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The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory
Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
www.elsevier.com/locate/diagmicrobio
Mycology
The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory Eileen Lees*, Richard C. Barton Mycology Reference Centre, General Infirmary at Leeds, Leeds LS1 3EX, UK Received 23 August 2002; accepted 19 November 2002
Abstract Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37°C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species. © 2003 Elsevier Inc. All rights reserved. Keywords: Niger seed agar; Germ tube; Candida dubliniensis
1. Introduction Candida dubliniensis is closely related to C. albicans and was first described in 1995 (Sullivan et al., 1995). Although C. albicans is commonly found in clinical specimens either as pathogen or commensal, C. dubliniensis is far less common and has mainly been isolated from the oral cavity of HIV positive patients (Sullivan et al., 1995) but occasionally from other sites (Sullivan and Coleman, 1998). It has also been isolated from specimens from various sites in non-HIV positive people. The overall incidence of this species is currently not clear and assessment of its clinical significance is difficult and will depend upon an easy method for the screening of this species in the routine microbiology laboratory. There has been a proliferation of methods in the literature for the differentiation of C. albicans and C. dubliniensis including carbohydrate assimilation profile (Salkin et al., 1998), appearance on chromagar (Schoofs et al., 1997) ability to grow at 45°C (Pinjon et al., 1998), restriction * Corresponding author. Tel.: ⫹0113-392-6787; fax: ⫹0113-3435640. E-mail address: [email protected] (E. Lees). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(02)00551-5
fragment length polymorphisms (McCullough et al., 1999), hybridization with oligonuncleotide probes (Sullivan et al., 1995), ribosomal DNA sequence (Gilfillan et al., 1998), specific polymerase chain reactions (PCR) (Kurzai et al., 1999) and mini and microsatellite PCR fingerprinting (Meyer et al., 2001). These tests are either not completely discrimatory or are unlikely to be feasible for primary screening purposes in the routine microbiology laboratory. Until the recognition of C. dubliniensis as a separate species, a positive germ tube test was considered to be a unique marker for C. albicans and is used as a diagnostic tool in mycology laboratories. It is known that C. dubliniensis may also produce a positive, though often weak, result. A further marker for C. albicans, also shared only by C. dubliniensis is the production of chlamydospores on certain media. Whereas C. albicans usually takes two or more days to produce chlamydospores in Dalmau plates, C. dubliniensis differs in that it will often produce them in greater abundance and after 24 h (Sullivan et al., 1995; Sullivan and Coleman, 1998). However this difference has not been found to be sufficiently reliable to be used to distinguish these two species (Kirkpatrick et al., 1998). Colony appearance on Niger Seed agar has been suggested as a method for the differentiation of the two species.
14
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
Staib and Morschhauser (1999) found that colonies of C. albicans grown on this medium were ‘smooth’ when examined microscopically, i.e., growth was entire with no encroachment into the agar by hyphae. In comparison, C. dubliniensis produced ‘rough’ colonies whose edges were fringed with hyphae. In 2001 Al Mosaid et al. confirmed the value of this as a cheap, rapid and effective test on culture collection isolates from around the world, with 97.7% of 130 C. dubliniensis strains growing as “rough” colonies on Niger seed (Staib) agar and none of 166 strains of C. albicans. We decided to evaluate the use of this characteristic as a routine screening test for all yeasts isolated in a clinical laboratory in order to assess its usefulness and to study the numbers and sources of C. dubliniensis isolated.
