Use of a Commercial Reagent Leads to Reduced Germ Tube Production by Candida dubliniensis
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微生物临床杂志 2005年第5期
Microbiology Division, Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-7093
ABSTRACT
The goal of this study was to determine the factor(s) explaining our inability to detect Candida dubliniensis. When germ tube-positive yeasts were tested for C. dubliniensis, no C. dubliniensis was detected; however, 58 C. dubliniensis strains were detected when germ tube-negative Candida albicans strains were tested further. Since all 58 C. dubliniensis strains detected were germ tube negative, these data implied that false-negative germ tube tests occurred with germ tube solution (GTS; Remel, Lenexa, KS). All 41 known C. dubliniensis strains tested were negative with GTS, whereas 40 were positive with rabbit serum (RS; Sigma-Aldrich, St. Louis, MO). Results for C. albicans were equivalent in GTS and RS. In conclusion, GTS cannot be used for the detection of C. dubliniensis, and switching from yeast to hyphae in C. dubliniensis is more restricted than in C. albicans.
TEXT
Candida dubliniensis is a relatively new species resembling Candida albicans that was first detected and characterized by molecular genetic assays in 1995 (16). This species is worldwide and is associated with oropharyngeal candidiasis or colonization in human immunodeficiency virus-positive individuals more than in normal individuals. Like C. albicans, it can develop fluconazole resistance during the treatment of oropharyngeal infections in human immunodeficiency virus-positive individuals. More-serious infections (candidemia) can also be caused by C. dubliniensis, but this occurs less often than with C. albicans. It had not been recognized as a separate species earlier because this species shares important phenotypic characteristics with C. albicans. Both species produce germ tubes and chlamydospores.
Algorithms have been designed to identify C. dubliniensis recovered from clinical specimens. A commonly used algorithm is to screen all germ tube-positive isolates for growth/no growth at 45°C; C. albicans grows, whereas C. dubliniensis does not. Other phenotypic assays used for screening or confirming the identification of C. dubliniensis include phenotypic assays based on growth, colony morphology/color, chlamydospore production, and carbohydrate assimilation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17). Molecular assays have been shown to be important in detecting this species, but such tests may not be possible in many clinical laboratories at the present time.
C. dubliniensis was not detected in clinical specimens in our mycology laboratory when germ tube-positive isolates (over 600) were tested. The purpose of this study was to identify the factor(s) responsible for our lack of detection of C. dubliniensis from clinical specimens.
Germ tube testing was performed with germ tube solution (GTS; Remel, Lenexa, KS). Yeast cells were inoculated into 0.5 ml of GTS and incubated in a 37°C heat block for 2.5 to 3 h, and aliquots were removed for microscopic examination. Temperature studies were performed to screen for C. dubliniensis by assessing growth at 45°C after 48 h of incubation (C. albicans grows, C. dubliniensis should not). In subsequent studies, xylose and methyl-D-glucose (Mdg) assimilation was used to screen for C. dubliniensis, since C. albicans is positive for them at 89% and 85%, respectively, and negative for C. dubliniensis (API-20C database). Xylose assimilation agar contained 3.0 g xylose, 3.4 g yeast nitrogen base without glucose, 1 ml of a 1.6% bromcreosol purple, and 16 g of purified agar per liter. Mdg agar was the same except that 3.0 g methyl-D-glucoside replaced the xylose. Three milliliters was dispensed into tubes 13- by 100-mm and stored at 4°C until used. Both media were inoculated and incubated at 30°C for 48 h. A color change from a purple to yellow due to a lowering of the pH was considered positive assimilation. In addition, chlamydospore production on casein agar (Remel, Lenexa, KS), was also used to screen for C. dubliniensis since "superchlamydospore" production is more indicative of C. dubliniensis than C. albicans (11). Slants were melted and transferred to petri dishes; isolates were cut into the agar, and the plates were incubated for 44 to 48 h at 25°C. Slides were made and examined microscopically for assessment of chlamydospore production compared to that of C. dubliniensis and C. albicans controls. Definitive identification of C. dubliniensis was made with the API-20C AUX kit (bioMerieux, Inc., Durham, NC), used according to the manufacturer's instructions.
