P-9. Direct Identification of Gram-Positive Bacteria Culture: Analytical Performance of a Multi-pathogen Molecular Assay

TL;DR

A new rapid molecular test can identify gram-positive bacteria and resistance markers directly from blood cultures in about 2 hours, improving diagnosis of bloodstream infections.

Contribution

A novel, amplification-free, multiplexed molecular assay for direct gram-positive pathogen and resistance marker detection from blood cultures is developed and evaluated.

Findings

The assay achieved 100% positivity for gram-positive targets and 0% for negative cultures in growth and detection studies.

It demonstrated 99.7% reproducibility across three sites and 100% inclusivity for 183 on-panel strains.

The test maintained sensitivity with low pathogen concentrations and co-infecting bacteria.

Abstract

Bloodstream infection (BSI) is a serious and life-threatening condition requiring timely diagnosis and treatment. This study evaluated a multi-target molecular assay under development for rapid and accurate identification of gram-positive (GP) bacteria directly from blood culture specimens. Here, we report progress in developing a multiplexed gram-positive blood culture assay in development to identify 13 pathogens and 3 resistance markers in approximately 2 hours.

The assay was performed directly on blood culture positive specimens determined to contain a GP organism. A 300 µL aliquot was processed in a single-use cartridge, utilizing a sensitive hybridization-based assay for rapid, direct detection without the need for target amplification. This fully automated system performs nucleic acid extraction followed by hybridization to a microarray with capture probes specifically designed to detect the clinically relevant gram-positive bacteria and resistance genes via gold nanoparticle chemistry with silver-enhanced signal amplification.

The prototype assay demonstrated 100% positivity for GP targets and 0% for negative blood cultures in a growth and detection study from ring positive bottles and bottles grown up to 8 hours past ring positivity. Over 1,500 samples were tested as part of a site-to-site reproducibility study including 16 blood culture samples comprised of 7 on-panel organisms, one off-panel organism, and one negative across 3 sites resulting in 99.7% assay reproducibility. It accurately detected all targets, including resistance markers, across 13 media types and demonstrated 100% inclusivity for 183 on-panel strains, including resistance markers. The prototype assay maintained sensitivity even with low concentrations of on-panel GP organisms in the presence of high levels of other co-infecting GP or gram-negative bacteria. Clinical validation is underway with over 600 prospective samples, supporting the ongoing assessment of the assay's real-world performance.

This rapid, highly sensitive prototype assay accurately detects GP pathogens and resistance markers, supporting timely BSI management directly from positive blood culture samples and without target amplification.

Christine Pepich, BS, Diasorin: Employee Samantha Coons, BS, DiaSorin: Employee Seema Shaikh, MS, Diasorin: Employee Brian Bernier, PhD, Diasorin: Employee Amanda Seyfert, MS, Diasorin: Employee Janet Farhang, PhD, Diasorin: Employee|Diasorin: Employee Sundaresh Brahmasandra, PhD, Diasorin: Employee