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Pseudooutbreak of Candida versatilitis fungemia in a microbiology laboratory
Diagnostic Microbiology and Infectious Disease 46 (2003) 73–75
www.elsevier.com/locate/diagmicrobio
Pseudooutbreak of Candida versatilitis fungemia in a microbiology laboratory Mary E. Brandta,*, Lynette E. Benjamina, Gregory E. Steinkrausb a
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Department of Pathology and Laboratory Medicine, New Hanover Regional Medical Center, Wilmington, NC 28402, USA
Abstract Candida versatilitis was isolated from 10 blood cultures that had been supplemented with olive oil to promote the growth of Malassezia spp., and from the stock olive oil bottle in the laboratory. This unusual non-pathogenic yeast isolate was readily identified by DNA sequencing methodology. This report also points out that care must be taken to ensure the sterility of supplements added to blood culture media. © 2003 Elsevier Inc. All rights reserved.
Case Report During a period of approximately 10 days from late November till early December 2001, blood cultures were drawn from 10 infants in the neonatal intensive care unit. In 5 cases, blood was drawn into a Pediatric BacT-ALERT bottle (bioMerieux, Durham, NC). When each bottle arrived in the laboratory, olive oil was added as enrichment for Malassezia species. In the other 5 cases, blood was drawn into a pediatric Isolator tube (Wampole Laboratories, Cranbury, NJ). Upon receipt in the laboratory, the tubes were processed and duplicate sets of media were inoculated, one overlaid with olive oil and one without olive oil. The oil had been purchased from a nearby grocery store, and was used without further treatment. This bottle was the only one purchased at the time, and had been stored in the laboratory for approximately 8 months before use in this particular group of cultures. Within a week, all of the BacT-ALERT bottles displayed an elevated growth index (GI) and were subcultured. In addition, visible growth could be seen on all of the plates on which olive oil had been overlaid, but not on the plates without olive oil. Gram stain and subculture of each sample revealed small budding yeasts which were not morphologically similar to any Malassezia species. Culture of the stock olive oil bottle showed growth of a similar organism. All isolates showed similar codes with the * Corresponding author. Tel.: ⫹1-404-639-0281; fax: ⫹1-404-6393546. E-mail address: [email protected] 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(02)00573-4
API20C identification system (bioMerieux, St. Louis, MO). One of the blood culture isolates and the olive oil isolate were sent for identification to the Fungus Reference Unit, Mycotic Diseases Branch, CDC. The organism formed colonies after 3-5 days incubation on phytone agar, malt agar and peptone-yeast extract agar at 25°C and 30°C, but not at 35°C or 40°C. Colonies could be seen on Sabouraud dextrose agar only after at least 7-10 days of incubation at 25°C or 30°C. On phytone agar, the isolates initially were cream in color, developing a tannish color with age. On a cornmeal Dalmau plate after 10 days at 30°C, small globose to subglobose budding yeasts 3.0-6.0 M in size were found, with no pseudohyphae, chlamydospores, arthrospores, or true hyphae. No ascospores were found after 14 days of incubation on malt agar at 30°C. Urease production was negative. A RapID Yeast Plus panel (Innovative Diagnostic Systems, Norcross, GA) gave a code of 106000, which was positive for glucose assimilation and production of ␣- and -gluconases. This code was not found in the codebook. An API20C gave a result of 604000 at 72 h (positive for assimilation of glucose, glycerol, and galactose); this code was also not found in the codebook. In both cases, these profiles were also not found in the extended databases accessed by telephone. DNA was isolated and sequenced on an ABI 3100 instrument using the Big Dye Terminator Kit (Applied Biosystems, Foster City, CA). Both strands were sequenced twice, each time in duplicate. A 506-nucleotide sequence was generated using primers NL-1, NL-4, and the internal primers NL-2A and NL-3A, which each amplified a portion
74
M.E. Brandt et al. / Diagnostic Microbiology and Infectious Disease 46 (2003) 73–75