2. Materials and methods 2.1. Culture Niger Seed (Staib) agar was prepared as described by Staib and Morschha¨ user (1999) with 50g Guizotia abyssinica seed (from local pet store), 1g glucose, 1g potassium dihydrogen phosphate and 1g creatinine in 1 liter, but using 2% (rather than 1.5%) Bacto Agar. Niger seed cultures were incubated at 37°C and read at 24 and 48 h. Dalmau plates were made using Czapek Dox agar (Oxoid, Basingstoke, UK) plus 1% Tween 80 and were inoculated using a drop of a suspension of yeast cells and covered with a clean coverslip. Dalmau plates were incubated at 27°C and scored for the presence of chlamydospores and or hyphae or pseudohyphae after 24 h. 2.2. Strains tested Five C. albicans strains (NCPF3153A, NCPF3940, NCPF3939, NCPF3281, ATCC90028), the C. dubliniensis type strain, NCPF3949, 6 C. dubliniensis isolates 99.096, 90.015, 90.013, 88.029, 90.006, 89.014 (first analyzed by Schmid et al., 1992 and confirmed by molecular methods by Joly et al., 1999) and a C. dubliniensis laboratory isolate identified by API32C were analyzed initially. Subsequently all 495 yeasts isolated in the clinical laboratory or sent to the laboratory for identification over a period of three months were analyzed. 2.3. Testing procedure Fresh isolates or subcultures on Sabouraud dextrose agar less than 48 h old were used in all cases. All yeasts were screened by germ tube test (incubation in horse serum at 37°C for 1.75 h). All yeasts were inoculated onto Niger Seed agar with C. albicans 3153A and C. dubliniensis NCPF 3949 as control strains with each batch of tests. After 24 h the entire growth streak on the Niger Seed agar was scanned using ⫻100 magnification and a positive result was
indicated by the production of multiple hyphal or pseudohyphal outgrowths from the growth streak into the agar. The absence of multiple hyphal or pseudohyphal cell outgrowths was considered negative (Fig. 1). Germ tube positive, Niger seed negative isolates were considered to be C. albicans. Any yeast that was germ tube positive and positive on Niger Seed agar was identified by API 32C (BioMerieux, Basingstoke, UK) and a Dalmau plate. All germ tube negative isolates were identified using Auxacolor (Biorad, Marnes La Coquette, France) and a Dalmau plate and, if this failed to provide a clear identification, API 32C.
3. Results A total of 495 yeast isolates were tested, and 419 (84.6%) were germ tube positive and failed to produce filamentation on Niger Seed agar after 24 h and were thus considered to be C. albicans. Of the germ tube positive, Niger Seed positive isolates 4 out of 7 were later identified as C. dubliniensis (Table 1). Sixty nine isolates were germ tube negative and of these 20 produced filamentation on Niger Seed agar. Four were subsequently identified as C. dubliniensis the rest were other species, almost half of which were C. tropicalis but none were identified as C. albicans. Of the 49 germ tube negative, Niger Seed agar negative yeasts there was a single C. dubliniensis and no C. albicans. Thus among all the yeast examined a total of 9 were identified as C. dubliniensis (1.8% of total tested). In relation to the Niger Seed test the negative predictive value was 99.8% and positive predictive value 72.7%. Of the 9 isolates of C. dubliniensis, three (8.3%) were from oral sources (Table 2). and four were from vaginal sites, reflecting the large proportion of the isolates from high vaginal swab specimens. The HIV status of most of the patients was unknown.
4. Discussion The aim of this study was to find out if the Niger Seed agar test is a useful tool for the identification of C. dubliniensis. In our preliminary tests of reference isolates all eight strains of C. dubliniensis were positive by Niger Seed agar. Our approach differs from that described by Al Mosaid et al. (2001) who incubated their cultures at 30°C while in this study the cultures were incubated at 37°C. In addition the cultures were examined microscopically, allowing us to obtain a result in 24 rather than 48 or 72 h. During the routine screening, our results showed that a positive result in the test is a good indication for further identification procedures. Of those isolates positive by both germ tube and niger seed test more than half were C. dubliniensis. One out of nine C. dubliniensis strains was Niger Seed agar negative. This isolate was also germ tube negative and since further identification tests were carried out on all germ tube negative yeasts, was correctly identified. This study was based
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
15
Fig. 1. a) Candida albicans and b) C. dubliniensis growing on Niger seed agar after 24 h at 37°C. Photographed at ⫻100.
