Parallel susceptibility testing of bacteria through culture-quantitative PCR in 96-well plates

Abstract

With rapidness to obtain results and the capabilities for automation and multiple-sample testing, the parallel culture-qPCR method would have great potentials in clinical labs.

Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction

“…The use of the universal BactQuant 16S amplification system simplified the assay, requiring only a single set of primers and probes for testing of all bacterial pathogens. Further improvement of this assay could be achieved through the application of recently reported thermo-cold lysis for DNA extraction, potentially decreasing hands-on time and operator error [21]. Overall, our assay showed high categorical agreement at 96.3% while representing a significant decrease in total assay time compared to a similar qPCR AST assay from blood culture.…”

“…Bacterial strains susceptible to the given antibiotic will display growth inhibition resulting in significantly lower concentrations of nucleic acid, measurable by quantitative PCR (qPCR) or other amplification technologies [18]. Previous studies utilizing this approach showed promising results with high levels of categorical agreement with gold standard assays [18–21]. In fact, the method adequately determined susceptibility across multiple classes of antibiotics and was applicable to a variety of bacterial pathogens depending on the primer (and probe) design [19, 21].…”

“…Previous studies utilizing this approach showed promising results with high levels of categorical agreement with gold standard assays [18–21]. In fact, the method adequately determined susceptibility across multiple classes of antibiotics and was applicable to a variety of bacterial pathogens depending on the primer (and probe) design [19, 21]. More recent studies focused on reducing the overall assay time-to-answer, with specific focus on rapid results obtained through the use of isothermal amplification technologies [20].…”

Rapid ultrasensitive diagnosis of pneumonia caused by Acinetobacter baumannii using a combination of enrichment and phage-based qPCR assay

“…Otherwise, the method can be applied for antibiotics-resistant detection through directly culturing the sputum samples with antibiotics for 1 or 2 hours, [28,29] and then the amount of the bacteria will be different for drug-sensitive and drug-resistant bacteria. Otherwise, there cases of pulmonary infection based on phage therapy are increasing [30,31] and this method provides ideas for phage screening and monitoring in phage therapy.…”

A Fundamental Change in Antibiotic Susceptibility Testing Would Better Prevent Therapeutic Failure: From Individual to Population-Based Analysis

“…A new generation of nucleic acid amplification tests (NAATs) can measure species-specific growth rates of bacteria in the original sample in response to different antibiotics (Rolain et al, 2004;Maxson et al, 2018). These tests require ∼2-4 h of in vitro incubation, which would be a significant improvement in turnaround time (Luo et al, 2018). Furthermore, the current operative cost of these PCRbased tests can be below equivalent cost related to traditional plate-based tests.…”