الخلاصـة: المبيضَّات الدَّبْلينيَّة Candida dubliniensis نوعٌ من الفطريات الخميرية المستجدَّة يمكن استفراده بصفة أساسية من المرضى الـمُنْتَقَصِي المناعة. وبالنظر إلى أن الاختبارات التي تعتمد فحص الجزيئات لم تعد ملائمة في الوقت الحاضر للاستخدام في المختبرات التشخيصية، فقد قام الباحثان بمقارنة طائفة من الطرق التي تعتمد على النمط الظاهري phenotype للتفريق بين المبيضَّات البيضاء والمبيضَّات الدَّبْلينيَّة. وقد شملت هذه الاختبارات لون المستعمرات على مستنبت الكروماغار للمبيضَّات، وملاحظة النمو في درجتَيْ حرارة 37 مئوية و45 مئوية، وكذلك القدرة على إنتاج أنابيب إنتاشيَّة وأبواغ متدثرة، إضافة إلى الاختبار بجهاز "أوكساكولور". وقد تضمنت الكائنات الدقيقة التي أمكن استفرادُها 105 مستفرَدَةً سَبَقَ تصنيفُها على أنها مبيضَّات بيضاء و10 ذَرَارٍ مرجعية من المبيضَّات البيضاء وذُرِّيَّتَيْن مرجعيَّتين من المبيضَّات الدَّبْلينيَّة، و102 مستفرَدَةً سريرية جديدة صُنفت على أنها مبيضَّات بيضاء. ولم يكن أي من هذه الاختبارات على حِدَة كافياً للتوصُّل إلى نتيجة يُعتمد عليها، ولكنَّ إشراك الاختبارات الثلاثة يمكن أن يكون ملائماً لأغراض الاستعراف الظنِّي للمبيضَّات الدَّبْلينيَّة.

ABSTRACT: Candida dubliniensis is an emerging yeast pathogen isolated mainly from immunocompromised patients. As molecular tests are currently unsuitable for use in routine diagnostic laboratories, we compared a variety of phenotypic techniques for differentiating C. albicans and C. dubliniensis. The tests included: colony colour on CHROMagar Candida medium; growth at 37 °C and 45 °C; ability to produce germ tubes and chlamydospores; and the Auxacolor® system. The organisms included 105 isolates previously identified as C. albicans, 10 reference strains of C. albicans, 2 reference strains of C. dubliniensis and 102 fresh clinical isolates identified as C. albicans. None of the tests alone was satisfactory but a combination of 3 tests may be suitable for presumptive identification of C. dubliniensis.

Identification de Candida dubliniensis dans un laboratoire de microbiologie diagnostique

RÉSUMÉ: Candida dubliniensis est un nouvelle levure pathogène isolée principalement chez des patients immunodéprimés. Les tests moléculaires ne convenant pas actuellement pour être utilisés dans les laboratoires de diagnostic de routine, nous avons comparé diverses techniques phénotypiques pour différencier C. albicans et C. dubliniensis, dont : la couleur des colonies sur milieu CHROMagarTM Candida ; la culture à 37 °C et 45 °C ; la capacité de produire des tubes de germes et des chlamydospores ; et le système Auxacolor®. Les micro-organismes comprenaient 105 isolats identifiés auparavant comme C. albicans, 10 souches de référence de C. albicans, 2 souches de référence de C. dubliniensis et 102 isolats cliniques frais identifiés comme C. albicans. Aucun des tests seul n’était satisfaisant mais une association de trois tests peut convenir pour une identification présomptive de C. dubliniensis..

¹Department of Microbiology, Omar Medical Laboratories, Amman, Jordan (Correspondence to O.M. Momani: momaniomar@hotmail.com). ¹King Hussein Medical Centre, Amman, Jordan. Received: 09/10/02; accepted: 14/09/03

 

Introduction

Over the past 10 years there have been frequent reports describing “atypical” isolates of Candida albicans [1–5]. In 1995, Sullivan et al. suggested that these isolates comprised a new species and named it C. dubliniensis, after Dublin, the capital city of Ireland, where the new species was first identified [2,6].

The organism has been recovered from the oral cavity in healthy people, HIV-     infected patients and AIDS patients [7,8]. The greatest concern about C. dubliniensis is the potential for development of antifungal drug resistance, especially in HIV-     infected patients [9,10].

