Rapid UTI, Sepsis Tests Uses Digital PCR

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs.

Sepsis can be caused by a very low abundance of pathogens in the bloodstream, and most diagnostic technologies require pathogen concentration, a days-long culture step, and pathogen extraction steps that have the potential to reduce sensitivity. Time to diagnosis is also critical in cases of suspected sepsis, as mortality increases 4% for every hour delay until treatment.

Scientists at the University of California, Irvine (Irvine, CA, USA) and their associates have developed a rapid diagnostic platform that integrates a novel one-step blood droplet digital polymerase chain reaction (PCR) assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps.

The team used the cefotaximase-M-9 (CTX-M-9) family extended-spectrum β-lactamases (ESBLs) as a model system, and demonstrated that their technology developed by Velox Biosystems (Irvine, CA, USA) can simultaneously achieve unprecedented high sensitivity (10 CFU per mL) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and Bio-Rad ddPCR (Bio-Rad, Hercules, CA, USA) only exhibited a limit of detection of 1,000 CFU per mL and 50–100 CFU per mL, respectively.

In the study, the team spiked whole-blood samples with a strain of Escherichia coli containing a synthetic resistance gene and was able to detect bacteria and resistance within one hour. The detection required no culture or sample processing, with a limit of detection of less than 10 colony-forming units per milliliter. The group was also able to demonstrate multiplexing ability for two targets per droplet. The study also detailed strategies to overcome the PCR inhibitory effects common to whole blood, a method to enhance the ability to partition blood into droplets by diluting it, and determined that the ideal fluorophore to get past the autofluorescence of blood was a dye called Quasar 670 from LGC Biosearch Technologies (Teddington, UK).

The team also demonstrated that the technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (bla_CTX-M-1 and bla_CTX-M-2 families) and CREs (bla_OXA-48 and bla_KPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. The study was first published on December 16, 2019 in the journal Lab on a Chip.

Related Links:
University of California, Irvine
Velox Biosystems
LGC Biosearch Technologies