D-763
Identification of Unusual Clinical Yeast Isolates: A 2 Year Review of
Internal Transcribed Spacer (ITS) Region Sequence Analysis
E. Susan Slechta
ARUP Institute for Clinical and Experimental Pathology
500 Chipeta Way
Salt Lake City, UT 84108
(801) 583-2772 x3223
email: Susan.Slechta@aruplab.com
E. Susan Slechta1,Sheri H. Hohmann2, and Kimberly E. Hanson2,3
1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT , 2Associated Regional and University Pathologists, Inc., Salt Lake City, UT,
3Department of Pathology, University of Utah, Salt Lake City, UT
RESULTS
REVISED ABSTRACT
Background:
More than 150 different yeasts are recognized as human pathogens.
.
Identification (ID) of these diverse organisms by conventional methods is time consuming and
often inconclusive for atypical species. We implemented nucleic acid sequencing for yeasts
that could not be identified by phenotypic testing. Our aim was to asses the utility of ITS
sequence analysis using SmartGene IDNS, a curated microbial sequence database and
software program.
Methods: Yeast isolates submitted to the ARUP between Nov 2007 and Feb 2010 were
evaluated using the following: germ tube formation, morphology on corn meal agar, color on
CHROMagar, urease production, rapid trehalose assimilation and/or the API 20C AUX
system. Fungal ITS 1 and 2 sequences were interrogated when API failed to yield a definitive
ID. A sequence homology of ≥ 99% with a > 0.8% difference between species was required
for species level ID, 97-99% identity for genus ID, and < 97% was considered unable to ID.
Figure 1. ITS Region
Figure 3. Phylogenetic Tree*
18S rRNA
ITS1
5.8S
Sequencing Identification
ITS2
ITS4
ARUP045 Trichosporon montevideense
ARUP055 Trichosporon mycotoxinivorans
ARUP027 Cryptococcus sp.
Phenotypic Identification (n=2776)
ARUP136 Rhodotorula nothofagi
Body fluid (209)
ARUP041 Pseudozyma rugulosa
Ocular (8)
Respiratory (500)
ARUP005 Ustilago sp.
ARUP099 Ustilago sp. (2)
Oral/GI (60)
Superficial (58)
ARUP017 Unable to ID
Wound (108)
Not specified (244)
ARUP144 Malassezia sp. (4)
Sequencing Identification (n=158)
ARUP025 Malassezia pachydermatis (5)
Blood (70)
Body fluid (18)
ARUP130 Unable to ID
Tissue (9)
ARUP096 Candida stellimalicola (2)
Respiratory (18)
ARUP060 Candida nivariensis (5)
The spectrum of yeast species identified as opportunistic human pathogens is continually
expanding as a result of the growing number of critically ill and immunocompromised
patients. The widespread use of broad spectrum antibiotics, invasive procedures and longterm indwelling catheters are also associated with increased risk for invasive yeast
infections. Early initiation of appropriate antifungal therapy improves clinical outcomes and
informed therapeutic decisions increasingly require a rapid and accurate species level
identification. Current phenotypic identification systems, however, are often unable to
correctly identify less common or newly described pathogens and may fail to differentiate
common organisms with rare or unique biochemical profiles. Furthermore, conventional
methods are subjective, labor intensive and time consuming.
Oral/GI (2)
ARUP059 Unable to ID
Susceptibility testing Susceptibility testing was performed upon request using the Sensititre®
YeastOne panel (Trek Diagnostic Systems), a colorimetric microtitre broth dilution method
based on the Clinical and Laboratory Standards Institute (CLSI) M27-A2 standard. The
antifungal drugs tested included amphotericin B (AMB), flucytosine (5FC), fluconazole
(FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS) and caspofungin (CAS).
CLSI breakpoints were applied to interpret susceptibility results.
Candida haemulonii
C. krusei (4)
0.5-1
(0.71)
Superficial (2)
ARUP102 Saccharomyces cerevisiae (5)
Wound (10)
ARUP067 Candida colliculosa
Not specified (13)
ARUP153 Candida kefyr
ARUP069 Candida catenulata (4)
Species Identification
C. glabrata
C. albicans
C. albicans/dubliniensis
C. parapsilosis
C. tropicalis
C. lusitaniae
Cryptococcus neoformans
C. guilliermondii
C. krusei
Saccharomyces cerevisiae
C. dubliniensis
C. kefyr
C. famata
Trichosporon asahii
Rhodotorula mucilaginosa
C. pelliculosa
Candida sp.