Candida dubliniensis shares many phenotypic similarities with C. albicans, resulting in significant problems in differentiation between the 2 species. Currently, there is a real need for a rapid and simple test for use in routine clinical laboratories to distinguish isolates of C. dubliniensis and C. albicans. Definitive identification of C. dubliniensis currently relies on molecular methods; however, these have high running costs and require well-trained laboratory personnel, who might not be available in routine diagnostic laboratories.

Several methods have been proposed for differentiating between the strains. The use of CHROMagar chromogenic culture medium has been proposed as a means of recognizing colonies of C. dubliniensis [11,12]. Several researchers have conducted carbohydrate assimilation studies [2,13, 14]; some kits contain individual tests that can be utilized for C. dubliniensis. Pinjon et al. suggested culture at 45 °C, concluding that C. dubliniensis is unable to grow at such high temperatures while C. albicans grows well [5]. A recent study however showed that some C. albicans strains are unable to grow at 45 °C [11].

The aim of this study was to assess the usefulness of different phenotypic techniques for differentiating between isolates of the 2 species, C. albicans and C. dubliniensis.

Methods

Organisms used

Two C. dubliniensis reference strains were used in this study. One strain (coded NCPF3108) was originally identified as C. stellatoidea and deposited in the British collection of pathogenic fungi; it has since been re-identified as C. dubliniensis [6]. The other strain (the Bristol strain) was supplied by Dr Colin Campbell from the Bristol Mycology Reference Laboratory at the Public Health Laboratory Service, Bristol, United Kingdom. The 2 strains were identified by molecular methods.

Ten C. albicans reference strains were used: 324/94RA, WK1, 122/94Rgl, 684/93, 455/94rgh, 455/94sm, ATCC 3516, Y01.544, LSHTM3153 and 91L.

Stock Candida spp. strains supplied by the mycology laboratory of the Department of Microbiolgy, University of Wales College of Medicine were used (n = 105). These had previously been identified as C. albicans using the germ tube test, chlamydospore formation and Auxacolor^ yeast identification system.

A total of 158 clinical specimens were studied, comprising 91 urine samples, 66 genital samples (high vaginal, penile and vulvo-vaginal swabs) and 1 blood culture sample, resulting in the identification of 102 isolates of C. albicans by the germ tube and Auxacolor^ tests.

Culture media

CHROMagar Candida medium (CHROMagar Microbiology, Paris, France) was prepared according to the manufacturer’s instructions.

Sabouraud agar + chloramphenicol and corn meal agar were supplied by the Department of Microbiolgy, University of Wales College of Medicine. Sabouraud agar + chloramphenicol was supplied as ready-to-use agar plates in packs of 15 plates. Corn meal agar was supplied ready made as 100 mL prepared solid medium in a flask. The agar was melted and distributed into 5 sterile Petri dishes.

The germ tube test for the production of germ tubes was done using horse serum (TCS Biologicals Ltd, Buckingham, UK).

Organisms were cultured on CHROM-agar Candida medium and Sabouraud agar + chloramphenicol for 48 hrs at 37 °C and 45 °C to determine the ability to grow at both temperatures and to study colony appearance and colour. To study the formation of chlamydospores, cultures on corn meal agar were incubated at room temperature. Identification was carried out using the Auxacolor^ system (Sanofi Diagnostics Pasteur, Marnes La Coquette, France). The Auxacolor^ test procedures were done in accordance with the manufacturer’s instructions.

Results

Six of the 105 stock strains of C. albicans failed to grow on both the Sabouraud and the CHROMagar media and 1 showed mould contamination. These were excluded from the study. The remaining 98 grew equally well on both media.

On CHROMagar Candida medium, all 10 references strains of C. albicans grew well, forming medium-sized, 3–5 mm smooth, entire colonies. The colony colour ranged from light green to green, most often with lighter edges, sometimes with the intense colour at the edge of the colony and sometimes in the centre. The 2 C. dubliniensis reference strains formed colonies similar to those of C. albicans; 1 was light green and the other dark green.

The conventional method using the germ tube test and chlamydospore formation identified 9 of the 10 C. albicans reference strains, and misidentified the C. dubliniensis strains as C. albicans; these were also misidentified by the Auxacolor^ system. The Auxacolor^ system correctly identified the 1 strain of C. albicans that was germ-tube negative.

Of the 219 cultures investigated, all remaining 212 which were viable grew well at 37 °C on both CHROMagar and Sabouraud agar + chloramphenicol. Growth was consistent at 48 hours.

