Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 172-177

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 172-177

Brief Communication

Yeast identification in routine clinical microbiology laboratory and its clinical relevance

S Agarwal¹, V Manchanda¹, N Verma¹, P Bhalla²

¹ Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, New Delhi - 110 031, India
² Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 031, India

Correspondence Address: V Manchanda Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, New Delhi - 110 031 India microcnbc@gmail.com

Date of Submission: 12-Dec-2010
Date of Acceptance: 23-Mar-2010

Code Number: mb11040

PMID: 21654115

DOI: 10.4103/0255-0857.81794

Abstract

Keywords: Candida, chrome agar, corn meal agar, slide culture, Trichosporon, Vitek

Introduction

Yeast infections other than those caused by Candida albicans have been increasingly reported worldwide. ^{[1],[2],[3],[4],[5],[6],[7]} The emergence of such infections has been attributed to the advancement in medical management and increasing load of severely ill patients. Because of the delay in obtaining identification by conventional methods as well as the close relationship between species and difference in fluconazole susceptibility, early speciation of positive clinical specimens has immense potential to impact the therapeutic decisions regarding empirical antifungal therapy. ^[8]

In routine diagnostic laboratories, conventionally, Sabouraud′s Dextrose Agar (SDA) is widely used for the isolation of all yeasts from clinical specimens. Most clinically relevant yeast species grow on SDA within 48 h, although few others (e.g., Cryptococcus neoformans) may require longer incubation. Moreover, identification of multiple yeast species infections is difficult during routine isolation procedures, which can have serious clinical implications. Many clinical laboratories use the formation of germ tube in serum as the initial test to differentiate C. albicans from other yeast species, although it proves to be a subjective test sometimes. ^[9] This is then followed by more time-consuming methods including microscopic identification based on morphology of growth on corn meal-tween 80 agar (CMA) and biochemical tests. Automation in yeast identification methods has been increasingly employed. ^[10],[11] Full identification of yeast may take up to 72 h or more for primary isolation of the organism.

The three Candida species viz., C. tropicalis, C. krusei and C. (torulopsis) glabrata constitute the majority of clinically significant yeasts other than C. albicans isolated in most of the clinical laboratories. ^[12],[13] These species have been observed to be 32-fold less-susceptible to fluconazole than C. albicans.^[12],[13] Given the potential for selection of these less-susceptible species by empirical antifungal treatment or prophylaxis, clinical laboratories should be able to characterize such yeast isolates in routine specimen processing. The presence of more than one yeast species in clinical samples (especially from neonates) is not uncommon.

The prompt detection of such clinical scenarios of multiple yeast infections may be an aid for early appropriate treatment decisions.

The need for rapid identification of the pathogen and the difficulty in detecting mixed cultures on the traditional SDA have led to the development of commercial isolation media intended to differentiate yeast species on the basis of colony colour. Many chromogenic agar media have been evaluated and are being used for the detection and presumptive identification of Candida species. ^{[5],[6],[14],[15],[16],[17],[18]}

The present study evaluates suitable methods and schemes for the rapid identification of yeast species other than capsulated yeasts in a routine microbiology laboratory using chromogenic agar medium, microscopic morphology on CMA and Vitek-2-compact (V2C) identification system (BioMerieux, Marcy, France).

Materials and Methods

A total of 173 yeasts isolated from various clinical specimens (blood, clean-voided urine and tracheal aspirates) submitted to the laboratory from different clinical units of Chacha Nehru Bal Chikitsalaya (CNBC), an exclusive pediatric tertiary care hospital, were included in the study. All yeast isolates that grew on Columbia blood agar (Oxoid, Basingstoke, UK), after Gram staining were sub-cultured on chromogenic medium (HiChrome agar; HiMedia, Mumbai, India), which was used as a follow-up medium. Yeast isolates sub-cultured on chromogenic medium were incubated overnight at 35°C. All yeast isolates grew well and developed distinctive coloured colonies after overnight incubation. The plates were further incubated for a total incubation of 48 h to get better-developed coloured colonies. Presumptive identification was made by colour and morphology of the colonies as per the manufacturer′s instructions. These isolates were also further identified on the basis of microscopic morphological features of the growth obtained through slide cultures on CMA. ^[19],[20] Briefly, pure distinctive colonies obtained from Columbia blood agar or chromogenic medium were inoculated through sterile straight wire on the margins of the 1 cm x 1 cm block of corn meal agar placed on a sterilized glass slide. The agar block was then covered with a sterile coverslip and incubated in a sterile petridish having moistened filter paper. The assembly was then incubated at 25°C. The slide cultures were observed after overnight incubation and after 48 h of incubation. Finally, all the yeast isolates were subjected to identification using the V2C system using protocols prescribed by the manufacturer. Purity check on chromogenic medium was performed for all V2C identifications. Quality control was achieved using C. parapsilosis ATCC 22019, C. albicans ATCC 611098, C. glabrata ATCC myA2950 and C. tropicalis ATCC 13803.