Ustilago sp.
C. pararugosa
C. guilliermondii/famata
Malassezia sp.
C. nivariensis
C. rugosa
Geotrichum candidum
Malassezia pachydermitis
C. catenulata
C. krusei/C. inconspicua
Pichia farinosa
Unable to ID
C. blankii
C. haemulonii
Malassezia furfur
Trichosporon inkin
Trichosporon sp.
Zygosaccharomyces fermentati
Total*
aOrganisms
Total No. (% of
total isolates)
677 (23.1)
520 (17.7)
516 (17.5)
422 (14.4)
305 (10.4)
67 (2.3)
62 (2.1)
50 (1.7)
50 (1.7)
34 (1.2)
28 (1.0)
20 (0.68)
13 (0.44)
13 (0.44)
12 (0.41)
11 (0.37)
11 (0.37)
9 (0.31)
7 (0.24)
6 (0.2)
6 (0.2)
5 (0.17)
5 (0.17)
4 (0.13)
4 (0.13)
4 (0.13)
4 (0.13)
4 (0.13)
4 (0.13)
3 (0.10)
3 (0.10)
3 (0.10)
3 (0.10)
3 (0.10)
3 (0.10)
2934
No. by
Phenotypic ID
(% of Species)
666 (98.4)
516 (99.2)
516 (100)
419 (99.3)
298 (97.7)
54 (80.6)
58 (93.5)
38 (76.0)
40 (80.0)
28 (82.4)
25 (89.3)
19 (95.0)
12 (92.3)
13 (100)
12 (100)
11 (100)
3 (27.3)
6 (66.7)
0 (0)
6 (100)
2 (33.3)
0 (0)
3 (60.0)
2 (50.0)
0 (0)
0 (0)
4 (100)
0 (0)
1 (25.0)
0 (0)
0 (0)
0 (0)
2 (66.7)
2 (66.7)
0 (0)
2776
No. by
Sequencing ID
(% of Species)
11 (1.6)
4 (0.8)
0 (0)
3 (0.7)
7 (2.3)
13 (19.4)
4 (6.5)
12 (24.0)
10 (20.0)
6 (17.6)
3 (10.7)
1 (5.0)
1 (7.7)
0 (0)
0 (0)
0 (0)
8 (72.7)
3 (33.3)
7 (100)
0 (0)
4 (66.7)
5 (100)
2 (40.0)
2 (50.0)
4 (100)
4 (100)
0 (0)
4 (100)
3 (75.0)
3 (100)
3 (100)
3 (100)
1 (33.3)
1 (33.3)
3 (100)
158
identified less than 3 times were removed from this table for readability. They represent 44 specimens out of the total
2985, 21 identified by classical methods and 23 by ITS sequencing. The full table is available for viewing upon request.
0.5-1
0.25-1
(0.42)
0.5
0.06-8
(0.58)
ND
1-32
(3.37)
4
0.5-1
(0.63)
0.5
1
(2)
0.5-1
(0.79)
4
4
1
Candida sp . (4)
ARUP118 Zygosaccharomyces fermentati (3)
Table 1 Yeast Identification Resultsa
32-64 0.12-0.5
(45.3)
(0.29)
0.030.5-1
0.06
(0.57)
(0.04)
4
0.12
16
0.5
32
0.12
0.5-1
(0.57)
0.5
0.5
0.5
C. membranifaciens
C. nivariensis
C. palmioleophila
C. parapsilosis (3)
0.06
0.03
8-16
(9.5)
0.030.06
(0.04)
0.06
0.06
0.06
0.060.25
(0.12)
0.06
0.125
0.060.12
C. lusitaniae (5)
C. pararugosa
C. rugosa
Candida tropicalis
Trichosporon sp.
POS
0.03
0.03
0.008
0.5-2
(0.87)
(6)
ARUP120 Candida glabrata (12)
INTRODUCTION
DNA Amplification and ITS sequencing. Fungal ITS 1 and 2 sequences were interrogated
for organisms isolated from significant sites when the API failed to yield a definitive ID.
