Multicenter Assessment of a Rapid PNA FISH™ Method for the Identification of
Candida Species from Blood Culture Bottles
C-2073
E. M. Marlowe1, M. Morgan2, S. M. Novak-Weekley1, J. M. Miller1, T. M. Painter3, H. Salimnia3, B. Crystal4
ASM 2010
San Diego, CA
1Southern
California Permanente Med. Grp., North Hollywood, CA, 2Cedars Sinai Med. Ctr., Los Angeles, CA, 3Detroit Med. Ctr., Detroit, MI, 4AdvanDx, Woburn, MA.
Abstract
Candida species are the fourth most common cause of blood stream infections and are
associated with significant mortality and health care costs (1,2,3). The C. albicans/C. glabrata
PNA FISH™ assay (AdvanDx, Woburn, MA) offers a 2.5 hr result after a blood culture flags
positive with the potential of providing important information for species specific treatment.
Multicenter studies were undertaken with the objective of evaluating the sensitivity and
specificity of a modified, shortened (1.5 hr) procedure for the C. albicans/C. glabrata PNA FISH
assay compared to the original (2.5 hr) procedure. This multicenter trial utilized positive blood
culture bottles (BACTEC, Becton Dickinson and BacT/ALERT, bioMérieux) which were deidentified clinical discards. A total of 150 samples (125 clinical and 25 seeded) were tested by
the C. albicans/C. glabrata PNA FISH assay. There was 100% agreement between the rapid
and standard PNA FISH methods as well as with routine identification methods. Specimens
included: 61 C. albicans, 31 C. glabrata (including 1 mixed culture positive for C. albicans/C.
glabrata) and 59 other yeasts (C. parapsilosis, C. neoformans, C. tropicalis and C.
guilliermondii). The new rapid procedure for the C. albicans/C. glabrata PNA FISH provides a
faster method for identifying yeast from positive blood culture bottles than the original
procedure without effecting the sensitivity or specificity of the assay. The reduced time to
results could have an impact on targeted treatment of yeast related blood stream infections.
Background
Candida species are a common cause of blood stream infections. While
C. albicans remains the most commonly isolated, other non-albicans
species such as C. glabrata, C. parapsilosis have more recently shown
an increased prevalence (2). Appropriate and timely antifungal therapy
has been shown to have a significant impact on hospital mortality
compared to empiric treatment.(2). Conventional culture schemes often
take more than 1 day for species identification. C. albicans/C. glabrata
PNA FISH (Peptide Nucleic Acid Fluorescent In-Situ Hybridization) assay
(AdvanDx, Woburn, MA) labels specific C. albicans and C. glabrata rRNA
identifying these species in approximately 2.5hrs. (Fig.1). The ability to
provide even faster results with a Rapid PNA FISH procedure (1.5 hours)
may be of even greater utility.
Multicenter studies were undertaken with the objective of evaluating the
sensitivity and specificity of a modified, rapid procedure for the
C.albicans/C. glabrata PNA assay compared to the original procedure
and to routine identification methods.
Materials and Methods
Results
Blood Cultures: Blood cultures (clinical discards) submitted to the
Southern California Permanente Regional Laboratories, Detroit Medical
Center and Cedars Sinai Microbiology Laboratory were used for testing in
this study. Blood was inoculated into either; bioMérieux Standard Blood
Culture bottles and incubated the BacT/Alert® 3D automated microbial
detection system (bioMerieux, Durham, NC), or BACTEC Standard
aerobic and anaerobic media and incubated in the BACTEC 9240®
automated detection system (BD, Cockeysville, MD). A total of 125 yeast
positive blood cultures were included in the study. Standard culture
methods as well as the API 20C were used to identify the yeast in all
positive blood cultures and compared to the PNA FISH results.
Additionally, 25 BACTEC blood culture bottles were seeded with known
ATCC strains.
PNA FISH: C. albicans/C. glabrata PNA FISH was performed directly
from yeast positive blood cultures. On all samples, both the standard
FDA cleared procedure and the new rapid method, which reduces the 90
minute hybridization step to 30 minutes and eliminates a 10 ethanol soak,
was performed (Fig.2). The operator was blinded to the results of routine
identifications and the 2 PNA FISH methods were read independently.
Expected are illustrated in figure 3.
Figure 1: Illustration of C. albicans/C. glabrata PNA FISH Principle
Table 1: Identification of Enrolled Blood Cultures
Organism Identified (Routine Methods)
Number
Table 2: Performance Data for Rapid C. albicans/C. glabrata PNA FISH
vs Standard PNA FISH Procedure (results where the same vs. routine
identification)
C. albicans
46
C. glabrata
27
C. albicans + C. glabrata
1
Study Site
C. albicans
C. tropicalis
6
A
C. neoformans
5
C. parapsilosis
40
C. albicans (ATCC #28815) seeded
14
C. glabrata (ATCC #2001) seeded
3
C. parapsilosis (ATCC #34136) seeded
2
C. guilliermondii (ATCC #6260) seeded
3
C. tropicalis (ATCC # 13803) seeded
1
C. krusei (ATCC # 6258) seeded
2
Total
150
C. glabrata
Other Yeast
Species
Blood
Culture
System
21/21
9/9
20/20
BACTEC
B
21/21
13/13
16/16
BacT/ALERT
C
19/191
9/91
23/23
BACTEC
Totals
Sensitivity
100% (61/61)
95% CI
(95.2-100)
Sensitivity
100% (31/31)
95% CI
(90.8-100)
Specificity
100% (59/59)
95% CI
(95.1-100)
1One
bottle was mixed C. albicans and C. glabrata
Summary
The Rapid procedure for the C. albicans/C. glabrata PNA FISH provides
a faster method for identifying yeast from positive blood culture bottles
than the original procedure without effecting the sensitivity or specificity of
the assay. Additional studies are needed to determine the impact
reduced time to results could have on lab workflow and improved patient
outcomes for blood stream infections due to yeast.
Figure 3: Fluorescence Microscopy Results
Bibliography
Left to right: C. albicans, C. glabrata, both C. albicans & C. glabrata, Negative
Figure 2: Standard vs. Rapid PNA FISH Protocol
X
Fixation solution
One Drop BC
Heat fix 20 min
Ethanol 10 min
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Probe/Coverslip
Hybridize 90 min
REDUCED TO
30 MIN
Incubate 30 min
Mount coverslip
Examine results
3. Zaoutis, T., Argon, J., Chu, J., Berlin, J., Walsh, T., Feudtner, C. The Epidemiology and
Attributable Outcomes of Canidemia in Adults and Children Hospitalized in the United States:
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