PCR and sequencing of independent genetic targets for the diagnosis of culture negative bacterial endocarditis

Molecular methods utilizing broad-range primers for 16S rDNA PCR and sequencing have been widely evaluated for their utility in culture negative diagnostic bacteriology. Difficulties in determining the incidence of false positive PCR results, especially in the absence of an equally sensitive confirmatory method however, have prevented wide clinical use of this sensitive technology. Here we report two cases of culture-negative endocarditis, in which PCR using 16S rDNA broad-range primers generated sequences specific for Bartonella spp. and Streptococcus oralis, respectively. To confirm these results, a second species- or genus-specific molecular target was chosen for each organism and detected in the split samples sequencially. Thus, molecular detection of a second species-specific target can be used to confirm PCR results generated from 16S rDNA broad-range primers and to control for potential false positive results due to environmental and amplicon carry-over contamination during specimen processing and testing in the laboratory.