eliminated from the study due to likelihoodof random sampling. The 1-h RAT test was performed simultaneouslyon isolates from CAC and SDA for identification of C. glabrata.Briefly, 100 µl of RAT broth, prepared in our laboratory(7) and containing yeast nitrogen base, trehalose, bromcresolgreen, and cycloheximide, was placed into microtiter wells;a heavy inoculum of yeast colonies was emulsified in the brothand incubated uncovered at 37°C in ambient air for 1 h.A positive result was indicated by the blue solution turningyellow to greenish yellow. The solution remaining blue to blue-greenindicated a negative result. Confirmatory identification wasbased on assimilation patterns on API 20C (bioMerieux, Marcyl'Etoile, France) and by microscopic morphology on cornmeal-Tween80 agar (prepared in lab) (7).

RESULTS

Top

Results

A total of 41% (112 of 270) of TUG cultures and 100% of thesmear-positive blood cultures yielded yeasts on at least twoinoculated media. From the 112 positive TUG specimens, 122 isolateswere detected on CAC, 105 were detected on IMA, 103 were detectedon SDA, and 91 were detected on MYC. Eleven mixed cultures weredetected only on CAC; mixed characteristics were unrecognizedon other media. The mixed cultures consisted of C. albicans-C.glabrata, C. albicans-C. tropicalis, and C. tropicalis-C. glabrata.The species recovered from TUG and blood culture specimens arelisted in Table 1. All isolates from both TUG and blood culturesproduced the predicted identifiable colors on CAC. C. albicansand C. dubliniensis colonies appeared light to medium green.These two species were definitively differentiated by abilityto grow at 42 to 45°C, by formation of single versus clusteredterminal chlamydospores on cornmeal-Tween 80 agar, and by API20C assimilation patterns. C. tropicalis colonies appeared darkblue to metallic blue, and C. krusei appeared dry and lightpink with a whitish border. C. glabrata produced smallish, smooth,light-pink colonies. The appearance of other species rangedfrom cream to light pink colonies, some resembling the appearanceof C. glabrata.

View this table:
[in this window]
[in a new window]
TABLE 1. Yeasts recovered from TUG and blood cultures

A total of 32 colonies suspected of being C. glabrata on CACplates were tested for RAT, and all were positive and simultaneouslyconfirmed to be C. glabrata; of 59 colonies with various colorand morphology characteristics recovered on CAC, none were RATpositive, and all were further identified as yeasts other thanC. glabrata (sensitivity and specificity = 100%). The same isolatesfrom SDA tested for RAT produced 6 false negatives and no falsepositives (sensitivity, 81%; specificity, 100%) (Table 2).

View this table:
[in this window]
[in a new window]
TABLE 2. Comparison of the RAT test results for yeast isolates from CAC medium versus those from SDA

DISCUSSION

Top

Discussion

More TUG specimens yielded yeast on CAC than on IMA, SDA, orMYC. When yeast was detected on any of the test media, it grewon CAC in all but one case (sensitivity = 99%). The one missedcase was probably due to random distribution at the time ofinoculation, as only sparse colonies were seen on the othermedia. Sensitivity values for recovery of yeast on IMA, SDA,and MYC were 96, 93, and 83%, respectively, but no identificationto species level or determination of mixed cultures could bedirectly made on these media. Only CAC was able to detect 11specimens with mixed cultures of yeasts. The mixed cultureswere unrecognized on the other media either because of a lownumber of the minority species or because of a similarity incolony appearance.

In our study, as in numerous others, C. albicans, C. krusei,and C. tropicalis were readily identifiable on CAC. C. glabratausually produced relatively small, light-pink colonies thatwith practice could be presumptively differentiated from othernon-albicans Candida species on CAC at $≤$ 48 h. We found that our1-h RAT test was 100% sensitive and specific for colonies testeddirectly from CAC but had a reduced sensitivity of 81% for thesame isolates tested from SDA (the precise explanation for thisdisparity is not known). The combination of growth on CAC andthe 1-h RAT test allowed for the identification of C. glabratain as early as 48 h following the initial culture setup.

C. glabrata accounted for 20% of the yeasts isolated from TUGspecimens and 29% of the yeasts isolated from blood cultures.When the other species widely acknowledged as identifiable onCAC are included, this medium paired with the 1-h RAT test identified87 and 84% of the yeasts from TUG and blood cultures, respectively,on the same day that mature growth occurred. An additional fortuitousbenefit is that although the price of one CAC plate was slightlyhigher than that of any other single medium tested, it was lessthan that of the combination of any two media that are commonlyused in tandem.

Of note is the issue that other brands of CHROMagar and othermethods of testing for RAT may not yield the successful resultswe have found with the combination of products used in thisstudy.

In conclusion, CAC can be used as the sole primary medium forfungus cultures of specimens in which yeasts are the main fungussought. When compared to other media commonly used, CAC wassuperior in the primary isolation of yeasts and in identifyingmixed cultures of yeasts. The use of BD/BBL CHROMagar Candidawith the 1-h RAT test formulation of Stockman and Roberts allowsfor extremely rapid, same-day identification of colonies ofC. glabrata. The use of CAC and RAT facilitates increased recoveryof yeasts, decreases identification turnaround time, and streamlinesthe overall workflow in a simple and cost-effective manner.

FOOTNOTES

* Corresponding author: Mailing address: Weill Cornell Medical Center, Clinical Microbiology—Starr 737, 525 East 68th St., New York, NY 10021. Phone: (212) 746-2405. Fax: (212) 746-8945. E-mail: dhlarone{at}med.cornell.edu.

REFERENCES

Top