of the D1-D2 region of the large ribosomal subunit (Kurtzman and Robnett, 1997). The two isolates had identical sequences, each displaying 99% homology (1 mismatch) in GenBank to the 26S ribosomal gene of Candida versatilitis NRRL Y-6652, a sequence previously deposited by Kurtzman and Robnett (Kurtzman and Robnett, 1997). Our sequences also showed 99% homology, but with 2 to 3 mismatches, to each of three additional sequences: the 26S ribosomal RNA genes of Debaromyces tamarii NRRL Y-6665, Candida mannitofaciens NRRL Y-7226, and Candida halophila NRRL Y-2483. These three species have since been designated as synonyms of Candida versatilitis (Meyer et al., 1998). The partial 26S nucleotide sequence of our isolate has been deposited in GenBank as AF526266. Sequencing with primers ITS-1 and ITS-4, which amplified the ITS region of ribosomal DNA, generated a sequence that showed no relevant matches to any sequences in GenBank. Furthermore, no Candida versatilitis ITS sequences could be found in the GenBank database. Antifungal susceptibility testing was attempted using the E-test method (AB Biodisk), but the organisms failed to grow on the testing medium. Candida versatilitis is a halophilic ascosporogenous yeast perhaps best known in the production of soy sauce and fermenting cucumber brine (Meyer et al., 1998). It has not been reported as a human pathogen (Meyer et al., 1998). This study shows that phylogenetic methods can be used to identify unusual, rarely encountered organisms in a straightforward manner, without dependence on conventional carbohydrate assimilations that can take up to 30 days for incubation and that require considerable experience to interpret. We were unable to identify these isolates by kitbased methods because this species is not included in the databases of phenotype-based identification systems such as API 20C and RAP-ID. The phenotypic characteristics of C. versatilitis, such as failure to grow above 30°C and failure to produce hyphae/pseudohyphae, are consistent with the features we noted in these isolates. However, these characteristics were not specific enough to identify these isolates to species without additional information. Kurtzman has previously shown that two ascosporogenous yeasts can be designated members of the same species if they show no more than 0 to 6 nucleotide substitutions (less than 1%) in the DNA sequence of the ca. 600 nucleotide D1-D2 variable region at the 5⬘ end of the large ribosomal subunit (Kurtzman and Robnett, 1997). We were able to designate these unknown isolates as Candida versatilitis because they showed only 1-3 mismatches (less than 1% divergence) in this region when their sequences were compared to those in the GenBank database. Malassezia species are basidiomycetous yeasts that cause a catheter-associated sepsis in individuals, primarily infants, who receive lipid-containing hyperalimentation solutions through a central line. Most members of this genus require lipid supplementation for growth (Guillot et al., 1996). In this instance, olive oil supplementation was added
to the blood cultures to facilitate recovery of Malassezia spp. from these samples. Detection of Malassezia fungemia can be accomplished in several ways. Blood can be collected through Isolator tubes and then plated directly on to lipid-containing media. Alternatively, in many laboratories lipid supplementation is added directly to the blood culture bottle upon receipt in the laboratory. The rationale for this action seems to be that a blood sample containing Malassezia organisms may be undetected by automated blood culture systems unless sufficient growth raises the GI of the bottle to a level that can be detected by the sensor in the time during which the bottle is being incubated. Historically, olive oil has been added as the lipid source. However, Nelson et al. have shown that 3% palmitic acid added to Peds Plus bottles improved recovery of Malassezia species in the Bactec NR 660 system when compared to olive oil (Nelson et al., 1995). The practice of adding exogenous lipid supplementation to blood culture bottles has been questioned as unnecessary and inhibitory to growth of Malassezia (Geha and Roberts, 1994). On the other hand, Nelson et al. showed that, when not supplemented with lipid, Malassezia cells lost viability rapidly after inoculation into Bactec Peds Plus bottles, and that the presence of blood appeared to accelerate this loss of viability (Nelson et al., 1995). Unfortunately, the addition of any solution to a blood culture bottle offers the possibility for contamination of that bottle, if the solution is not sterile and if the solution is not added aseptically. In this case, Candida versatilitis organisms were isolated from the stock bottle of olive oil as well as from the olive oil-supplemented blood cultures, thus implicating the olive oil as the source of contamination. Investigation showed that the olive oil was not sterilized prior to addition to blood culture bottles. We recommend that all solutions to be added to blood culture bottles be sterilized prior to use, either by autoclaving or by filter sterilization when appropriate. Small portions of the sterile olive oil should then be aliquotted into separate tubes or containers to prevent mass contamination of the entire stock solution. Furthermore, a portion of the solution should be streaked to an agar plate each time it is used, to verify continued sterility of the stock solution.
