Gram Typing Bacteria Panels in Whole Blood Using a Biphasic Duplex-Loop-Mediated Isothermal Amplification Assay

Timely identification of bacteria in bloodstream infections is critical for guiding appropriate antibiotic treatment. However, current clinical workflows entail blood culture (1-5 days), followed by Gram staining, PCR, and antibiotic susceptibility testing. These steps delay actionable results, often leading clinicians to prescribe broad-spectrum antibiotics without results from the above tests, contributing to the rising threat of antimicrobial resistance. Specifically, rapid information of even presence of Gram-positive and/or Gram-negative bacteria would help clinicians choose a specific antibiotic regimen after bacteremia is suspected. Here, we developed a rapid, culture-free method that identifies bacterial Gram type within a panel of 6 bacteria from whole blood at a sensitivity of 1-5 CFU/μL within 1.5 h. The assay features a duplex probe-based detection of amplification by release of quenching (DARQ) loop-mediated isothermal amplification (LAMP) system targeting six of the most common bloodstream pathogens in blood cultures in published hospital reports. The two DARQ probes distinguish a panel of four Gram-negative bacteria (E. coli, S. marcescens, P. mirabilis, and K. pneumoniae) from two Gram-positive bacteria (methicillin-susceptible S. aureus/methicillin-resistant S. aureus and S. epidermidis). Coupled with our "biphasic" sample preparation technique (reported earlier) in a 4 μL sample volume, the assay could eliminate the need for blood culture, extraction & purification, providing Gram type information to guide clinical treatments.