on the assumption that all germ tube positive—Niger Seed agar negative yeasts were C. albicans. In the three studies to date 11/11 (Staib and Morschhauser, 1999), 166/166 (Al Mosaid et al., 2001) and 5/5 (this study, reference isolates) of C. albicans isolates were Niger seed negative. We cannot be completely sure that some of the 419 Niger seed negative, germ tube positive isolates in this study were not C. dubliniensis. However, Al Mosaid et al., 2001 found 97.7% of 130 C. dubliniensis strains appeared as “rough” colonies,
i.e., produced filamentation on Niger seed (Staib) agar. While the method described here differs in some aspects from that of Al Mosaid et al., 2001, it is likely that the vast majority of C. dubliniensis strains will produce hyphae or pseudohyphae on this medium. All clinical isolates were screened and 1.8% of the total were C. dubliniensis. Isolation of C. dubliniensis in this study did not necessarily indicate active infection. In studies of the proportion of patients with candidosis yielding C.
16
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
Table 1 Germ tube and Niger seed results on study yeasts Germ tube (GT) and Niger seed (NS) result
Total isolates (%)
Identification
GT⫹ NS⫺ GT⫹ NS⫹ GT⫺ NS⫺
419 (84.6%)
419 C. albicans
GT⫺ NS⫹
7 (1.4%) 49 (10%)
20 (4%)
3 C. albicans 4 C. dubliniensis 1 C. dubliniensis 48 Candida species (none C. albicans) 4 C. dubliniensis 9 C. tropicalis 2 C. krusei 1 C. lusitaniae 2 C. parapsilosis
dubliniensis there is variation according to underlying condition and site of sampling. C. dubliniensis has been isolated from 25-30% of HIV⫹/AIDS patients with oral candidosis, and from 18-25% HIV⫹/AIDS patients without symptoms. In HIV negative patients C. dubliniensis is recovered from 10-14% of cases of oral candidosis and carried orally in 2-3% of people without any symptoms (Ponton et al., 2000). Our figure of 8.3% of oral Candida isolates being C. dublinensis suggests that we have probably not missed many, particularly since it was not known whether the patients concerned had candidosis or were simply colonized. In our Study 1% of vaginal Candida isolates were C. dubliniensis. In a single cohort of 110 HIV negative patients with candida vaginitis C. dubliniensis was isolated in 2.7% of cases (Ponton et al., 2000). Preparation of the Niger seed agar is the most labor intensive aspect of the study as it involves grinding and filtering the seeds. However, once made and autoclaved, the agar can be kept for many months before being melted and poured and the cost of ingredients is low. Recently the use of hypotonic broth culture has been suggested as a method for screening (Alvesa et al., 2002) with similar advantages. Following this trial the test has been used as a routine aid in this laboratory, as a quick and simple way to screen selected germ tube positive yeasts, including isolates giving Table 2 Site of isolation of study yeasts Site
No. specimens
Vaginal Blood culture Oral Respiratory (sputum/Brochoalveolar lavage) Other1
339 19 36 29
4 (1.1) 0 3 (8.3) 0
72
22 (2.8)
1 2
No. C. dubliniensis (%)
Including, urine, nail, drain fluids, wound swabs etc. One each from abdominal wound and peritoneal fluid.
a weak germ tube positive result, germ tube positive isolates from HIV ⫹ ve and AIDS patients and any isolates from oral specimens. Testing on Niger seed agar, results in very little delay and involves little expense. If isolates are found to be Niger seed test positive, further identification is sought in all cases by API32C and Dalmau plate. Isolates that are strongly suspected for any reason of being C. dubliniensis are tested directly by API32C in addition to the Niger seed test. This study did not set out to look for a totally sensitive test for C. dubliniensis but to find a rapid, inexpensive and easy screening tool to detect most of the isolates of this species in a clinical microbiology laboratory. This method will permit investigation of the extent of involvement of C. dubliniensis in the various forms of candidosis and thus enable the analysis of outcomes in infected patients as compared to C. albicans.
Acknowledgments We thank the staff of the Mycology Reference Centre for their work and Dr Ruth Ashbee for critical reading of the manuscript.