Initially, our protocol was that all germ tube-positive yeast isolates were screened for growth/no growth at 45°C for up to 48 h; C. dubliniensis was not detected among the >600 tested. With our second protocol, both germ tube-positive and germ tube-negative C. albicans strains (germ tube-negative C. albicans identification was established by carbohydrate fermentation and chlamydospore production on corn meal agar with caffeic acid) were evaluated further. Isolates that produced superchlamydospores on casein agar and did not assimilate xylose or Mdg were considered putative C. dubliniensis isolates. A subset of 58 putative isolates was confirmed as C. dubliniensis by the API-20C AUX test. Interestingly, all 58 C. dubliniensis isolates were germ tube negative initially and when they were retested. The data implied that our germ tube results for C. dubliniensis were most likely false negatives.
To test the hypothesis that C. dubliniensis was not detected because of false-negative germ tube production with GTS, we performed a study comparing levels of germ tube production in GTS and RS (Sigma-Aldrich, St. Louis, MO). We tested a panel of 41 isolates of C. dubliniensis, including control strains, an ATCC strain, MYA 179, a Johns Hopkins control isolate, CD-12, seven well-characterized strains kindly provided by M. Jabra-Rizk of the University of Maryland, and 32 of the 58 isolates recovered from clinical specimens (Table 1). The 41 C. dubliniensis isolates and 10 C. albicans isolates (randomly included) were coded to leave the readers unaware of their identities. Germ tube production was determined independently by two highly trained individuals. No germ tubes were produced by the 41 C. dubliniensis isolates in GTS within 3 h of incubation; three isolates (7%) had very few and short germ tubes seen after >3 h of incubation. In contrast, 40/41 (98%) were clearly positive using RS within 3 h. Levels of germ tube production by C. albicans were equivalent on GTS and RS when 50 clinical isolates were compared. This lack of germ tube production has been confirmed with more than five different lots of the GTS.
These germ tube results explain our inability to identify C. dubliniensis recovered from clinical specimens. Since the germ tube test was the initial screening test, germ tube-negative samples (false negatives) were not worked up further. These data and the literature (15) also support the possibility that there are differences in levels of induction of germ tube production between C. albicans and C. dubliniensis. This switch from yeast to hyphal growth seems to be under more control in C. dubliniensis than in C. albicans. Since the production of hyphae is involved in virulence, this restriction of hyphal formation might contribute to the reduced virulence of C. dubliniensis compared to that of C. albicans.
ACKNOWLEDGMENTS
We thank the staff of the Clinical Mycology Laboratory for their cooperation and M. Jabra-Rizk, University of Maryland, for kindly providing well-characterized strains of C. dubliniensis.
REFERENCES
Al Mosaid, A., D. Sullivan, I. F. Salkin, D. Shanley, and D. C. Coleman. 2001. Differentiation of Candida dubliniensis from Candida albicans on staib agar and caffeic acid-ferric citrate agar. J. Clin. Microbiol. 39:323-327.
Al Mosaid, A., D. J. Sullivan, and D. C. Coleman. 2003. Differentiation of Candida dubliniensis from Candida albicans on Pal's agar. J. Clin. Microbiol. 41:4787-4789.
Arikan, S., O. Darka, G. Hascelik, and A. Gunalp. 2003. Identification of Candida dubliniensis strains using heat tolerance tests, morphological characteristics and molecular methods. Mikrobiyol. Bul. 37:49-57. (Abstract in English.)
Fotedar, R., and S. S. Al Hedaithy. 2004. Prevalence of Candida dubliniensis among germ tube positive yeasts recovered from the respiratory specimens in HIV-negative patients. Mycoses 47:150-155.
Gales, A. C., M. A. Pfaller, A. K. Houston, S. Joly, D. J. Sullivan, D. C. Coleman, and D. R. Soll. 1999. Identification of Candida dubliniensis based on temperature and utilization of xylose and alpha-methyl-D-glucoside as determined with the API 20C AUX and Vitek YBC systems. J. Clin. Microbiol. 37:3804-3808.
Kim, D., W. S. Shin, K. H. Lee, K. Kim, J. Y. Park, and C. M. Kob. 2002. Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 degrees C. Yeast 19:957-962.
