Identifying viable Neisseria gonorrhoeae through validation and application of viability RT-PCR

Gonorrhea, caused by Neisseria gonorrhoeae (NG), is rising globally alongside increasing antimicrobial resistance, including to first-line treatment. Standard diagnostics, such as nucleic acid amplification tests, cannot distinguish viable from non-viable bacteria, while culture-an inherent viability assessment-is challenged by the organism's fastidious nature and environmental sensitivity. Molecular viability assays have not been applied to NG. This study aims to validate and apply a viability PCR (V-PCR) method for NG and perform an exploratory application on clinical samples to assess concordance with culture. Viability was assessed using PMAxx, a membrane-impermeable dye that binds nucleic acids in non-viable cells, enabling discrimination from viable cells. Technical validation used mixtures of viable and heat-killed NG with paired PMAxx-treated/PMAxx-untreated aliquots and subsequent porA qPCR to derive ΔCt values. In an exploratory application, clinical samples (anorectal, oropharyngeal, and urogenital) were tested using V-PCR and compared with culture. V-PCR eliminated 99.998% of DNA from non-viable NG, with a strong correlation (R²=0.96) across dilution series. Culture-positive clinical samples had a higher median viable load (3.92 log₁₀ copies/mL, interquartile range [IQR]: 1.71-5.23) than culture-negative samples (0.87 log₁₀ copies/mL, IQR: 0.38-2.69, P = 0.0003). Median viability percentage was significantly higher in culture-positive samples (16.92%, IQR: 0.41%-42.45%) versus culture-negative samples (1.23%, IQR: 0.00%-9.38%, P = 0.01). Overall, V-PCR reliably discriminated viable from non-viable NG in controlled experiments. In an exploratory application on clinical specimens, higher viable loads and viability percentages were associated with culture positivity. These findings support further evaluation of V-PCR as a tool to study factors influencing culture recovery; formal validation would be required before clinical implementation.IMPORTANCEGonorrhea affects an estimated 82 million people annually, with rising antimicrobial resistance threatening its treatability. Accurate diagnostics are essential, yet a diagnostic gap persists: standard PCR cannot distinguish viable from non-viable Neisseria gonorrhoeae, while culture-an inherent viability assessment-is challenged by the organism's fastidious nature and environmental sensitivity. This limits accurate viability assessment, which is relevant to diagnostic practice, antimicrobial resistance surveillance, and broader research into gonococcal infection.PMAxx-based viability PCR (V-PCR) has shown promise in other pathogens, including Chlamydia trachomatis and SARS-CoV-2, but had not been applied to N. gonorrhoeae. Here, V-PCR is validated for N. gonorrhoeae for the first time, demonstrating strong discrimination between viable and non-viable organisms with consistent performance across predefined viability ratios, and in an exploratory clinical application, viable load and viability percentage were significantly higher in culture-positive specimens. These findings establish a foundation for more accurate viability assessment in gonococcal diagnostics and research.