References Al Mosaid, A., Sullivan, D., Salkin, I. F., Shanley, D., & Sullivan, D. C. (2001). Differentiation of Candida dubliniensis from Candida albicans on Staib agar and caffeic acid-ferric citrate agar. J Clin Microbiol, 39, 323–327. Alvesa, S. H., Milan, E. P., Sant’Ana, P de L., Oliveira, L. O., Santurioa, J. M., & Colombo, A. L. (2002). Hypertonic Sabouraud broth as a simple and powerful test for Candida dubliniensis screening. Diag Microbiol Infect Dis, 43, 85– 86. Gilfillan, G. D., Sullivan, D. J., Haynes, K., Parkinson, T., Coleman, D. C., & Gow, N. A. R. (1998). Candida dubliniensis: phylogeny and putative virulence factors. Microbiology, 144, 829 – 838. Joly, S., Pujol, C., Rysz, M., Vargas, K., & Soll, D. R. (1999). Development and characterization of complex DNA fingerprinting probes for the infectious yeast Candida dubliniensis. J Clin Microbiol, 37, 1035– 1044. Kirkpatrick, W. R., Revankar, S. G., McAtee, R. K., Lopez-Ribot, J. L., Fothergill, A. W., McCarthy, D. I., Sanche, S. E., Cantu, R. A., Rinaldi, M. G., & Patterson, T. F. (1998). Dection 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. Kurzai, O., Heinz, W. J., Sullivan, D. J., Coleman, D. C., Frosch, M., & Mu¨ hlschlegel, F. A. (1999). Rapid PCR test for discriminating between Candida albicans and Candida dubliniensis isolates using primers derived from the pH-regulated PHR1 and PHR2 genes of C. albicans. J Clin Microbiol, 37, 1587–1590. McCullough, M. J., Clemons, K. V., & Stevens, D. A. (1999). Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J Clin Microbiol, 37, 417– 421. Meyer, W., Maszewska, K., & Sorrell, T. C. (2001). PCR fingerprinting: a convenient molecular tool to distinguish between Candida dubliniensis and Candida albicans. Med Mycol, 39, 185–193.
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17 Pinjon, E., Sullivan, D., Salkin, I., Shanley, D., & Coleman, D. (1998). Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J Clin Microbiol, 36, 2093–2095. Ponton, J., Ruchel, R., Clemons, K. V., Coleman, D. C., Grillot, R., Guarro, J., Aldebert, D., Ambroise-Thomas, P., Cano, J., CarilloMunoz, A. J., Gene, J., Pinel, C., Stevens, D. A., & Sullivan, D. J. (2000). Emerging pathogens. Med Mycol, 38 (Suppl. 1), 225–236. Salkin, I. F., Pruitt, W. R., Padhye, A. A., Sullivan, D., Coleman, D., & Pincus, D. H. (1998). Distinctive carbohydrate assimilation profiles used to identify the first clinical isolates of Candida dubliniensis recovered from the United States. J Clin Microbiol, 36, 1467. Schmid, J., Odds, F. C., Wiselka, M. J., Nicholson, K. G., & Soll, D. R. (1992). Genetic similarity and maintenance of Candida albicans strains from a group of AIDS patients, demonstrated by DNA fingerprinting. J Clin Microbiol, 30, 935–941.
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Schoofs, A., Odds, F. C., Colebunders, R., Ieven, M., & Goosens, H. (1997). Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis, 16, 296 –300. Staib, P., & Morschhauser, J. (1999). Chlamydospore formation on Staib agar as a species specific characteristic of C.dubliniensis. Mycoses, 42, 521–524. Sullivan, D. J., & Coleman, D. C. (1998). Candida dubliniensis: characteristics and identification. J Clin Microbiol, 36, 329 –332. Sullivan, D. J., Westerneng, T. J., Haynes, K. A., Bennet, D. E., & Coleman, D. C. (1995). Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candiosis in HIV-infected individuals. Microbiology, 141, 1507– 1521.