Kirkpatrick, W. R., S. G. Revankar, R. K. Mcatee, J. L. Lopez-Ribot, A. W. Fothergill, D. I. McCarthy, S. E. Sanche, R. A. Cantu, M. G. Rinaldi, and T. F. Patterson. 1998. Detection 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.
Lees, E., and R. C. Barton. 2003. The use of niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory. Diagn. Microbiol. Infect. Dis. 46:13-17.
McCullough, M. J., K. V. Clemons, and D. A. Stevens. 1999. Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J. Clin. Microbiol. 37:417-421.
Mosca, C. O., M. D. Moragues, J. Llovo, A. Al Mosaid, D. C. Coleman, and J. Ponton. 2003. Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 41:1259-1262.
Pincus, D. H., D. C. Coleman, W. R. Pruitt, A A. Padhye, I. F. Salkin, M. Geimer, A. Bassel, D. J. Sullivan, M. Clarke, and V. Hearn. 1999. Rapid identification of Candida dubliniensis with commercial yeast identification systems. J. Clin. Microbiol. 37:3533-3539.
Pinjon, E., D. Sullivan, I. Salkin, D. Shanley, and D. Coleman. 1998. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 36:2093-2095.
Sancak, B., J. H. Rex, V. Paetznick, E. Chen, and J. Rodriguez. 2003. Evaluation of a method for identification of Candida dubliniensis bloodstream isolates. J. Clin. Microbiol. 41:489-491.
Staib, P., and J. R. Morschhause. 1999. Chlamydospore formation on staib agar as a species-specific characteristic of Candida dubliniensis. Mycoses 42:521-524.
Sullivan, D. J., G. P. Moran, E. Pinjon, A. Al-Mosaid, C. Stokes, C. Vaughan, and D. C. Coleman. 2004. Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans. FEMS Yeast Res. 4:369-376.
Sullivan, D. J., T. J. Westerneng, K. A. Haynes, D. E. Bennett, and D. C. Coleman. 1995. Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141:1507-1521.
Tintelnot, K., G. Haase, M. Seibold, F. Bergmann, M. Staemmler, T. Franz, and D. Naumann. 2000. Evaluation of phenotypic markers for selection and identification of Candida dubliniensis. J. Clin. Microbiol. 38:1599-1608.(Leigh E. Davis, Christine)
ABSTRACT
The goal of this study was to determine the factor(s) explaining our inability to detect Candida dubliniensis. When germ tube-positive yeasts were tested for C. dubliniensis, no C. dubliniensis was detected; however, 58 C. dubliniensis strains were detected when germ tube-negative Candida albicans strains were tested further. Since all 58 C. dubliniensis strains detected were germ tube negative, these data implied that false-negative germ tube tests occurred with germ tube solution (GTS; Remel, Lenexa, KS). All 41 known C. dubliniensis strains tested were negative with GTS, whereas 40 were positive with rabbit serum (RS; Sigma-Aldrich, St. Louis, MO). Results for C. albicans were equivalent in GTS and RS. In conclusion, GTS cannot be used for the detection of C. dubliniensis, and switching from yeast to hyphae in C. dubliniensis is more restricted than in C. albicans.
TEXT
Candida dubliniensis is a relatively new species resembling Candida albicans that was first detected and characterized by molecular genetic assays in 1995 (16). This species is worldwide and is associated with oropharyngeal candidiasis or colonization in human immunodeficiency virus-positive individuals more than in normal individuals. Like C. albicans, it can develop fluconazole resistance during the treatment of oropharyngeal infections in human immunodeficiency virus-positive individuals. More-serious infections (candidemia) can also be caused by C. dubliniensis, but this occurs less often than with C. albicans. It had not been recognized as a separate species earlier because this species shares important phenotypic characteristics with C. albicans. Both species produce germ tubes and chlamydospores.
Algorithms have been designed to identify C. dubliniensis recovered from clinical specimens. A commonly used algorithm is to screen all germ tube-positive isolates for growth/no growth at 45°C; C. albicans grows, whereas C. dubliniensis does not. Other phenotypic assays used for screening or confirming the identification of C. dubliniensis include phenotypic assays based on growth, colony morphology/color, chlamydospore production, and carbohydrate assimilation (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17). Molecular assays have been shown to be important in detecting this species, but such tests may not be possible in many clinical laboratories at the present time.
