Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain...

Area of Science:

• Microbiology
• Molecular Diagnostics
• Clinical Laboratory Science

Background:

• Bloodstream infections (BSI) caused by gram-positive bacteria present a significant clinical challenge.
• Rapid and accurate identification of causative pathogens and antibiotic resistance markers is crucial for effective patient management.

Purpose of the Study:

• To develop and validate the iC-GPC Assay, a novel multiplexed in vitro diagnostic test.
• To assess the assay's performance in identifying common gram-positive bacteria and key resistance markers associated with BSIs.

Main Methods:

• Utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization for assay development.
• Validated the iC-GPC Assay using limit of detection, inclusivity, cross-reactivity, and reproducibility studies.

Main Results:

• The iC-GPC Assay demonstrated limits of detection between 3.0 × 10⁵-1.7 × 10⁷ CFU/mL.
• Achieved high performance with 99.4% inclusivity and 98.9% specificity (95/96 non-target organisms tested negative).
• Reproducibility studies across multiple sites, operators, and lots showed overall performance above 99%.

Conclusions:

• The iC-GPC Assay accurately and rapidly identifies gram-positive bacteria and resistance determinants in positive blood cultures.
• This molecular diagnostic tool has the potential to decrease time to treatment, healthcare costs, and BSI-related mortality.