Genomic DNA was obtained using the PrepMan Lysis Kit (ABI). DNA amplification was
achieved with ITS primers {ITS5 forward 5’GGAAGTAAAAGTCGTAACAAGG and ITS4
reverse 5’TCCTCCGCTTATTGATATGC} (FIGURE 1) on an ABI 9700 thermal cycler.
Sequences were analyzed with SmartGene IDNS web based software. Reference sequences
submitted from type or reference strains and/or GenBank entries published in the peer
reviewed literature were selected for comparison to the clinical isolate. A sequence homology
of ≥ 99% with > 0.8% difference between species was required for species level identification
(ID), 97 to 99% identity for genus ID and <97% was considered unidentifiable.
C. guilliermondii
ARUP047 Malassezia furfur (3)
Ocular (4)
Conventional testing. Phenotypic assessments included: germ tube formation, morphology
on corn meal agar, color on CHROMagar, urease production, rapid trehalose assimilation
and/or analysis using the API 20C AUX system.
C. blankii
C. dubliniensis
C. glabrata (5)
ARUP093 Cryptococcus neoformans (4)
Genital/Urinary (401)
4-128 0.12-0.5
(11.3)
(0.27)
32
0.5
0.5
1
0.25
ARUP063 Cryptococcus sp.
Tissue (69)
0.5-1
(0.62)
1
AMB
ARUP095 Cryptococcus albidus
Blood (1101)
0.5-1
(0.57)
MIC ranges (geometric mean)
5FC
FLC
ITC
VRC
0.060.120.05
0.25
0.06
0.008
0.06
1
0.015
0.015
0.06
0.25
0.016
0.008
0.030.060.06 4-128
0.5-4
0.25
(0.039) (16)
(0.76)
(0.18)
C. albicans (2)
ARUP107 Trichosporon inkin
Genital/Urinary (12)
Yeast Specimens. From September 2007 to February 2010, a total of 2934 clinical yeast
isolates were submitted to the ARUP Mycology Reference Laboratory for identification.
(No. of Isolates)
ARUP007 Trichosporon sp.
Figure 2. Specimen Sources by Method of Identification
Conclusions: Analysis of fungal ITS 1 and 2 regions using SmartGene software
unambiguously identified 87% of yeast isolates not categorized by API. Our experience
supports the use of molecular techniques as an adjunct to conventional methods for the
identification of medically important yeasts.
METHODS
Table 2 Susceptibility Test Results for Selected Sequenced Isolates.a,b
28S rRNA
ITS5
Results: A total of 2934 isolates were evaluated. Overall, 95% (2776), encompassing 68
species, were fully identified by classical methods or API. Candida spp. accounted for the
majority (95%, 2687) of phenotypic IDs. The remaining 158 isolates required sequence
analysis, with 87% (137) identified to species, 11% (18) to genus, and 2% (3) remained
unidentified. A total of 73 unique species were discerned by sequencing. Of these, 48 were
common pathogens with atypical biochemical profiles and 25 were rarer yeasts not included
in the API database. The genus level IDs included: 7 Candida spp., 3 Ustilago spp., 2
Cryptococcus spp., 2 Pichia spp., 1 Trichosporon spp. and 1 Sporopachydermia spp.
To facilitate the identification (ID) of unusual yeasts, the ARUP Mycology Laboratory
implemented sequence analysis of the Internal Transcribed Spacer (ITS) region using
SmartGene IDNS (SmartGene Inc), a web-based database of approximately 109,000 ITS
refernce sequences derived from Genbank. The aim of this study was to evaluate the use of
SmartGene as a tool to classify ITS 1 and 2 nucleic acid sequences from clinically significant
yeast isolates that could not be definitively characterized by classical methods. The
antifungal susceptibility profiles of the isolates requiring molecular identification were also
reviewed.