www.elsevier.com/locate/diagmicrobio
Mycology
The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory Eileen Lees*, Richard C. Barton Mycology Reference Centre, General Infirmary at Leeds, Leeds LS1 3EX, UK Received 23 August 2002; accepted 19 November 2002
Abstract Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37°C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species. © 2003 Elsevier Inc. All rights reserved. Keywords: Niger seed agar; Germ tube; Candida dubliniensis
1. Introduction Candida dubliniensis is closely related to C. albicans and was first described in 1995 (Sullivan et al., 1995). Although C. albicans is commonly found in clinical specimens either as pathogen or commensal, C. dubliniensis is far less common and has mainly been isolated from the oral cavity of HIV positive patients (Sullivan et al., 1995) but occasionally from other sites (Sullivan and Coleman, 1998). It has also been isolated from specimens from various sites in non-HIV positive people. The overall incidence of this species is currently not clear and assessment of its clinical significance is difficult and will depend upon an easy method for the screening of this species in the routine microbiology laboratory. There has been a proliferation of methods in the literature for the differentiation of C. albicans and C. dubliniensis including carbohydrate assimilation profile (Salkin et al., 1998), appearance on chromagar (Schoofs et al., 1997) ability to grow at 45°C (Pinjon et al., 1998), restriction * Corresponding author. Tel.: ⫹0113-392-6787; fax: ⫹0113-3435640. E-mail address: [email protected] (E. Lees). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(02)00551-5
fragment length polymorphisms (McCullough et al., 1999), hybridization with oligonuncleotide probes (Sullivan et al., 1995), ribosomal DNA sequence (Gilfillan et al., 1998), specific polymerase chain reactions (PCR) (Kurzai et al., 1999) and mini and microsatellite PCR fingerprinting (Meyer et al., 2001). These tests are either not completely discrimatory or are unlikely to be feasible for primary screening purposes in the routine microbiology laboratory. Until the recognition of C. dubliniensis as a separate species, a positive germ tube test was considered to be a unique marker for C. albicans and is used as a diagnostic tool in mycology laboratories. It is known that C. dubliniensis may also produce a positive, though often weak, result. A further marker for C. albicans, also shared only by C. dubliniensis is the production of chlamydospores on certain media. Whereas C. albicans usually takes two or more days to produce chlamydospores in Dalmau plates, C. dubliniensis differs in that it will often produce them in greater abundance and after 24 h (Sullivan et al., 1995; Sullivan and Coleman, 1998). However this difference has not been found to be sufficiently reliable to be used to distinguish these two species (Kirkpatrick et al., 1998). Colony appearance on Niger Seed agar has been suggested as a method for the differentiation of the two species.
14
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
Staib and Morschhauser (1999) found that colonies of C. albicans grown on this medium were ‘smooth’ when examined microscopically, i.e., growth was entire with no encroachment into the agar by hyphae. In comparison, C. dubliniensis produced ‘rough’ colonies whose edges were fringed with hyphae. In 2001 Al Mosaid et al. confirmed the value of this as a cheap, rapid and effective test on culture collection isolates from around the world, with 97.7% of 130 C. dubliniensis strains growing as “rough” colonies on Niger seed (Staib) agar and none of 166 strains of C. albicans. We decided to evaluate the use of this characteristic as a routine screening test for all yeasts isolated in a clinical laboratory in order to assess its usefulness and to study the numbers and sources of C. dubliniensis isolated.