Table 1 shows a summary of colony colours and growth at 45 °C of the strains investigated. None of the C. albicans stock culture strains produced a deep green colour on CHROMagar but 15 clinical isolates as well as the Bristol strain of C. dubliniensis produced colonies of a deep bluish-green colour. The germ tube test was positive for these strains, as was chlamydospore formation. The deep green clinical isolates were identified by the    Auxacolor^ system as C. albicans with biocodes of 7145207 or 7143207. The Bristol reference strain had a biocode of 7141207 and the NCPF3108 reference strain had a biocode of 7143207.

All reference strains of C. albicans grew at 45 °C. The 2 C. dubliniensis strains did not grow at all at 45 °C on either CHROMagar or Sabouraud agar + chloramphenicol. Among the stock culture strains, only 2 failed to grow at 45 °C. Among the clinical isolates, 1 from a urine sample and 1 from a genital sample did not grow at this temperature.

Specimens that did not grow at 45 °C and/or showed dark green colonies on CHROMagar and showed a biocode of 7143207 or 714107 were re-identified as C. dubliniensis.

Discussion

Over the past 2 decades, there has been a rapid growth in clinical microbiology technology. In the past, test results were available only after several days owing to the labour-intensive methods. With the emergence of new pathogens, especially the drug-resistant Candida spp., it has become necessary for laboratories to seek more efficient and cost-effective methods of identification. Therefore, it is essential that the available diagnostic tools be constantly modified to keep abreast of the ever- changing spectrum of pathogens.

A new emerging yeast pathogen, C. dubliniensis, was the focus of this study. It is closely related to C. albicans. Microbiological information about C. dubliniensis shows similarities with C. albicans; using the currently available conventional methods it is difficult to discriminate between them. The colony appearance on CHROM-agar Candida medium makes it extremely useful for rapid presumptive identification of the most common Candida spp. As reported originally by Odds and Bernaert, the green colour is unique to C. albicans [15]. Casal described the deep green colour as characteristic of C. zeylanoides, a species not mentioned in the study of Odds and Bernaert [16]. In our study, C. dubliniensis could show the same colour as C. albicans. However, the colour was less intense in older cultures and in freshly cultured clinical specimens it appeared dark green. Sullivan and Coleman described this pheno- menon as a way of differentiating between the 2 species on primary culture. In our study, 2 of the stock cultures and 1 C. dubliniensis reference strain appeared light green and failed to grow at 45 °C while 2 of the fresh clinical isolates and the Bristol reference strain (all dark green) failed to grow at 45 °C. Thirteen C. albicans strains appeared deep green on primary isolation. These findings suggest that CHROMagar medium alone is not suitable for discriminating between C. albicans and C. dubliniensis on primary isolation.

Pinjon et al. reported that C. dubliniensis could be discriminated from C. albicans by the ability of the latter to grow at 45 °C [5]. This test is unsatisfactory because some C. albicans strains are unable to grow at this elevated temperature and this cannot, therefore, be used as a discriminatory test [11]. The results obtained in our study are consistent with Pinjon’s criterion, as the 2 reference strains of C. dubliniensis failed to grow at 45 °C. Two stock culture strains and 2 clinical isolates failed to grow at this temperature, although all 10 C. albicans reference strains did grow. Those strains are considered suspect for C. dubliniensis.

Gales et al. studied the use of xylose and α-methyl-glucose as determined with the API 20C AUX and Vitek YBC systems for the identification of C. dubliniensis [17], The Auxacolor^ system used in this study contained xylose. The inability of C. dubliniensis to utilize xylose is reflected by specific biocodes.

Conclusion

None of the tests alone—CHROMagar Candida media, growth at 45 °C or Auxacolor^—was a satisfactory discriminatory test. A combination of the 3 tests used in this study may be a good tool for presumptive identification of C. dubliniensis. The 4 strains (2 stock culture strains and 2 clinical isolates) that appeared to be presumptive of C. dubliniensis need to be confirmed by further testing. Recognition of C. dubliniensis provides valuable information regarding its epidemiology to help establish its clinical significance.

Acknowledgements

This work was done at the Department of Microbiology, University of Wales College of Medicine, United Kingdom, as a part of an MSc degree project. This study was sponsored by the Royal Jordanian Medical Services and supervised by Dr Rosemary Barnes. Special thanks go to Professor Sheikh Mahjoub for revising this manuscript.

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