Rapid molecular phenotypic antimicrobial susceptibility test for Neisseria gonorrhoeae based on propidium monoazide...

Area of Science:

• Microbiology
• Molecular Biology
• Antimicrobial Resistance

Background:

• Neisseria gonorrhoeae (NG) poses a significant global threat due to rapidly acquired antimicrobial resistance (AMR).
• Existing treatments are failing, leaving few options and highlighting the need for rapid susceptibility testing to guide therapy and preserve current antibiotics.
• Current methods for determining NG antibiotic susceptibility are slow, taking up to 3 days, which is inadequate for timely clinical decision-making.

Approach:

• Developed a novel PCR-based assay utilizing a DNA-intercalating dye and NG-specific primers/probes.
• Generated quantitative PCR (qPCR) cycle threshold (Ct) values to assess bacterial response to varying concentrations of ceftriaxone and ciprofloxacin.
• Constructed a dual-antimicrobial logistic regression model using differential Ct measurements to predict antibiotic susceptibility.

Key Points:

• The proof-of-concept model achieved an AUC of 0.93 and 84.6% categorical agreement for susceptibility.
• Individual antimicrobial predictions showed high accuracy for ceftriaxone (90% susceptible, 75% resistant) and reasonable accuracy for ciprofloxacin (66.7% susceptible, 83.3% resistant).
• The PCR assay demonstrated reliable susceptibility classification irrespective of bacterial inoculum size and provided results in under 30 minutes.

Conclusions:

• This novel PCR approach offers a rapid and accurate method for determining phenotypic susceptibility of Neisseria gonorrhoeae to key antibiotics.
• The assay significantly outperforms conventional susceptibility testing in terms of speed, providing crucial information in under 30 minutes versus 3 days.
• This rapid diagnostic tool has the potential to aid in preserving last-line antibiotics like ceftriaxone by enabling timely use of alternative treatments based on susceptibility data.