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

Abstract

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.Bacteremia has become a major nosocomial infection in most medical centers and represents a formidable source of morbidity and mortality (1,14,19,26). Patients with bloodstream infections have a three-times-greater risk of dying than that expected from the underlying diseases alone (27). It has been estimated that there are 500,000 cases of bacteremia each year, with an associated crude mortality of 35% (28). The rapid and reliable detection and subsequent identification of microorganisms from blood remain critical so that the appropriate antimicrobial therapy can be provided.DNA sequencing of conserved regions within phylogenetically informative genetic targets, such as the small-subunit (16S) rRNA gene, is promising as a means for identifying bacte...

Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles

“…The rates of sequencing failures and no matches were low (1% each). Like other investigators (5,12,22), we also found this approach to be useful in confirming false-positive blood culture results when Gram staining and subculture onto solid media yielded no organisms (data not shown). It is a good idea to incorporate more than one ribosomal gene target into the testing algorithm, as it increases the number of DNA sequences one has to analyze, which can provide a cross check on the data generated and increase your confidence in the accuracy of the organism's identification, if concordance occurs.…”

“…Several sequence-based approaches have been successfully used to identify bacteria directly from positive blood culture bottles. Qian et al successfully used the MicroSeq 500 kit (Perkin-Elmer Applied Biosystems, Foster City, CA), a commercially available method that sequences the first 527 bases of the amplified 16S rRNA gene, for this purpose (22). Turenne et al used single-stranded conformation polymorphism analysis of PCR amplicons to distinguish between organisms (23), while Peters et al used fluorescence in situ hybridization to identify pathogens out of positive blood cultures (19).…”

A Sample Extraction Method for Faster, More Sensitive PCR-Based Detection of Pathogens in Blood Culture

“…Although it is considered the gold standard, culturing blood can generate false-negative results up to 31% of the time and false-positive results up to 10% of the time, [1][2][3][4] making confirmation by PCR desirable. Achieving accurate PCR results from blood culture samples is difficult, however, because of the presence of sodium polyanetholsulfonate (SPS), a powerful PCR inhibitor that is added routinely to blood culture media to i) lower the barrier for bacterial growth by inhibiting the complement pathway, ii) prevent blood coagulation, and iii) interfere with the activity of some antibiotics.…”

Distribution, Optimum Detection Time and Antimicrobial Susceptibility Rates of the Microorganisms Isolated from Blood Cultures over a 4-Year Time Period in a Turkish University Hospital and a Review of the International Literature

“…The contamination rate in the present study was 2.8% which lies within the accepted contamination value and is low in comparison with contamination rates of 1.21 -13.6% reported by other studies. 11,13,16,21,22 Mortality from fungal infections in intensive care units is reported to be 38 -75% and, in recent years, there has been an increase in the rates of fungal infections with nosocomial origins. 41 -43 Most of these involve Candida spp, of which 48% of are Candida albicans.…”