A.M. Freydiere 1 and R. Robert 2

1 Hôpital Debrousse, Hospices Civils de Lyon, 2Parasitologie-Mycologie, Angers, France

Rapid Candida glabrata identification with a commercial trehalase-maltase detection test

Rapid Candida glabrata identification with a commercial trehalase-maltase detection test

Increasing numbers of immuno-compromised patients and certain current medical and surgical management practices have favoured the emergence of normally commensal yeast species as life-threatening pathogens. These yeasts differ in their antifungal drug susceptibilities, so that rapid species-level identification of yeasts isolated from clinical material is imperative for prompt institution of appropriate antifungal therapy (12), since susceptibility data for the isolate may not be immediately available. Candida glabrata, one of these emerging pathogens (3, 11, 13), is the second-most-commonly-isolated yeast species in clinical laboratories (2, 10) and some strains readily show a reduced susceptibility to several azole antifungal agents and notably to fluconazole. Thus because of its frequency of isolation and decreased susceptibility, laboratories should be able to identify this Candida species rapidly. The GLABRATA RTT® test (Fumouze Diagnostics, France) based on the ability of C. glabrata to hydrolyse trehalose but not maltose allows the identification within 20 minutes. It uses a glucose-oxidase reaction for the detection of glucose and requires only 4 to 6 colonies. The purpose of this work was (i) to assess the performance of this rapid trehalase test for C. glabrata identification applied to colonies grown on four different mycological isolation media (ii) to compare these performance to those of the strip test previously described by Parant et al. (5, 8, 9)

IntroductionIntroduction

Material and methodsMaterial and methodsStrains: 125 stock isolates, most commonly encountered in clinical samples and representing a total of 14 yeast species were kept on glass beads at –20°C and passaged through one subculture on Sabouraud agar (bioMerieux) to check for purity. Then they were streaked to single colonies on the four different media including chromogenic and non-chromogenic media and incubated at 32 °C for 24 h.Confirmation of the identification of all isolates tested was done with ID 32C identification strips (bioMérieux Marcy l'Etoile, France).The GLABRATA RTT® test: Principle, description, methodology and interpretation.

ConclusionConclusion The GLABRATA RTT® has been shown to be simple, rapid, and reliable. Due to the low inoculum density required (4 to 6 colonies), it should allow the identification directly from the primary isolation medium.An ideal approach to efficient identification of clinical yeast isolates is the use of preliminary presumptive identifications from a polyvalent chromogenic medium that reduces the need for trehalase testing only to those colonies that did not form the characteristic colours.The GLABRATA RTT is a useful adjunct to the work of the routine medical mycology laboratory.

Chromogen

C. glabrata colonies

H2O2

H2O

O2

Oxided chromogen(brown-orange)

Glucose

Rapid hydrolysis (10 min))

Glucose oxidase

Peroxidase

RE

AGENT

Trehalase

Trehalose

1- Principle

We thank our technicians for their assitanceand A. Frangin for her valuable help in producing this poster.

The slightly lower specificity of CandiSelect versus Candida ID concerns mainly Candida famata. As a matter of fact, C. famata strains yield pink colonies on Candida ID, and due to this pink coloration, these strains were excluded as C. glabrata.The GLABRATA RTT® showed higher sensitivity and specificity than the strip tests described by Parant and al. (5, 9) using commercially supplied glucose oxidase strips moistened with trehalose or maltose solutions and smeared with material taken from a yeast colony with an inoculating loop, for strains grown on CHROMagar Candida. Two reasons might be hypothesised  (i) the longer contact time with trehalose with the GLABRATA RTT® (ii) the formulation of CHROMagar Candida since this medium has recently been reformulated by Becton Dickinson (6).For yeasts isolated on chromogenic media, because some Candida species can be recognised directly, only the non-identifiable colonies need to be tested, reducing handling, reagent consumption, time and cost.

For strains grown on Candida ID, CandiSelect and Sabouraud gentamicin chloramphenicol, the results of the GLABRATA RTT® test were similar to those of the strip test.

Table 1. Results of the GLABRATA RTT® test for 125 yeast strains subcultured on 3 chromogenic media and 1 Sabouraud gentamicin chloramphenicol agar.

Table 2. Comparaison of the results GLABRATA RTT® test with those of the Strip trehalase- maltase test with 107 yeast strains subcultured on Candida ID, CandiSelect and one Sabouraud agar.

