Culture-free genotyping of Neisseria gonorrhoeae revealed distinct strains at different anatomical sites in a quarter of patients, the Netherlands, 2012 to 2016

Background Genotyping of Neisseria gonorrhoeae (NG) is essential for surveillance to monitor NG transmission and dissemination of resistant strains. Current genotyping methods rely on bacterial culture which frequently fails. Aim Our aim was to develop a culture-free genotyping method that is compatible with the widely used N. gonorrhoeae multi-antigen sequence typing (NG-MAST) database, which facilitates genotyping of NG detected at separate anatomical sites in individual patients. Methods Specific primers for both PCR targets porB and tbpB were designed and technically validated by assessing the analytical sensitivity, cross-reactivity with 32 non-gonoccocal Neisseria species, and concordance with NG-MAST. Clinical application was assessed on 205 paired samples from concurrent NG infections at different anatomical sites of 98 patients (81 men who have sex with men and 17 women) visiting our sexually transmitted infections clinic. Results Typing could be consistently performed on samples with a PCR quantification cycle (Cq) value <35. Furthermore, the method showed no cross-reactivity and was concordant with NG-MAST. Culture-free NG-MAST improved the typing rate from 62% (59/95) for cultured samples to 94% (89/95) compared with culture-dependent NG-MAST. Paired samples of 80 of 98 patients were genotyped, revealing distinct NG strains in separate anatomical sites in 25% (20/80) of the patients. Conclusions This NG-specific genotyping method can improve NG surveillance as it facilitates genotyping of non-culturable and extra-genital samples. Furthermore, 25% of patients were infected with multiple NG strains, which is missed in current culture-dependent surveillance. Including non-culturable and concurrent NG infections in surveillance informs actions on dissemination of multidrug-resistant NG strains.