Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene

Bacteremia continues to result in significant morbidity and mortality, part icularly in patients who are immunocompromised, Currently, patients with su spected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impos e significant delays in and limitations to pathogen identification. To addr ess the limitations of growth-based identification, the sequence variabilit y of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluoresc ence-based PCR-single-st and conformation polymorphism (SSCP) protocol. Spe cies-specific SSCP patterns were determined for 25 of the most common bacte rial species isolated from blood cultures; these isolates subsequently serv ed as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens contain ing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP pattern s distinct from those in the reference collection. Time to identification f rom blood culture positivity ranged from 1 to 8 days with biochemical testi ng, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) pe r specimen when tested in batches of 10. Limitations encountered included t he inability to consistently detect mixed cultures as,well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identifica tion would result in a timely diagnosis with possible implications for pati ent management costs and the mortality and morbidity of infections.