2. Materials and methods 2.1. Culture Niger Seed (Staib) agar was prepared as described by Staib and Morschha¨ user (1999) with 50g Guizotia abyssinica seed (from local pet store), 1g glucose, 1g potassium dihydrogen phosphate and 1g creatinine in 1 liter, but using 2% (rather than 1.5%) Bacto Agar. Niger seed cultures were incubated at 37°C and read at 24 and 48 h. Dalmau plates were made using Czapek Dox agar (Oxoid, Basingstoke, UK) plus 1% Tween 80 and were inoculated using a drop of a suspension of yeast cells and covered with a clean coverslip. Dalmau plates were incubated at 27°C and scored for the presence of chlamydospores and or hyphae or pseudohyphae after 24 h. 2.2. Strains tested Five C. albicans strains (NCPF3153A, NCPF3940, NCPF3939, NCPF3281, ATCC90028), the C. dubliniensis type strain, NCPF3949, 6 C. dubliniensis isolates 99.096, 90.015, 90.013, 88.029, 90.006, 89.014 (first analyzed by Schmid et al., 1992 and confirmed by molecular methods by Joly et al., 1999) and a C. dubliniensis laboratory isolate identified by API32C were analyzed initially. Subsequently all 495 yeasts isolated in the clinical laboratory or sent to the laboratory for identification over a period of three months were analyzed. 2.3. Testing procedure Fresh isolates or subcultures on Sabouraud dextrose agar less than 48 h old were used in all cases. All yeasts were screened by germ tube test (incubation in horse serum at 37°C for 1.75 h). All yeasts were inoculated onto Niger Seed agar with C. albicans 3153A and C. dubliniensis NCPF 3949 as control strains with each batch of tests. After 24 h the entire growth streak on the Niger Seed agar was scanned using ⫻100 magnification and a positive result was
indicated by the production of multiple hyphal or pseudohyphal outgrowths from the growth streak into the agar. The absence of multiple hyphal or pseudohyphal cell outgrowths was considered negative (Fig. 1). Germ tube positive, Niger seed negative isolates were considered to be C. albicans. Any yeast that was germ tube positive and positive on Niger Seed agar was identified by API 32C (BioMerieux, Basingstoke, UK) and a Dalmau plate. All germ tube negative isolates were identified using Auxacolor (Biorad, Marnes La Coquette, France) and a Dalmau plate and, if this failed to provide a clear identification, API 32C.
3. Results A total of 495 yeast isolates were tested, and 419 (84.6%) were germ tube positive and failed to produce filamentation on Niger Seed agar after 24 h and were thus considered to be C. albicans. Of the germ tube positive, Niger Seed positive isolates 4 out of 7 were later identified as C. dubliniensis (Table 1). Sixty nine isolates were germ tube negative and of these 20 produced filamentation on Niger Seed agar. Four were subsequently identified as C. dubliniensis the rest were other species, almost half of which were C. tropicalis but none were identified as C. albicans. Of the 49 germ tube negative, Niger Seed agar negative yeasts there was a single C. dubliniensis and no C. albicans. Thus among all the yeast examined a total of 9 were identified as C. dubliniensis (1.8% of total tested). In relation to the Niger Seed test the negative predictive value was 99.8% and positive predictive value 72.7%. Of the 9 isolates of C. dubliniensis, three (8.3%) were from oral sources (Table 2). and four were from vaginal sites, reflecting the large proportion of the isolates from high vaginal swab specimens. The HIV status of most of the patients was unknown.
4. Discussion The aim of this study was to find out if the Niger Seed agar test is a useful tool for the identification of C. dubliniensis. In our preliminary tests of reference isolates all eight strains of C. dubliniensis were positive by Niger Seed agar. Our approach differs from that described by Al Mosaid et al. (2001) who incubated their cultures at 30°C while in this study the cultures were incubated at 37°C. In addition the cultures were examined microscopically, allowing us to obtain a result in 24 rather than 48 or 72 h. During the routine screening, our results showed that a positive result in the test is a good indication for further identification procedures. Of those isolates positive by both germ tube and niger seed test more than half were C. dubliniensis. One out of nine C. dubliniensis strains was Niger Seed agar negative. This isolate was also germ tube negative and since further identification tests were carried out on all germ tube negative yeasts, was correctly identified. This study was based
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
15
Fig. 1. a) Candida albicans and b) C. dubliniensis growing on Niger seed agar after 24 h at 37°C. Photographed at ⫻100.
