Introduction The mosaic penA gene, partly derived from commensal Neisseria strains, is strongly associated with diminished susceptibility of Neisseria gonorrhoeae (Ng) against cephalosporins. We developed a direct PCR test for Ng-positive clinical specimens to detect the mosaic penA gene.
Methods Swabs and urines from patients with gonorrhoea were in medium for NAAT testing (Aptima Combo 2). Corresponding Ng strains were obtained by culture on selective GC agar plates and stored at –80°C. Presence of a mosaic penA gene in these strains was demonstrated by PCR.
Results Using one conserved forward primer and two reverse primers, specific for mosaic- and wild type PenA genes, and SYBR green as a fluorescing agent, two real-time PCRs were developed. Testing diluted DNA samples showed that the mosaic penA gene PCR was 10–100 fold more sensitive than the wild type gene PCR. Both PCRs were negative with strains belonging to N.meningitidis (n = 3), N.lactamica (n = 4), N.subflava (n = 2), N.cinerea (n = 1) and N.elongata (n = 1). Ten urine (U), 10 cervical (C), 10 rectal (R) and 10 tonsillar (T) samples, all negative in the NAAT for Ng, were negative in both PCRs. Testing paired samples from patients, who had a positive culture and NAAT (10 R, 9 U, 8 C, 9 T) showed concordant results in 35/36 samples: 4 pairs tested positive in the mosaic PCR and 31 in the wild type PCR. From one patient a wild type strain had been cultured from the throat, but both PenA PCRs on the swab were negative, possibly due to a low amount of DNA.
Conclusion We successfully developed discriminating PCRs with which the Ng mosaic penA gene can be detected without culture of Ng. This test can be used to estimate the prevalence of diminished susceptibility of Ng against cephalosporins in regions where culture is no longer performed.
- ceftriaxone resistance
- Neisseria gonorrhoeae