Results

A total of 173 yeast non-repeat isolates were recovered from 169 clinical specimens, viz. blood (58.5%), clean-voided urine (26%), tracheal aspirates (10%) and cerebrospinal fluid (0.60%). Among these 173 yeast isolates, 149 isolates (86.12%) were identifiable by observing colony colour on the chromogenic medium [Table - 1]. These clinical isolates, namely C. albicans, C. tropicalis, C. parapsilosis, C. krusei and T. asahii were presumptively identified to the species level by their colony morphology and colour using chromogenic medium [Figure - 1]. Among the remaining 24 isolates, (13.87%) 22 isolates (12.71%) produced shades of pink colour on the chromogenic medium and two isolates (1.41%) produced white pasty colonies with a blue tinge. These isolates could not be identified using chromogenic medium and, hence, were reported as "yeast under identification". The chromogenic medium detected the presence of multiple yeast species in six clinical specimens [Table - 2]. Interestingly, the present study found that yeasts appearing morphologically different on primary isolation media (Columbia blood agar) were later confirmed as single yeast species. Similarly, unsuspected cultures on the primary medium also revealed multiple yeast infections on the chromogenic medium in all six cases [Table - 2]. The findings were confirmed by repeat isolation of yeast species in repeat specimens. All isolates were inoculated on CMA blocks for slide culture and identification by V2C system was performed. Identification by observing microscopic morphology on CMA blocks was successful for the 149 (86.12%) isolates that were easily identified by colony colour on the chromogenic medium. All these isolates produced characteristic morphology distinct for that particular species and confirmed the identification by chromogenic medium. For the remaining 24 isolates, 22 isolates revealed presence of yeast cells only (no pseudohypahe formation). These isolates produced pink-coloured colonies on the chromogenic medium. Although there were a few differences in the microscopic morphology of the yeast cells, e.g. three isolates produced ovoid yeast cells, two isolates produced very small round yeast cells, but the difference was not easily discernible [Figure - 1] and all these isolates were labeled as pseudohyphae non-producing yeast species. Two isolates that produced white pasty colonies with a blue tinge on the chromogenic medium showed the presence of chain and bunches of blastoconidia, but the morphology was not distinctive for any particular species.

The V2C system identified 168 isolates (97.10%) to the species level and labeled five isolates (2.89%) as low discrimination. All low-discrimination isolates were sub-cultured on a chromogenic medium for purity check and subjected to a repeat identification attempt by the V2C system. All isolates were pure and were labeled as low-discrimination by the Vitek 2 Compact system. These low-discrimination isolates were identified as C. tropicalis (n = 3) and C. albicans (n = 2) by chromogenic medium and microscopic morphology on CMA. Among the 168 isolates that were identified to a species level by the V2C system, identification of 144 isolates (85.71%) was in complete agreement with the previous two methods. The remaining 24 isolates (14.28%) could not be identified by either of the earlier two methods and could be successfully identified to the species level only using the V2C system [Table - 1].

Discussion

Because of the increasing complexity in the management and disease profiles of patients, there has been a surge of infections due to yeast infections other than C. albicans. Over the past decade, there has been a significant increase in the number of reports of systemic and mucosal yeast infections with Candida species other than C. albicans.^{[3],[16],[21]} Infections with these yeast species also have a direct impact on the choice of empiric antifungal therapy and clinical outcome. The potential clinical importance of species-level identification has been recognized as Candida species differ in the expression of putative virulence factors and antifungal susceptibility. ^[6],[22] Rapid identification of yeast species also guides early appropriate antifungal therapy. Thus, it has become imperative to identify all yeast isolates up to the species level in routine microbiology laboratories. Newer techniques like real-time PCR, matrix-assisted laser desorption ionization-time of flight mass spectrometry and multiplex-tandem PCR are being increasing employed for the identification of yeast species. ^{[1],[23],[24],[25]} However, such methods are still at an experimental stage and are not yet suited for use in routine clinical microbiology laboratories.

