Rapid molecular phenotypic antimicrobial susceptibility test forNeisseria gonorrhoeaebased on propidium monoazide viability PCR

Abstract

Neisseria gonorrhoeae(NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof of concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90% and 75% susceptible and resistant strains respectively to ceftriaxone and 66.7% and 83.3% susceptible and resistant strains respectively to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy in under 30 min, a significant improvement compared to the conventional method which takes 3 days.Table of Content Graphic

Effect of Propidium Monoazide in the Detection of Enterotoxigenic Escherichia coli in the Pediatric

“…Previous research has indicated that chronic infection can persist despite antibiotic use and negative culture results due to the presence of VBNC bacteria (33). These findings highlight the limitations of culture techniques and the need for more advanced molecular techniques, which offer greater sensitivity, specificity, and efficiency (34,35). While some studies continue to advocate for the culture approach, many confirm the precision and effectiveness of the PCR method.…”

“…Real-time PCR can detect both the genus and species of bacteria and provide a precise quantification, while the culture method only detects the genus, and further biochemical and sugar fermentation studies are required for species identification, leading to longer turnaround times (33,34). To reliably detect a signal, a sufficient concentration of DNA is necessary, and the limit of detection indicates the lowest DNA concentration that can be reliably distinguished from a blank sample (35). The sensitivity of a test reflects its ability to correctly identify a patient, with higher sensitivity leading to a lower false negative rate and better identification of disease cases (36).…”