C. dubliniensis was not detected in clinical specimens in our mycology laboratory when germ tube-positive isolates (over 600) were tested. The purpose of this study was to identify the factor(s) responsible for our lack of detection of C. dubliniensis from clinical specimens.
Germ tube testing was performed with germ tube solution (GTS; Remel, Lenexa, KS). Yeast cells were inoculated into 0.5 ml of GTS and incubated in a 37°C heat block for 2.5 to 3 h, and aliquots were removed for microscopic examination. Temperature studies were performed to screen for C. dubliniensis by assessing growth at 45°C after 48 h of incubation (C. albicans grows, C. dubliniensis should not). In subsequent studies, xylose and methyl-D-glucose (Mdg) assimilation was used to screen for C. dubliniensis, since C. albicans is positive for them at 89% and 85%, respectively, and negative for C. dubliniensis (API-20C database). Xylose assimilation agar contained 3.0 g xylose, 3.4 g yeast nitrogen base without glucose, 1 ml of a 1.6% bromcreosol purple, and 16 g of purified agar per liter. Mdg agar was the same except that 3.0 g methyl-D-glucoside replaced the xylose. Three milliliters was dispensed into tubes 13- by 100-mm and stored at 4°C until used. Both media were inoculated and incubated at 30°C for 48 h. A color change from a purple to yellow due to a lowering of the pH was considered positive assimilation. In addition, chlamydospore production on casein agar (Remel, Lenexa, KS), was also used to screen for C. dubliniensis since "superchlamydospore" production is more indicative of C. dubliniensis than C. albicans (11). Slants were melted and transferred to petri dishes; isolates were cut into the agar, and the plates were incubated for 44 to 48 h at 25°C. Slides were made and examined microscopically for assessment of chlamydospore production compared to that of C. dubliniensis and C. albicans controls. Definitive identification of C. dubliniensis was made with the API-20C AUX kit (bioMerieux, Inc., Durham, NC), used according to the manufacturer's instructions.
Initially, our protocol was that all germ tube-positive yeast isolates were screened for growth/no growth at 45°C for up to 48 h; C. dubliniensis was not detected among the >600 tested. With our second protocol, both germ tube-positive and germ tube-negative C. albicans strains (germ tube-negative C. albicans identification was established by carbohydrate fermentation and chlamydospore production on corn meal agar with caffeic acid) were evaluated further. Isolates that produced superchlamydospores on casein agar and did not assimilate xylose or Mdg were considered putative C. dubliniensis isolates. A subset of 58 putative isolates was confirmed as C. dubliniensis by the API-20C AUX test. Interestingly, all 58 C. dubliniensis isolates were germ tube negative initially and when they were retested. The data implied that our germ tube results for C. dubliniensis were most likely false negatives.
To test the hypothesis that C. dubliniensis was not detected because of false-negative germ tube production with GTS, we performed a study comparing levels of germ tube production in GTS and RS (Sigma-Aldrich, St. Louis, MO). We tested a panel of 41 isolates of C. dubliniensis, including control strains, an ATCC strain, MYA 179, a Johns Hopkins control isolate, CD-12, seven well-characterized strains kindly provided by M. Jabra-Rizk of the University of Maryland, and 32 of the 58 isolates recovered from clinical specimens (Table 1). The 41 C. dubliniensis isolates and 10 C. albicans isolates (randomly included) were coded to leave the readers unaware of their identities. Germ tube production was determined independently by two highly trained individuals. No germ tubes were produced by the 41 C. dubliniensis isolates in GTS within 3 h of incubation; three isolates (7%) had very few and short germ tubes seen after >3 h of incubation. In contrast, 40/41 (98%) were clearly positive using RS within 3 h. Levels of germ tube production by C. albicans were equivalent on GTS and RS when 50 clinical isolates were compared. This lack of germ tube production has been confirmed with more than five different lots of the GTS.
These germ tube results explain our inability to identify C. dubliniensis recovered from clinical specimens. Since the germ tube test was the initial screening test, germ tube-negative samples (false negatives) were not worked up further. These data and the literature (15) also support the possibility that there are differences in levels of induction of germ tube production between C. albicans and C. dubliniensis. This switch from yeast to hyphal growth seems to be under more control in C. dubliniensis than in C. albicans. Since the production of hyphae is involved in virulence, this restriction of hyphal formation might contribute to the reduced virulence of C. dubliniensis compared to that of C. albicans.