The results of the present study confirm that a chromogenic medium helps in the presumptive identification of C. albicans, C. tropicalis, C. krusei and C. parapsilosis, as reported in earlier studies. ^{[15],[16],[17],[18]} Additionally, in the present study, the chromogenic medium also allowed the identification of T. asahii at a similar level of accuracy. Also, the chromogenic medium provides an opportunity to detect "multi-species" yeast infections as observed in previous studies. ^{[4],[17],[21],[24]} In the present study, six cases (3.5%) of "multi-species" yeast infections were identified among 169 positive cultures in contrast to the previous studies, which report an 18% occurrence of multi-species yeast infections. The difference in the results of multi-species yeast infections could be attributed to the difference in the study population characteristics. Furthermore, chromogenic medium facilitates presumptive identification of yeast isolates to the species level of isolates within 24 h of incubation. Primary inoculation of clinical specimens showing yeast cells on direct Gram′s stain examination on the chromogenic medium further hastens the presumptive species identification of yeast in clinical specimens (data not shown), allowing early initiation of appropriate therapy.

CMA morphology revealed similar results for the 149 isolates (100% agreement) and V2C system for the 144 isolates (94% agreement). In the present study, chromogenic medium and morphology on CMA did not enable the differentiation of C. glabrata (n = 2), C. pelliculosa (n = 13), C. utilis (n = 3) and C. hemulonii (n = 1). However, all these isolates could be clustered as those producing pink to purple colonies producing only yeast cells (yeast cells without psudohyphae) [Figure - 1]. White colonies with a blue hue on the chromogenic medium could be observed with two C. rugosa isolates, and were unique to the species [Figure - 1]. All such isolates (yeast cells without pseudohyphae) could be successfully identified only through automated systems like the V2C system. None of the methods was self-sufficient in identifying all yeast species in all clinical specimens. Thus, a judicious and cost-effective workflow needs to applied for faster and accurate identification of such isolates. Based on the current study findings, an algorithm is proposed that may be followed in routine microbiology laboratory for rapid and accurate identification of clinical yeast isolates [Figure - 2]. Early identification will facilitate early appropriate therapy, which in turn reduces the cost of antimicrobial therapy, morbidity and mortality in patients infected with yeast. For non-resolving identification issues of yeast, automated systems may be helpful.

The study has a limitation: that many other clinically significant yeast species that are not prevalent in our setting need to be assessed using the proposed algorithm and with a larger number of yeast isolates. However, the present algorithm helped our laboratory to improve upon yeast identification and confidence in establishing clinical diagnosis.

Given the fact that many clinical microbiology laboratories do not perform identification beyond a germ tube test, the use of chromogenic medium with morphology on CMA provides rapid and accurate identification of commonly isolated single or multispecies yeast infections. Identification of yeast by the V2C system has its own limitations; however, it was most appropriate for yeast isolates that do no produce characteristic pseudohyphae along with chlamydoconidia or blastoconidia. Presumptive identification followed by confirmation of yeast species is a useful strategy to initiate early appropriate antifungal therapy, reducing morbidity and mortality in patients infected with such infections.

