Detection of Bacteremia in Emergency Department Patients at Risk for Infective Endocarditis Using Universal 16S rRNA Primers in a Decontaminated Polymerase Chain Reaction Assay

Abstract

Prompt definitive diagnosis of acute bacterial endocarditis in febrile injection drug users (IDUs) remains problematic because of delays associated with blood culture. Rapid detection of bacteremia by polymerase chain reaction (PCR) by use of "universal" primers has been hampered by background bacterial contamination. Broad-range eubacterial primers selected from the 16S rRNA gene were used in a PCR assay coupled with a simple pre-PCR decontamination step. All PCR reagents were pretreated with the restriction enzyme AluI, which has multiple digestion sites in the amplicon but none in the primer sets. When 4 different bacterial species were spiked into healthy human blood specimens, the assay identified each pathogen with an analytic sensitivity of 5 bacteria/PCR reaction. A clinical trial with 51 febrile IDUs revealed that PCR had a sensitivity and specificity of 86.7% and 86.9%, respectively, versus blood culture. Importantly, all (8/8) patients with blood culture-positive infective endocarditis were determined to be positive by PCR. This assay provides a promising diagnostic for rapid identification of bacteremia, particularly valuable in acute care settings.

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

“…BC has been most widely used as a "gold standard" for statistical evaluation of new PCR tests, and we therefore include these parameters to compare with other studies. However, PCR can detect more cases of bacteremia than culture, since it can detect the DNA of nonviable microorganisms (28). Thus, we also calculated the concordance, both positive and negative, of PCR and BC, which was 86.0%.…”

“…The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4,39), have been counteracted by the development of purification methods (12,28) and the availability of commercial products (22).…”

Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis

“…Moreover, there is a high risk of contamination by skin saprophytes such as coagulase-negative staphylococci (CoNS) and streptococci, which makes it difficult to implicate them as agents of catheterassociated bacteremia (9). All these issues can be overcome using PCR, because it is based on the direct detection of the microbe without relying on its growth curve or without suffering the bacteriostatic effect of antimicrobial therapy (12,20). In the present study, we evaluated a commercially available multiplex real-time PCR assay (the LightCycler SeptiFast test) for the direct detection of bacteria and fungi.…”

Polymerase Chain Reaction to Diagnose Infective Endocarditis

“…All 8 patients who had definite IE (infected with S aureus, streptococci, or both) in this group also were positive by broad-range PCR. 11 Despite the lower sensitivity reported by Breitkopf et al, 1 A disadvantage of these approaches is that DNA from the causative agent(s) must be present at a relatively high concentration to be properly detected. The general result has been that sensitivity achieved by broad-range PCR strategies, particularly for the more common pathogenic agents of IE, often is no better than that with blood culture.…”