Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain

Abstract

Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

Preparing for an era of untreatable gonorrhea

“…Some public health programs and STD clinics have chosen to culture all symptomatic urethritis as well as screen-positive sites in yet untreated persons. Additionally, many research groups are working on molecular methods to identify resistant gonococcal strains directly from the NAAT-based sample;[54] however, none are commercially available yet.…”

World Health Organization Global Gonococcal Antimicrobial Surveillance Program (WHO GASP): review of new data and evidence to inform international collaborative actions and research efforts

“…77 PCRs detecting specific ceftriaxone-resistant strains such as H041, 9 F89 10 and FC428 15 have been developed. [82][83][84] The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR; https://ngstar.canada.ca, accessed 28 June 2019), examining seven AMR determinants, has standardised the nomenclature of AMR determinants and can be used to track AMR strains and indirectly AMR prediction. 73 Whole-genome sequencing (WGS) can elucidate the emergence, transmission and evolution of AMR and national and international spread of AMR gonococcal strains, but can also be used to predict AMR with relatively adequate accuracy.…”

Molecular tests for the detection of antimicrobial resistant Neisseria gonorrhoeae

“…Molecular tests for surveillance can also be used to monitor specific strains. For example, real-time PCR assays have been developed to screen clinical specimens for extensively drug resistant gonococcal strains ('superbugs') that were first detected in Japan (H041) [29] and subsequently in Europe (F89) [24].…”

“…An antibiogram gives information about susceptibility to multiple antimicrobials. Most molecular assays detect resistance determinants and relate to a single antibiotic (penicillin [19,30,44 & ]; ; or a single strain [24,29]. Molecular assays aiming to detect resistance to several antimicrobials have been run in series [26] or as multiplex reactions [27 & ,36], but their analytical sensitivity was too low to be used in clinical specimens [36] or, if assessed on clinical specimens, was not validated against a culture-based MIC reference standard [26,27 & ].…”