The culture-free, “biphasic” approach enables pathogen DNA to be amplified directly from just 1 ml of whole blood, reducing the time needed for a result to less than 2.5 hours compared with more than 20 hours using conventional methods.
The system offers single-molecule sensitivity in detecting pathogens including methicillin-resistant and methicillin-sensitive Staphylococcus aureus, Escherichia coli, and Candida albicans.
Validation studies using 63 whole-blood samples showed total agreement in sensitivity and specificity with clinical laboratory results that used blood culture and polymerase chain reaction (PCR).
The new system, described in the Proceedings of the National Academy of Sciences, involves rapidly drying blood and creating a porous microfluidic and nanofluidic network within this.
DNA amplification enzymes and primers are then able to diffuse into the dried blood matrix to access any pathogen DNA within, initiating its amplification without the need for conventional nucleic acid purification.
Researcher Rashid Bashir, professor of bioengineering at the University of Illinois at Urbana-Champaign, U.S.A., told Inside Precision Medicine that traditional blood culture could take many days to provide enough bacteria for subsequent PCR detection.
“Alternatively, our approach using a new blood drying technique can be used to detect pathogens in less than a few hours. This can potentially be very important for rapid and early detection of onset of sepsis caused by bacteremia.”
Antibiotic therapy within three hours of initial, symptom-based recognition can significantly reduce the risk of death from bloodstream infections and bacteremia, the researchers note.
However, the current clinical gold standard for diagnosing sepsis and bloodstream infections remains blood culture followed by nucleic acid amplification and detection using PCR.
“The blood culture step is too slow and cumbersome to allow for initial management of patients and thus contributes to high mortality,” the authors explain.
“Moreover, in the absence of timely results from robust diagnostic tests, the patients are administered highly potent broad-spectrum antibiotics without any patient stratification, increasing antimicrobial resistance and emergence of drug-resistant and atypical pathogens.”
The new platform uses whole blood, which can be dried in as little as 10 minutes using high temperatures of 95 °C. The dried blood then acts as a substrate that does not take part in the reaction, and inhibitory elements such as platelets, cells and proteins are neutralized to form part of this substrate.
Thermal lysis improves the porosity of microfluidic and nanofluidic networks within the dried blood matrix, which allows enzymes to access pathogen DNA and initiate amplification with single-molecule sensitivity, thereby bypassing the need for conventional DNA purification.
The dried blood solid phase does not re-mix with the supernatant and keeps the high heme locked in red blood cells in the background while amplicons diffuse out and bind to fluorescent dye in the clear supernatant phase, leading the researchers to term this “biphasic amplification”.
Reporting their findings, they propose: “The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.”