Direct Screening of Blood by PCR and Pyrosequencing for a 16S rDNA Target from ED and ICU XXXX Being Evaluated for Bloodstream Infection.

@entity1 @entity582 @entity2286
UNASSIGNED: Here we compared results of PCR/pyrosequencing to culture for detecting bacteria directly from blood. DNA was extracted from 1130 bloods on 913 @entity1 suspected of @entity582 (enrollment criteria; physician-ordered blood culture and CBC), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rDNA targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rDNA PCR/pyrosequencing as containing Staphylococci, Streptococci, or enteric gram-negative rod had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (Tm; 87.2  C   0.2  C) occurred in 99.1% (1120/1130) of @entity1 extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1085/1120) for Universal 16S rDNA target, with positivity rates of 9.4% (105/1120) and 11.3% (126/1120), respectively. Bacteria cultured included: Staphylococci (59/126, 46.8%), gram-negative rods (34/126, 27%), Streptococci (32/126, 25.4%), and Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% CI) for Universal 16S rDNA PCR/pyrosequencing included sensitivity: 77.8% (69.5-84.7), specificity: 99.3% (98.6-99.7), @entity2286 : 93.3% (86.8-97.3), and NPV: 97.2% (96.0-98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. Initial PCR/pyrosequencing took    5.5 hours to complete, or    7.5 hours when including target-specific PCR/pyrosequencing compared to 27.9 +/- 13.6 hours for Gram stain or 81.6 +/- 24.0 hours for phenotypic identification. In summary, this molecular-based approach detected the causative bacteria in over three-quarters of all culture-confirmed cases of @entity582 directly from blood in significantly less time than standard culture, but cannot be used to rule-out infection.
