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Journal of Clinical Microbiology, November 2003, p. 5127-5133, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5127-5133.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Application of an rRNA Probe Matrix for Rapid Identification of Bacteria and Fungi from Routine Blood Cultures

Elizabeth M. Marlowe,1* James J. Hogan,2 Janet F. Hindler,1 Irene Andruszkiewicz,2,{dagger} Pat Gordon,2 and David A. Bruckner1

Department of Pathology and Laboratory Medicine, UCLA Medical Center, Los Angeles,1 Gen-Probe Incorporated, San Diego, California2

Received 1 May 2003/ Returned for modification 11 June 2003/ Accepted 6 August 2003

One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at -70°C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).

* Corresponding author. Present address: Gen-Probe Incorporated, 10210 Genetic Center Dr., San Diego, CA 92121-4362. Phone: (858) 410-8692. Fax: (858) 410-8870. E-mail: bethm{at}gen-probe.com.

{dagger} Present address: Diversa Incorporated, San Diego, Calif.

Journal of Clinical Microbiology, November 2003, p. 5127-5133, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5127-5133.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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