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Journal of Clinical Microbiology, February 2000, p. 513-520, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid Identification of Bacteria from Positive
Blood Cultures by Fluorescence-Based PCR-Single-Strand Conformation
Polymorphism Analysis of the 16S rRNA Gene
Christine Y.
Turenne,1,*
Evelyn
Witwicki,2
Daryl J.
Hoban,1,2
James A.
Karlowsky,1,2,3 and
Amin M.
Kabani1,2,4
Department of Medical Microbiology, Faculty
of Medicine,1 and Faculty of
Pharmacy,3 University of Manitoba, and
Departments of Clinical Microbiology2
and Child Health,4 Health Sciences
Centre, Winnipeg, Manitoba R3A 1R9, Canada
Received 12 July 1999/Returned for modification 3 September
1999/Accepted 5 November 1999
Bacteremia continues to result in significant morbidity and
mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies
upon the use of blood cultures, which impose significant delays in and
limitations to pathogen identification. To address the limitations of
growth-based identification, the sequence variability of the 16S rRNA
gene of bacteria was targeted for rapid identification of bacterial
pathogens isolated directly from blood cultures using a
fluorescence-based PCR-single-strand conformation polymorphism (SSCP)
protocol. Species-specific SSCP patterns were determined for 25 of the
most common bacterial species isolated from blood cultures; these
isolates subsequently served as a reference collection for bacterial
identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were
tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns
distinct from those in the reference collection. Time to identification
from blood culture positivity ranged from 1 to 8 days with biochemical
testing, whereas identification by fluorescence-based capillary
electrophoresis was obtained as early as 7 h at a calculated cost
of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect
mixed cultures as well as some species demonstrating identical SSCP
patterns. This method can be applied directly to blood cultures or
whole-blood specimens, where early pathogen identification would result
in a timely diagnosis with possible implications for patient management
costs and the mortality and morbidity of infections.
*
Corresponding author. Present address: Department of
Clinical Microbiology, Health Sciences Centre, MS6-820 Sherbrook St., Winnipeg, Manitoba R3A 1R9, Canada. Phone: (204) 787-6038. Fax: (204)
789-2036. E-mail: cturenne{at}hc-sc.gc.ca.
Journal of Clinical Microbiology, February 2000, p. 513-520, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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