ACKNOWLEDGMENTS
We thank the staff of the Clinical Mycology Laboratory for their cooperation and M. Jabra-Rizk, University of Maryland, for kindly providing well-characterized strains of C. dubliniensis.
REFERENCES
Al Mosaid, A., D. Sullivan, I. F. Salkin, D. Shanley, and D. C. Coleman. 2001. Differentiation of Candida dubliniensis from Candida albicans on staib agar and caffeic acid-ferric citrate agar. J. Clin. Microbiol. 39:323-327.
Al Mosaid, A., D. J. Sullivan, and D. C. Coleman. 2003. Differentiation of Candida dubliniensis from Candida albicans on Pal's agar. J. Clin. Microbiol. 41:4787-4789.
Arikan, S., O. Darka, G. Hascelik, and A. Gunalp. 2003. Identification of Candida dubliniensis strains using heat tolerance tests, morphological characteristics and molecular methods. Mikrobiyol. Bul. 37:49-57. (Abstract in English.)
Fotedar, R., and S. S. Al Hedaithy. 2004. Prevalence of Candida dubliniensis among germ tube positive yeasts recovered from the respiratory specimens in HIV-negative patients. Mycoses 47:150-155.
Gales, A. C., M. A. Pfaller, A. K. Houston, S. Joly, D. J. Sullivan, D. C. Coleman, and D. R. Soll. 1999. Identification of Candida dubliniensis based on temperature and utilization of xylose and alpha-methyl-D-glucoside as determined with the API 20C AUX and Vitek YBC systems. J. Clin. Microbiol. 37:3804-3808.
Kim, D., W. S. Shin, K. H. Lee, K. Kim, J. Y. Park, and C. M. Kob. 2002. Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 degrees C. Yeast 19:957-962.
Kirkpatrick, W. R., S. G. Revankar, R. K. Mcatee, J. L. Lopez-Ribot, A. W. Fothergill, D. I. McCarthy, S. E. Sanche, R. A. Cantu, M. G. Rinaldi, and T. F. Patterson. 1998. Detection 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.
Lees, E., and R. C. Barton. 2003. The use of niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory. Diagn. Microbiol. Infect. Dis. 46:13-17.
McCullough, M. J., K. V. Clemons, and D. A. Stevens. 1999. Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J. Clin. Microbiol. 37:417-421.
Mosca, C. O., M. D. Moragues, J. Llovo, A. Al Mosaid, D. C. Coleman, and J. Ponton. 2003. Casein agar: a useful medium for differentiating Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 41:1259-1262.
Pincus, D. H., D. C. Coleman, W. R. Pruitt, A A. Padhye, I. F. Salkin, M. Geimer, A. Bassel, D. J. Sullivan, M. Clarke, and V. Hearn. 1999. Rapid identification of Candida dubliniensis with commercial yeast identification systems. J. Clin. Microbiol. 37:3533-3539.
Pinjon, E., D. Sullivan, I. Salkin, D. Shanley, and D. Coleman. 1998. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 36:2093-2095.
Sancak, B., J. H. Rex, V. Paetznick, E. Chen, and J. Rodriguez. 2003. Evaluation of a method for identification of Candida dubliniensis bloodstream isolates. J. Clin. Microbiol. 41:489-491.
Staib, P., and J. R. Morschhause. 1999. Chlamydospore formation on staib agar as a species-specific characteristic of Candida dubliniensis. Mycoses 42:521-524.
Sullivan, D. J., G. P. Moran, E. Pinjon, A. Al-Mosaid, C. Stokes, C. Vaughan, and D. C. Coleman. 2004. Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans. FEMS Yeast Res. 4:369-376.
Sullivan, D. J., T. J. Westerneng, K. A. Haynes, D. E. Bennett, and D. C. Coleman. 1995. Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141:1507-1521.
Tintelnot, K., G. Haase, M. Seibold, F. Bergmann, M. Staemmler, T. Franz, and D. Naumann. 2000. Evaluation of phenotypic markers for selection and identification of Candida dubliniensis. J. Clin. Microbiol. 38:1599-1608.(Leigh E. Davis, Christine)