References

1.	Khan Z, Mustafa AS, Alam FF. Real-time LightCycler polymerase chain reaction and melting temperature analysis for identification of clinically important Candida spp. J Microbiol Immunol Infect 2009;42:290-5.  Back to cited text no. 1  [PUBMED]
2.	Kossoff EH, Buescher ES, Karlowicz MG . Candidemia in a neonatal intensive care unit: Trends during fifteen years and clinical features of 111 cases. Pediatr Infect Dis J 1998;17:504-8.  Back to cited text no. 2
3.	Kothavade RJ, Kura MM, Valand AG, Panthaki MH . Candida tropicalis: Its prevalence, pathogenicity and increasing resistance to fluconazole. J Med Microbiol 2010;59:873-80.  Back to cited text no. 3
4.	Levy I, Rubin LG, Vasishtha S, Tucci V, Sood SK . Emergence of Candida parapsilosis as the predominant species causing candidemia in children. Clin Infect Dis 1998;26:1086-8.  Back to cited text no. 4
5.	Moyer GJ, Romagnoli M, Merz WG . CHROMagar for presumptive identification and detection of multiple yeast species in oncology surveillance, In Abstracts on the 95 th General Meeting of Americal society for Microbiology. American Society for Microbiology, Washington, D.C: Abstr. F-117 1995. p. 107.  Back to cited text no. 5
6.	Murray MP, Zinchuk R, Larone DH . CHROMagar Candida as the sole primary medium for isolation of yeasts and as a source medium for the rapid-assimilation-of-trehalose test. J Clin Microbiol 2005;43:1210-2.  Back to cited text no. 6
7.	Pfaller MA, Jones RN, Doern GV, Sader HS, Messer SA, Houston A, et al. Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, 1997-1998. Antimicrob Agents Chemother 2000;44:747-51.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.	Zilberberg MD, Kollef MH, Arnold H, Labelle A, Micek ST, Kothari S, et al. Inappropriate empiric antifungal therapy for candidemia in the ICU and hospital resource utilization: A retrospective cohort study. BMC Infect Dis 2010;10:150.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.	Sheppard DC, Locas MC, Restieri C, Laverdiere M . Utility of the germ tube test for direct identification of Candida albicans from positive blood culture bottles. J Clin Microbiol 2008;46:3508-9.  Back to cited text no. 9
10.	Fenn JP, Segal H, Barland B, Denton D, Whisenant J, Chun H, et al. Comparison of updated Vitek Yeast Biochemical Card and API 20C yeast identification systems. J Clin Microbiol 1994;32:1184-7.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.	Graf B, Adam T, Zill E, Göbel UB . Evaluation of the VITEK 2 system for rapid identification of yeasts and yeast-like organisms. J Clin Microbiol 2000;38:1782-5.  Back to cited text no. 11
12.	Price MF, LaRocco MT, Gentry LO . Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period. Antimicrob Agents Chemother 1994;38:1422-4.  Back to cited text no. 12
13.	Rex JH, Pfaller MA, Barry AL, Nelson PW, Webb CD. Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia. NIAID Mycoses Study Group and the Candidemia Study Group. Antimicrob Agents Chemother 1995;39:40-4.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.	Louwagie B, Surmont I, Verhaegen J, Odds F. Differential and enrichment media for selective culture and recognition of yeast species from clinical material. Eur J Clin Microbiol Infect Dis 1995;14:406-11.  Back to cited text no. 14  [PUBMED]
15.	Odds FC, Bernaerts R. CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 1994;32:1923-9.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.	Pfaller MA, Houston A, Coffmann S. Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata. J Clin Microbiol 1996;34:58-61.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.	Baradkar VP, Mathur M, Kumar S. Hichrom candida agar for identification of Candida species. Indian J Pathol Microbiol 2010;53:93-5.  Back to cited text no. 17  [PUBMED]
18.	Raut SH, Varaiya A. Differentiation of Candida dubliniensis on chrom agar and Pal′s agar. Indian J Med Microbiol 2009;27:55-8.  Back to cited text no. 18  [PUBMED]
19.	Garcia J. Candida albicans budding on slide culture. Available from: http://www.microbelibrary.org/index.php/library/fungi/ 2799-candida-albicans-budding-on-slide-culture. [cited on 2004 Apr 7].  Back to cited text no. 19
20.	World Health Organisation. Updated on 27 April, 2006. Chapter 21, Mycological Techniques. In Blood safety and clinical technology: Guidelines on Standard Operating Procedures for Microbiology. Available from: http://www.searo.who.int/en/Section10/Section17/Section53/Section482_1805.htm. [cited in 2002].   Back to cited text no. 20
21.	Nguyen MH, Peacock JE Jr, Morris AJ, Tanner DC, Nguyen ML, Snydman DR, et al. The changing face of candidemia: Emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617-23.  Back to cited text no. 21  [PUBMED]  [FULLTEXT]
22.	Baillie GS, Douglas LJ. Iron-limited biofilms of Candida albicans and their susceptibility to amphotericin B. Antimicrob Agents Chemother 1998;42:2146-9.  Back to cited text no. 22  [PUBMED]  [FULLTEXT]
23.	Lau A, Halliday C, Chen SC, Playford EG, Stanley K, Sorrell TC. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J Clin Microbiol 2010;48:811-6.  Back to cited text no. 23  [PUBMED]  [FULLTEXT]
24.	Marklein G, Josten M, Klanke U, Müller E, Horré R, Maier T, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates. J Clin Microbiol 2009;47:2912-7.  Back to cited text no. 24
25.	van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol 2010;48:900-7.  Back to cited text no. 25  [PUBMED]  [FULLTEXT]

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