Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood.

Jörk Nölling,Joel I Amburg,Ranjit A Prakash,Arthur R Rabson,Srinivas Rapireddy,Elizabeth M Crawford,Yi-Wei Tang,Alon Singer,Martin J Blaser

doi:10.1128/mbio.00345-16

Abstract

ABSTRACTBloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention.

Highlights

RESULTS␥PNAs as novel sequence interrogation tools for highly sensitive pathogen profiling. We have developed a bead array-based assay that employs ␥PNAs, a newly developed class of duplex DNA-invading artificial nucleic acid oligomers that have significant advantages in kinetics, sensitivity, and specificity over DNA, RNA, and standard peptide nucleic acids; these capabilities are derived from the ␥PNA structure [15,16,17,18,19,20]
Bloodstream infections are a leading cause of morbidity and mortality
We evaluated the performance of our pathogen identification (PID) assay in comparison to conventional culturing using clinical specimens

Summary

RESULTS

␥PNAs as novel sequence interrogation tools for highly sensitive pathogen profiling. We have developed a bead array-based assay that employs ␥PNAs, a newly developed class of duplex DNA-invading artificial nucleic acid oligomers that have significant advantages in kinetics, sensitivity, and specificity over DNA, RNA, and standard peptide nucleic acids; these capabilities are derived from the ␥PNA structure [15,16,17,18,19,20]. We note that the detection sensitivity is directly related to both high target affinity as well as the selectivity of ␥PNAs; off-targets are effectively removed during wash steps, thereby significantly lowering the background commonly seen in DNA-based assays It is the combined performance characteristics of ␥PNAs that enable the development of a highly effective diagnostic method. The results of the PID assay suggest that high numbers of intact S. aureus cells (free pathogen DNA is removed during early PID assay processing steps) were still circulating in the patient’s bloodstream, possibly indicating an ongoing infection and that the antibiotic inhibited growth, a well-known problem with blood cultures [32]. Experiments were completed in triplicate; data are represented as means Ϯ SD

DISCUSSION

MATERIALS AND METHODS

Findings

FUNDING INFORMATION