Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n ‫؍‬ 34) and N. gonorrhoeae clinical isolates (n ‫؍‬ 176) but not isolates of the 13 different nongonococcal Neisseria species (n ‫؍‬ 68) that we tested. Furthermore, a panel of gram-negative bacterial (n ‫؍‬ 18), gram-positive bacterial (n ‫؍‬ 23), fungal (n ‫؍‬ 1), and viral (n ‫؍‬ 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/ PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.

The present porA pseudogene real-time PCR comprises a valuable supplement to the traditional culture techniques for diagnosis of N. gonorrheae, especially for samples from extragenital sites such as pharynx and rectum.

Culture is commonly regarded as the gold standard for diagnosis of Neisseria gonorrhoeae. However, nucleic acid amplification tests (NAATs) have rapidly replaced culture for diagnostics in many settings. The aim of the present study was to investigate the appropriate time for test-of-cure (TOC) when NAATs are used for diagnosis of gonorrhoea. In total, 30 patients (28 men and 2 women) provided urethral, cervical, rectal or pharyngeal specimens for TOC. All included patients, except one who did not return for second TOC before day 19, tested negative within 2 weeks after treatment with cefixime 400 mg × 1. Antimicrobial susceptibility testing showed that 68% of the culture-positive strains were resistant to ciprofloxacin. Thus, the recommended empirical treatment with ciprofloxacin in Norway should be changed immediately. TOC can be performed 2 weeks after treatment when NAATs are used for diagnosis of gonorrhoea.