Results - DiscussionResults - Discussion

1. Chang H.C., S.N. Leaw, A.H. Huang, T.L. Wu and T.C. Chang. 2001. Rapid identification of yeast in positive blood cultures by a multiplex PCR method. J. Clin. Microbiol. 39:3466-3471.2. Fenn J.P., E. Billetdeaux , H. Segal, L. Skodack-Jones, P.E. Padilla, M. Bale and K. Carroll. 1999. Comparison of four methodologies for rapid and cost-effective identification of Candida glabrata. J Clin Microbiol. 37:3387-3389.3. Fidel P.L., J.A.Vazquez and J.D. Sobel. 1999. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 12:80-96.4. Freydiere A.M., R. Guinet and P. Boiron. 2001. Yeast identification in the clinical microbiology laboratory: phenotypical methods. Med. Mycol. 39:9-335. Freydiere A.M., F. Parant, F. Noel-Baron, M. Crepy, A. Treny, H. Raberin, A. Davidson and F.C. Odds. 2002. Identification of Candida glabrata by a 30-second trehalase test. Submitted on the 8 th of January to the J. Clin. Microbiol. (in Press).6. Jabra-Rizk M.A., T.M. Brenner, M. Romagnoli, A.A. Baqui, W.G. Merz, W.A. Falkler and T.F. Meiller. 2001. Evaluation of a reformulated CHROMagar Candida. J. Clin. Microbiol. 39:2015-2016. 7. Land G., J. Burke, C. Shelby, J. Rhodes, J. Collett, I. Bennett and J. Johnson. 1996. Screening protocol for Torulopsis (Candida) glabrata. J. Clin. Microbiol. 34:2300-2303. 8. Parant F., M. Crepy, Y. Gille and A.M. Freydiere. 2000. A one minute trehalase detection test for Candida glabrata identification. 6th congress of the European Confederation of Medical Mycology (ECMM 2000). Barcelona 9-11 November. 9. Parant F, A.M. Freydiere, Y. Gille, P. Boiron and F.C. Odds. 2001. A one minute trehalase detection test for identification of Candida glabrata. Journal de Mycologie Médicale. 11(1): 26-31.10. Peltroche-Llacsahuanga H., A. Jenster , R Lütticken and G. Haase. 1999. Novel microtiter plate format for testing germ tube formation and proposal of a cost-effective scheme for yeast identification in a clinical laboratory. Diagn Microbiol Infect Dis 35:197-204 11. Price M.F, M.T. La Rocco and L.O. Gentry. 1994. Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period. Antimicrob Agents Chemother 38:1422-1424. 12. Rowen J.L, J.M. Tate, N. Nordoff, L. Passarell, M.R. McGinnis. 1999. Candida isolates from neonates: frequency of misidentification and reduced Fluconazole susceptibility. J Clin Microbiol. 37:3735-3737. 13. Wingard J.R, W.G. Merz, M.G. Rinaldi, C.B. Miller, J.E. Karp and R. Saral. 2000. Association of Torulopsis glabrata infections with Fluconazole prophylaxis in neutropenic bone marrow transplant patient. Antimicrob Agents Chemother 37:1847-9.

ReferencesReferences

2 - Description

Only Candida glabrata should give positive trehalase and negative maltase results.

Well T Well M Well B SpeciesTrehalase Maltase Control

+ - - C. glabrata - + - non C. glabrata + + + uninterpretable + + - non C. glabrata - - - non C. glabrata

1

2

3

4

1 2

3 4T

REALOSE

M

ALTOSE

C

ONTROL

Four tests per panel and three wells per test

Nb of strains identified with the GLABRATA RTT® test subcultured on :Species No. of

strains testedCHROMagar

Candida (Becton Dickinson)

Candida ID(bioMerieux)

CandiSelect(BioRad)

Sabouraud gentamicinchloramphenicol

(bioMerieux)Candia glabrata 54 53 51 53 52Test sensitivity (%) 98.1 94.4 98.1 96.3

Candida non-glabrata 71 1 1 4 1Candida famata 13 1 1 3* 1Candida lusitaniae 2 0 0 1 0Other species * 56 0 0 0 0

Test specificity (%) 98.6 98.6 94.4 98.6

No. of strains identified with the GLABRATA RTT® and the Strip test subcultured on :Species No. of

strainstested

Candida ID(bioMerieux)

CandiSelect(BioRad)

Sabouraud gentamicinchloramphenicol

(bioMerieux)GLABRATA

RTT® Strip

Trehalase/maltase

GLABRATARTT®

StripTrehalase/maltase

GLABRATARTT®

StripTrehalase/maltase

Candia glabrata 38 37 37 37 37 37 37Test sensitivity (%) 97.4 97.4 97.4 97.4 97.4 97.4

Candida non-glabrata 69 1 1 4 1 1 0Candida famata 13 1 1 3 1 1 0Candida lusitaniae 2 0 0 1 0 0 0Other species * 54 0 0 0 0 0 0

Test specificity (%) 98.5 98.5 94.2 98.5 98.5 100* 16 C. tropicalis, 13 C. parapsilosis, 5 C. guilliermondii, 4 C. kruseï, 4 C. inconspicua, 3 C. norvegensis, 3 C. lipolytica, 2 Saccharomyces cerevisiae, 2 C.

pelliculosa, 2 C. kefyr

4- Interpretation

A brown/orange coloration corresponds to a positive reaction while no coloration

corresponds to a negative result. + -

Of the 125 isolates tested with the GLABRATA RTT® test, 107 had been previously tested with the one minute strip trehalase and maltase tests described by Parant and al. (5, 9). The comparison of the results of the GLABRATA RTT ® test and the strip trehalase/maltase test is reported in Table 2.

In terms of sensitivity and specificity, the GLABRATA RTT® gives performance similar to those achieved with longer-duration (i) C. glabrata screening tests (2, 7, 10) and (ii) widely used commercial yeast identification systems such as API20CAUX and Vitek YBC (4). Multiplex PCR methods offers 100% sensitivity and specificity for identification of C. glabrata (1), but are less rapid and more expensive than the GLABRATA RTT® test.

The performance of the GLABRATA RTT® test for C. glabrata identification applied to colonies grown on four mycological isolation media are reported in Table 1.

AcknowledgementsAcknowledgements

* 17 C. tropicalis, 13 C. parapsilosis, 5 C. guilliermondii, 4 C. kruseï, 4 C. inconspicua, 3 C. norvegensis, 3 C. lipolytica, 2 Saccharomyces cerevisiae, 2 C. pelliculosa, 2 C. kefyr, 1 C. utilis

8th ECMMBudapest

25 -27 August 2002

4 to 6 colonies in 100 µl of distilled water

CHROMagar Candida Candida ID CandiSelect

Pink colonies White coloniesWhite colonies

3- Methodology

25 µl of suspension

12

3 4

10 mn

5 to 10 min

25 µl of reagent

am.freydiere@chu-lyon.fr

Sabouraud

White colonies