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Journal of Clinical Microbiology, October 2001, p. 3578-3582, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3578-3582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Direct Identification of Bacteria from Positive Blood Cultures by Amplification and Sequencing of the 16S rRNA Gene: Evaluation of BACTEC 9240 Instrument True- Positive and False-Positive Results

Qinfang Qian,1,dagger Yi-Wei Tang,1,Dagger Christopher P. Kolbert,1 Catherine A. Torgerson,1 John G. Hughes,1 Emily A. Vetter,1 W. Scott Harmsen,2 Stacy O. Montgomery,3 Franklin R. Cockerill III,1,* and David H. Persing1,§

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology,1 and Department of Health Sciences Research,2 Mayo Clinic, Rochester, Minnesota 55905, and PE Biosystems, Foster City, California 944043

Received 11 January 2001/Returned for modification 14 May 2001/Accepted 16 July 2001

In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% "instrument false-positive" rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (n = 45) were "instrument true positives"; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (n = 20) were "instrument true negatives"; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

* Corresponding author. Mailing address: Department of Laboratory Medicine and Pathology, Hilton 470B, Mayo Clinic, 200 First St., S.W., Rochester, MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail: cockerill.franklin{at}mayo.edu

dagger Present address: Department of Pathology, Yamins 309, Beth Israel Deaconess Medical Center, Boston, MA 02215.

Dagger Present address: Departments of Medicine and Pathology, Vanderbilt University Medical Center, Nashville, TN 37232-2605.

§ Present address: Infectious Disease Research Institute/Corixa Corporation, Seattle, WA 98104.

Journal of Clinical Microbiology, October 2001, p. 3578-3582, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3578-3582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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