Acknowledgments We thank Cletus Kurtzman for helpful comments, and Sally Meyer for critically reviewing the manuscript.
References Geha, D. J., & Roberts, G. D. (1994). Laboratory detection of fungemia. Clin Lab Med, 14, 83–97. Guillot, J., Gueho, G. D., Lesourd, M., Midgley, G., Chevrier, G., & Dupont, B. (1996). Identification of Malassezia species: a practical approach. J Mycol Med, 6, 103–110.
M.E. Brandt et al. / Diagnostic Microbiology and Infectious Disease 46 (2003) 73–75 Kurtzman, C. P., & Robnett, C. J. (1997). Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5⬘ end of the large subunit (26S) ribosomal DNA gene. J Clin Microbiol, 35, 1216 –1223. Meyer, S. A., Payne, R. W., & Yarrow, D. (1998). Candida Berkhout. In:
75
C. P. Kurtzman, & J. W. Fell, The Yeasts-A Taxonomic Study, 4th ed. Amsterdam: Elsevier Science, p. 454 –573. Nelson, S. C., Yau, Y. C. W., Richardson, S. E., & Matlow, A. G. (1995). Improved detection of Malassezia species in lipid-supplemented Peds Plus blood culture bottles. J Clin Microbiol, 33, 1005–1007.
www.elsevier.com/locate/diagmicrobio
Pseudooutbreak of Candida versatilitis fungemia in a microbiology laboratory Mary E. Brandta,*, Lynette E. Benjamina, Gregory E. Steinkrausb a
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Department of Pathology and Laboratory Medicine, New Hanover Regional Medical Center, Wilmington, NC 28402, USA
Abstract Candida versatilitis was isolated from 10 blood cultures that had been supplemented with olive oil to promote the growth of Malassezia spp., and from the stock olive oil bottle in the laboratory. This unusual non-pathogenic yeast isolate was readily identified by DNA sequencing methodology. This report also points out that care must be taken to ensure the sterility of supplements added to blood culture media. © 2003 Elsevier Inc. All rights reserved.