Sheri Hohmann
ARUP Laboratories
500 Chipeta Way
Salt Lake City, UT 84108
email: Sheri.Hohmann@aruplab.com
0.12
0.06
0.125
0.120.25
0.060.12
(0.07)
ND
CAS
0.030.25
0.03
0.03
0.030.25
(0.07)
0.030.25
(0.17)
0.03
0.120.25
(0.21)
0.03-1 0.06-0.5
(0.11)
(0.20)
0.25
5
0.120.25 0.12-0.5
(0.21)
(0.21)
0.0080.008.016
0.06 0.03-0.5
(0.009)
(.016)
(0.16)
0.03
0.06
0.06
0.25
1
0.06
0.12
0.06
0.03
0.008
0.03
0.008
0.060.12
0.0080.06
(0.03)
0.25
0.06
0.03
0.008
0.25
0.0080.06
(0.03)
0.25
0.5-1
(0.63)
0.03
0.25
0.060.25
0.030.25
(0.06)
16
aAbbreviations
used AMB= Amphotericin B, 5-FC = 5-Flucytosine, FLC = Fluconazole, ITC = Itraconazole , VRC
=Voriconazole , POS = Posaconazole, CAS = Caspofungin. ND = Not Done.
ARUP058 Pichia sp. (2)
ARUP074 Candida sp.
bYellow
boxes indicate dose dependent susceptibility and/or resistance to the indicated antifungal drug. for at least
one specimen.
ARUP083 Candida tropicalis (7)
ARUP081 Candida albicans (4)
CONCLUSIONS
ARUP032 Candida dublinensis (3)
ARUP080 Candida parapsilosis (3)
1. Despite the reference nature of our work, Candida albicans remained the most
common yeast identified during the study period.
ARUP011 Pichia farinosa (4)
ARUP141 Candida guillermondii (11)
ARUP084 Candida fermentati
2. The majority (95%) of clinical isolates were successfully identified using
routine phenotypic methods.
ARUP031 Candida palmioleophila
ARUP014 Candida famata
3. ITS sequencing was useful for a diverse group of rare organisms as well as
common species with atypical biochemical profiles, and has the potential to
discover novel species.
ARUP105 Candida insectorum/Pichia mexicana
ARUP085 Candida membranifaciens
ARUP104 Sporopachydermia sp.
ARUP023 Candida blankii (3)
ARUP020 Candida intermedia
A significant number of isolates requiring molecular identification were
Candida species with intrinsic antifungal resistance or potential for elevated
fluconazole MICs (22%).
For laboratories without timely access to
molecular identification methods, susceptibility testing alone may provide
valuable treatment information.
ARUP127 Candida haemulonii (3)
REFERENCES
ARUP140 Candida lipolytica
4.
ARUP097 Candida rugosa (2)
ARUP082 Candida sp. (3)
ARUP042 Candida lusitaniae/tropicalis
ARUP061 Candida lusitaniae (13)
ARUP116 Blastoschizomyces capitatus (2)
ARUP124 Candida pararugosa (6)
1. Iwen, P. C., S. H Hinrichs, and M. E. Rupp. 2002. Utilization of the Internal Transcribed Spacer
Regions as Molecular Targets to Detect and Identify Human Fungal Pathogens. Medical
Mycology 20:87-109.
2.
Leaw, S. N., H. C. Chang, H. F. Sun, R. Barton, J-P. Bouchara, and T. C. Chang. 2006.
Identification of Medically Important Yeast Species by Sequence Analysis of the Internal
Transcribed Spacer Regions. Journal of Clinical Microbiology 44:693-699.
3.
Linton, C.J., Borman, A.M., Cheung, G.C., Holmes, A.D., Szekely, A., Palmer, M.D., Bridge,
P.D., Campbell, C.K., and Johnson, E.M. 2007. Molceulat Identification of Unusual Pathogenic
Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United
Kingdom Mycology Reference Laboratory. Journal of Clinical Microbiology 45:1152-1158.
ARUP087 Geotrichum candidum (2)
ARUP101 Candida lambica (2)
ARUP150 Candida sp. (2)
ARUP117 Candida krusei (10)
0.05
*Phylogenetic tree built in Mega 4 by the neighbor-joining method.
Acknowledgements: We would like to thank Keith Simmon, June Pounder, and the Mycology
Laboratory at ARUP for their assistance in this project. This research was supported in part by the
ARUP Institute for Clinical and Experimental Pathology.