on the assumption that all germ tube positive—Niger Seed agar negative yeasts were C. albicans. In the three studies to date 11/11 (Staib and Morschhauser, 1999), 166/166 (Al Mosaid et al., 2001) and 5/5 (this study, reference isolates) of C. albicans isolates were Niger seed negative. We cannot be completely sure that some of the 419 Niger seed negative, germ tube positive isolates in this study were not C. dubliniensis. However, Al Mosaid et al., 2001 found 97.7% of 130 C. dubliniensis strains appeared as “rough” colonies,
i.e., produced filamentation on Niger seed (Staib) agar. While the method described here differs in some aspects from that of Al Mosaid et al., 2001, it is likely that the vast majority of C. dubliniensis strains will produce hyphae or pseudohyphae on this medium. All clinical isolates were screened and 1.8% of the total were C. dubliniensis. Isolation of C. dubliniensis in this study did not necessarily indicate active infection. In studies of the proportion of patients with candidosis yielding C.
16
E. Lees, R.C. Barton / Diagnostic Microbiology and Infectious Disease 46 (2003) 13–17
Table 1 Germ tube and Niger seed results on study yeasts Germ tube (GT) and Niger seed (NS) result
Total isolates (%)
Identification
GT⫹ NS⫺ GT⫹ NS⫹ GT⫺ NS⫺
419 (84.6%)
419 C. albicans
GT⫺ NS⫹
7 (1.4%) 49 (10%)
20 (4%)
3 C. albicans 4 C. dubliniensis 1 C. dubliniensis 48 Candida species (none C. albicans) 4 C. dubliniensis 9 C. tropicalis 2 C. krusei 1 C. lusitaniae 2 C. parapsilosis
dubliniensis there is variation according to underlying condition and site of sampling. C. dubliniensis has been isolated from 25-30% of HIV⫹/AIDS patients with oral candidosis, and from 18-25% HIV⫹/AIDS patients without symptoms. In HIV negative patients C. dubliniensis is recovered from 10-14% of cases of oral candidosis and carried orally in 2-3% of people without any symptoms (Ponton et al., 2000). Our figure of 8.3% of oral Candida isolates being C. dublinensis suggests that we have probably not missed many, particularly since it was not known whether the patients concerned had candidosis or were simply colonized. In our Study 1% of vaginal Candida isolates were C. dubliniensis. In a single cohort of 110 HIV negative patients with candida vaginitis C. dubliniensis was isolated in 2.7% of cases (Ponton et al., 2000). Preparation of the Niger seed agar is the most labor intensive aspect of the study as it involves grinding and filtering the seeds. However, once made and autoclaved, the agar can be kept for many months before being melted and poured and the cost of ingredients is low. Recently the use of hypotonic broth culture has been suggested as a method for screening (Alvesa et al., 2002) with similar advantages. Following this trial the test has been used as a routine aid in this laboratory, as a quick and simple way to screen selected germ tube positive yeasts, including isolates giving Table 2 Site of isolation of study yeasts Site
No. specimens
Vaginal Blood culture Oral Respiratory (sputum/Brochoalveolar lavage) Other1
339 19 36 29
4 (1.1) 0 3 (8.3) 0
72
22 (2.8)
1 2
No. C. dubliniensis (%)
Including, urine, nail, drain fluids, wound swabs etc. One each from abdominal wound and peritoneal fluid.
a weak germ tube positive result, germ tube positive isolates from HIV ⫹ ve and AIDS patients and any isolates from oral specimens. Testing on Niger seed agar, results in very little delay and involves little expense. If isolates are found to be Niger seed test positive, further identification is sought in all cases by API32C and Dalmau plate. Isolates that are strongly suspected for any reason of being C. dubliniensis are tested directly by API32C in addition to the Niger seed test. This study did not set out to look for a totally sensitive test for C. dubliniensis but to find a rapid, inexpensive and easy screening tool to detect most of the isolates of this species in a clinical microbiology laboratory. This method will permit investigation of the extent of involvement of C. dubliniensis in the various forms of candidosis and thus enable the analysis of outcomes in infected patients as compared to C. albicans.
Acknowledgments We thank the staff of the Mycology Reference Centre for their work and Dr Ruth Ashbee for critical reading of the manuscript.
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