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

doi:10.1128/JCM.39.10.3578-3582.2001

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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^, Yi-Wei Tang,¹^, 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 InstrumentSystems, Sparks, Md.), we noted a 1.3% "instrument false-positive"rate. That is, the BACTEC system signaled that a bottle (BACTECPlus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culturewas positive but a Gram stain was negative and there was no growthof bacteria or yeasts on subculture to chocolate agar. Furthermore,from the same sample of blood, cultures for fungi using the Isolatorblood culture system (Wampole Laboratories, Cranbury, N.J.) werenegative for growth. For the present study, we evaluated 76 instrumentfalse-positive samples for the presence of 16S ribosomal DNA usingthe MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). Thesesamples also were negative for fungi by the Isolator method. Thiskit has a PCR module and sequencing module for the amplificationand sequencing of the 16S RNA gene and provides a database forsequence alignment and identification of bacteria. To optimizethe assay, we evaluated the effect of adding 0.5% bovine serumalbumin to the sample from blood culture bottles and found thatit decreased the effects of inhibitors on the PCR. Two controlgroups of blood culture specimens were also evaluated. One group(n = 45) were "instrument true positives"; the instrument signaledpositive, and subsequent Gram stains were positive and subcultureson 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 agarand from the Isolator tube on fungal media showed no growth. Noneof the 76 instrument false-positive samples had evidence for 16SrRNA gene sequences. All of the instrument true-positive samplesand all of the instrument true-negative specimens were positiveand negative, respectively, using the MicroSeq 500 kit. Totalperipheral white blood cell counts were statistically significantlyhigher for patients who had instrument false-positive resultsthan for patients who had instrument true-positive or true-negativeresults (P = 0.001). We conclude that instrument false positivessignaled by the BACTEC 9240 system are not due to bacteria inthe blood culturesamples.

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

Present address: Department of Pathology, Yamins 309, Beth Israel Deaconess Medical Center, Boston, MA02215.

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

^§ Present address: Infectious Disease Research Institute/Corixa Corporation, Seattle, WA98104.

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