Enhanced gonococcal antimicrobial surveillance in the era of ceftriaxone resistance: a real-time PCR assay for direct detection of the Neisseria gonorrhoeae H041 strain

Recent emergence of the extensively drug-resistant Neisseria gonorrhoeae H041 strain in Japan raises concerns that gonorrhoea may soon become untreatable and emphasizes the need for enhanced surveillance. In this study we developed a real-time PCR assay for direct detection of the H041 strain. Two real-time PCR assays for detection of the penA gene of the H041 strain, H041-PCR1 and H041-PCR2, were developed and evaluated in parallel. Assay performance was assessed using a panel of pathogenic and commensal Neisseria species (n167 strains) including the N. gonorrhoeae H041 strain and clinical specimens (n252) submitted for sexual health screening. The detection limits of the assays were compared with a standard N. gonorrhoeae real-time PCR method. Both the H041-PCR1 and H041-PCR2 assays correctly detected the N. gonorrhoeae H041 strain and provided negative results for all other N. gonorrhoeae strains. However, only the H041-PCR2 assay proved to be specific when applied to the non-gonococcal Neisseria species and clinical samples. False-positive results in the H041-PCR1 included cross-reactions with two Neisseria subflava isolates and eight clinical specimens. DNA sequencing of these N. subflava strains revealed the presence of the penicillin-binding protein 2 Ala328Thr alteration previously only observed in the N. gonorrhoeae H041 strain. The H041-PCR2 assay is suitable for direct detection of the N. gonorrhoeae H041 ceftriaxone-resistant strain in cultured and non-cultured samples.