Case Report During a period of approximately 10 days from late November till early December 2001, blood cultures were drawn from 10 infants in the neonatal intensive care unit. In 5 cases, blood was drawn into a Pediatric BacT-ALERT bottle (bioMerieux, Durham, NC). When each bottle arrived in the laboratory, olive oil was added as enrichment for Malassezia species. In the other 5 cases, blood was drawn into a pediatric Isolator tube (Wampole Laboratories, Cranbury, NJ). Upon receipt in the laboratory, the tubes were processed and duplicate sets of media were inoculated, one overlaid with olive oil and one without olive oil. The oil had been purchased from a nearby grocery store, and was used without further treatment. This bottle was the only one purchased at the time, and had been stored in the laboratory for approximately 8 months before use in this particular group of cultures. Within a week, all of the BacT-ALERT bottles displayed an elevated growth index (GI) and were subcultured. In addition, visible growth could be seen on all of the plates on which olive oil had been overlaid, but not on the plates without olive oil. Gram stain and subculture of each sample revealed small budding yeasts which were not morphologically similar to any Malassezia species. Culture of the stock olive oil bottle showed growth of a similar organism. All isolates showed similar codes with the * Corresponding author. Tel.: ⫹1-404-639-0281; fax: ⫹1-404-6393546. E-mail address: [email protected] 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(02)00573-4
API20C identification system (bioMerieux, St. Louis, MO). One of the blood culture isolates and the olive oil isolate were sent for identification to the Fungus Reference Unit, Mycotic Diseases Branch, CDC. The organism formed colonies after 3-5 days incubation on phytone agar, malt agar and peptone-yeast extract agar at 25°C and 30°C, but not at 35°C or 40°C. Colonies could be seen on Sabouraud dextrose agar only after at least 7-10 days of incubation at 25°C or 30°C. On phytone agar, the isolates initially were cream in color, developing a tannish color with age. On a cornmeal Dalmau plate after 10 days at 30°C, small globose to subglobose budding yeasts 3.0-6.0 M in size were found, with no pseudohyphae, chlamydospores, arthrospores, or true hyphae. No ascospores were found after 14 days of incubation on malt agar at 30°C. Urease production was negative. A RapID Yeast Plus panel (Innovative Diagnostic Systems, Norcross, GA) gave a code of 106000, which was positive for glucose assimilation and production of ␣- and -gluconases. This code was not found in the codebook. An API20C gave a result of 604000 at 72 h (positive for assimilation of glucose, glycerol, and galactose); this code was also not found in the codebook. In both cases, these profiles were also not found in the extended databases accessed by telephone. DNA was isolated and sequenced on an ABI 3100 instrument using the Big Dye Terminator Kit (Applied Biosystems, Foster City, CA). Both strands were sequenced twice, each time in duplicate. A 506-nucleotide sequence was generated using primers NL-1, NL-4, and the internal primers NL-2A and NL-3A, which each amplified a portion
74
M.E. Brandt et al. / Diagnostic Microbiology and Infectious Disease 46 (2003) 73–75
of the D1-D2 region of the large ribosomal subunit (Kurtzman and Robnett, 1997). The two isolates had identical sequences, each displaying 99% homology (1 mismatch) in GenBank to the 26S ribosomal gene of Candida versatilitis NRRL Y-6652, a sequence previously deposited by Kurtzman and Robnett (Kurtzman and Robnett, 1997). Our sequences also showed 99% homology, but with 2 to 3 mismatches, to each of three additional sequences: the 26S ribosomal RNA genes of Debaromyces tamarii NRRL Y-6665, Candida mannitofaciens NRRL Y-7226, and Candida halophila NRRL Y-2483. These three species have since been designated as synonyms of Candida versatilitis (Meyer et al., 1998). The partial 26S nucleotide sequence of our isolate has been deposited in GenBank as AF526266. Sequencing with primers ITS-1 and ITS-4, which amplified the ITS region of ribosomal DNA, generated a sequence that showed no relevant matches to any sequences in GenBank. Furthermore, no Candida versatilitis ITS sequences could be found in the GenBank database. Antifungal susceptibility testing was attempted using the E-test method (AB Biodisk), but the organisms failed to grow on the testing medium. Candida versatilitis is a halophilic ascosporogenous yeast perhaps best known in the production of soy sauce and fermenting cucumber brine (Meyer et al., 1998). It has not been reported as a human pathogen (Meyer et al., 1998). This study shows that phylogenetic methods can be used to identify unusual, rarely encountered organisms in a straightforward manner, without dependence on conventional carbohydrate assimilations that can take up