Review on biphasic blood drying method for rapid pathogen detection in bloodstream infections

Rapid and accurate detection of pathogenic microorganisms in blood is critical for diagnosing life-threatening conditions such as bloodstream infections (BSIs). Current methods for the detection and identification of bacteria from large volumes of blood (5 mL) involve culture steps followed by DNA extraction/purification/concentration and Polymerase Chain Reaction (PCR)-based nucleic acid amplification. DNA extraction and amplification directly from blood samples is hampered by the complexity of the blood matrix, resulting in time-consuming and labor-intensive processes. This review delves into recent advancements in molecular diagnostics based on blood drying, coined as 'biphasic reaction', and highlights this new technique that attempts to overcome the limitations of traditional sample preparation and amplification processes. The biphasic blood drying method, in combination with isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA), has recently been shown to improve the sensitivity of detection of bacterial, viral, and fungal pathogens from ∼1 mL of whole blood, while minimizing DNA loss and avoiding the use of extraction/purification/concentration kits. Furthermore, the biphasic approach in combination with LAMP has been shown to be a culture-free method capable of detecting bacteria in clinical samples with a sensitivity of ∼1 CFU/mL in ∼2.5 h. This represents a significant reduction in detection and identification time compared to current clinical procedures based on bacterial culture prior to PCR amplification. This review paper aims to be a guide to identify new opportunities for future advancements and applications of the biphasic technology.