Tipificación de Bacterias por Tinción de Gram en Paneles de Sangre Completa Mediante un Ensayo de Amplificación...

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.