to 30 days for incubation and that require considerable experience to interpret. We were unable to identify these isolates by kitbased methods because this species is not included in the databases of phenotype-based identification systems such as API 20C and RAP-ID. The phenotypic characteristics of C. versatilitis, such as failure to grow above 30°C and failure to produce hyphae/pseudohyphae, are consistent with the features we noted in these isolates. However, these characteristics were not specific enough to identify these isolates to species without additional information. Kurtzman has previously shown that two ascosporogenous yeasts can be designated members of the same species if they show no more than 0 to 6 nucleotide substitutions (less than 1%) in the DNA sequence of the ca. 600 nucleotide D1-D2 variable region at the 5⬘ end of the large ribosomal subunit (Kurtzman and Robnett, 1997). We were able to designate these unknown isolates as Candida versatilitis because they showed only 1-3 mismatches (less than 1% divergence) in this region when their sequences were compared to those in the GenBank database. Malassezia species are basidiomycetous yeasts that cause a catheter-associated sepsis in individuals, primarily infants, who receive lipid-containing hyperalimentation solutions through a central line. Most members of this genus require lipid supplementation for growth (Guillot et al., 1996). In this instance, olive oil supplementation was added
to the blood cultures to facilitate recovery of Malassezia spp. from these samples. Detection of Malassezia fungemia can be accomplished in several ways. Blood can be collected through Isolator tubes and then plated directly on to lipid-containing media. Alternatively, in many laboratories lipid supplementation is added directly to the blood culture bottle upon receipt in the laboratory. The rationale for this action seems to be that a blood sample containing Malassezia organisms may be undetected by automated blood culture systems unless sufficient growth raises the GI of the bottle to a level that can be detected by the sensor in the time during which the bottle is being incubated. Historically, olive oil has been added as the lipid source. However, Nelson et al. have shown that 3% palmitic acid added to Peds Plus bottles improved recovery of Malassezia species in the Bactec NR 660 system when compared to olive oil (Nelson et al., 1995). The practice of adding exogenous lipid supplementation to blood culture bottles has been questioned as unnecessary and inhibitory to growth of Malassezia (Geha and Roberts, 1994). On the other hand, Nelson et al. showed that, when not supplemented with lipid, Malassezia cells lost viability rapidly after inoculation into Bactec Peds Plus bottles, and that the presence of blood appeared to accelerate this loss of viability (Nelson et al., 1995). Unfortunately, the addition of any solution to a blood culture bottle offers the possibility for contamination of that bottle, if the solution is not sterile and if the solution is not added aseptically. In this case, Candida versatilitis organisms were isolated from the stock bottle of olive oil as well as from the olive oil-supplemented blood cultures, thus implicating the olive oil as the source of contamination. Investigation showed that the olive oil was not sterilized prior to addition to blood culture bottles. We recommend that all solutions to be added to blood culture bottles be sterilized prior to use, either by autoclaving or by filter sterilization when appropriate. Small portions of the sterile olive oil should then be aliquotted into separate tubes or containers to prevent mass contamination of the entire stock solution. Furthermore, a portion of the solution should be streaked to an agar plate each time it is used, to verify continued sterility of the stock solution.
Acknowledgments We thank Cletus Kurtzman for helpful comments, and Sally Meyer for critically reviewing the manuscript.
References Geha, D. J., & Roberts, G. D. (1994). Laboratory detection of fungemia. Clin Lab Med, 14, 83–97. Guillot, J., Gueho, G. D., Lesourd, M., Midgley, G., Chevrier, G., & Dupont, B. (1996). Identification of Malassezia species: a practical approach. J Mycol Med, 6, 103–110.
M.E. Brandt et al. / Diagnostic Microbiology and Infectious Disease 46 (2003) 73–75 Kurtzman, C. P., & Robnett, C. J. (1997). Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5⬘ end of the large subunit (26S) ribosomal DNA gene. J Clin Microbiol, 35, 1216 –1223. Meyer, S. A., Payne, R. W., & Yarrow, D. (1998). Candida Berkhout. In:
75
C. P. Kurtzman, & J. W. Fell, The Yeasts-A Taxonomic Study, 4th ed. Amsterdam: Elsevier Science, p. 454 –573. Nelson, S. C., Yau, Y. C. W., Richardson, S. E., & Matlow, A. G. (1995). Improved detection of Malassezia species in lipid-supplemented Peds Plus blood culture bottles. J Clin Microbiol, 33, 1005–1007.