Rapid, Culture-Free Detection of Staphylococcus aureus Bacteremia

Abstract

S. aureus bacteremia (SAB) is a common condition with high rates of morbidity and mortality. Current methods used to diagnose SAB take at least a day, and often longer. Patients with suspected bacteremia must therefore be empirically treated, often unnecessarily, while assay results are pending. In this proof-of-concept study, we describe an inexpensive assay that detects SAB via the detection of micrococcal nuclease (an enzyme secreted by S. aureus) in patient plasma samples in less than three hours. In total, 17 patient plasma samples from culture-confirmed S. aureus bacteremic individuals were tested. 16 of these yielded greater nuclease assay signals than samples from uninfected controls or individuals with non-S. aureus bacteremia. These results suggest that a nuclease-detecting assay may enable the rapid and inexpensive diagnosis of SAB, which is expected to substantially reduce the mortality and morbidity that result from this condition.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity

“…10 We recently described rapid assays based on this same concept for focal S. aureus infections 12 and S. aureus bacteremia. 13 To the best of our knowledge, there has been no other description of a rapid assay that detects a disease by detecting the activity of a disease-associated nuclease. Our identification of endonuclease I as a novel biomarker for E. coli and our demonstration that its activity can be used to rapidly detect an E. coli infection therefore demonstrates the broader value and potential of nuclease-based assays for diagnosing infectious diseases.…”

“…Because nucleases are very diverse 14 and are essential for life, 14 nuclease probes that selectively detect alternative bacterial pathogens may also be developed. Moreover, antibodies that selectively inhibit 9 or immuno-precipitate 13 target nucleases can be incorporated into nuclease assays to yield detection and discrimination of multiple uropathogens. We envision a rapid multiplexed nuclease assay that incorporates such approaches for the differential diagnosis of UTI with coverage of several bacterial species as a rapid diagnostic alternative to the commonly used dipstick (which lacks sensitivity) and culture (which is time consuming).…”

“…Several groups have previously developed rapid methods for detecting and identifying S. aureus in cultured material that function by detecting the activity of micrococcal nuclease, a secreted nuclease of S. aureus. [9][10][11] More recently, we developed rapid, culture-independent methods that detect S. aureus via micrococcal nuclease activity in focal infections 12 (with noninvasive imaging) and in bacteremic patients 13 (with an ultrasensitive in vitro assay). Beyond detection of S. aureus, the value of disease-associated nuclease activities in rapid molecular diagnostics is uncertain, as there are no examples of such assays that target other nucleases, to the best of our knowledge.…”

Detection and treatment of critical illnesses using oligonucleotides

“…We demonstrated the sensitivity of our nuclease-activated probe technology by detecting attomolar (10 −18 molar) levels of a bacterial nuclease in the human plasma. (239) In the present study, we sought to exploit the potential of the nucleaseactivated probe technology to diagnose cancer. The challenge here was to reliably distinguish between mammalian nucleases expressed in cancer cells versus those expressed in leukocytes.…”

“…The assay steps include: (1) capture of cancer cells, which is performed within 10 min with commercially available microfiltration units from SCREENCELL, (2) lysis of cancer cells using an optimized nuclease digestion buffer that contains the chemically optimized nuclease probes, and (3) signal detection using standard fluorometry technology.We previously demonstrated the utility of quenched fluorescent nucleic acid probes for the detection of bacterial nucleases (237,238). In this initial study, we demonstrated a sensitivity in the low attomolar (10 −18 molar) range for the detection of a bacterial nuclease in human plasma (239). In this study, we performed key technical modifications for adapting this technology for the detection of endogenous nucleases in cancer patients.…”