Analytical evaluation of GeneXpert CT/NG, the first genetic point-of-care assay for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis.

GeneXpert CT/NG was evaluated with 372 characterized bacterial strains. Sensitivity of 10 genome copies/reaction was obtained for both agents. Four Neisseria mucosa and two Neisseria subflava isolates were positive for one of two gonococcal targets; however, the assay flagged all as negative. The assay was analytically highly sensitive and specific.

Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes.

OBJECTIVES: Neisseria gonorrhoeae multilocus sequence typing (MLST) is a key tool used to investigate the macroepidemiology of gonococci exhibiting antimicrobial resistance (AMR). However, the utility of MLST is undermined by the high workload and cost associated with DNA sequencing of seven housekeeping genes. In this study, we investigated single nucleotide polymorphism (SNP)-based profiling as a means of circumventing these problems. METHODS: A total of 14 SNPs were selected following in silico analysis of available N. gonorrhoeae MLST sequence data. Real-time PCR methods were developed for characterization of each SNP and applied to 86 N. gonorrhoeae isolates exhibiting a range of ceftriaxone MICs. Twenty-one isolates had previously been characterized by MLST. The ability of the real-time PCR methods to generate SNP profiles and of the 14 SNP profiles to predict MLST types were assessed. RESULTS: In silico analysis of the 217 different MLST types available on the Neisseria web site showed 181 different 14 SNP profiles (Simpson's index of diversity = 0.998). When the real-time PCR methods were applied to the isolates, 29 different 14 SNP profiles were obtained for 83 isolates. Predicted MLST types were consistent with those for the 21 isolates previously characterized by MLST. For 46 isolates with raised ceftriaxone MICs (≥ 0.03 mg/L), there were 14 different 14 SNP profiles observed, with two profiles accounting for more than half of these isolates. CONCLUSIONS: The 14 SNP real-time PCR profiling approach is a simple and cost-effective alternative to N. gonorrhoeae MLST and could be used to complement current typing schemes in N. gonorrhoeae AMR investigations.

The influence of target population on nonculture-based detection of markers of Neisseria gonorrhoeae antimicrobial resistance.

BACKGROUND: With treatment options for gonorrhoea (Neisseria gonorrhoeae) diminishing, strengthening antimicrobial resistance (AMR) surveillance is paramount. METHODS: In this study, we investigated polymerase chain reaction (PCR) based methods, in parallel with N. gonorrhoeae multi-antigen sequence typing (NG-MAST), for direct detection of four N. gonorrhoeae chromosomal mechanisms associated with emerging resistance to extended spectrum cephalosporins using noncultured samples: an adenine deletion in the mtrR promoter, a mosaic penicillin-binding protein (PBP) 2, an A501V PBP2 mutation, and alterations at positions 120 and 121 of the porB protein. The PCR assays were validated using a panel of characterised N. gonorrhoeae isolates (n=107) and commensal Neisseria (n=100) species. These PCR assays with NG-MAST were then applied to noncultured clinical specimens from distinct populations in Australia with differing levels of N. gonorrhoeae AMR: the Northern Territory (NT), where resistance has a low population prevalence, and Queensland (Qld), with higher AMR prevalence. RESULTS: The real-time PCR assays proved highly sensitive and specific. When applied to the noncultured samples, only 1 out of 50 (2%) samples from NT harboured a resistant mechanism, whereas the Qld samples (n=129) collected over different periods showed progressive acquisition of resistant mechanisms, and these were associated with specific NG-MAST types, including Type 225. CONCLUSIONS: The results suggest that our PCR-based methods could be used to rapidly pinpoint incursion of resistant strains into previously unaffected populations. Likewise, our results show that for molecular AMR surveillance, the population being investigated is as important as the genetic mechanisms being targeted.

The ticking time bomb: escalating antibiotic resistance in Neisseria gonorrhoeae is a public health disaster in waiting.

From a once easily treatable infection, gonorrhoea has evolved into a challenging disease, which in future may become untreatable in certain circumstances. International spread of extensively drug-resistant gonococci would have severe public health implications. It seems clear that under the current treatment pressure from extended-spectrum cephalosporins, and owing to Neisseria gonorrhoeae's remarkable evolutionary adaptability, further rise of ceftriaxone-resistant strains around the world is inevitable. Simply increasing the doses of extended-spectrum cephalosporins will likely prove ineffective in the long run, and has been a lesson learnt for all single-agent therapies used for gonorrhoea to date. We recommend that dual therapy, especially those consisting of extended-spectrum cephalosporins and azithromycin, be adopted more widely and complemented by strengthening of antimicrobial resistance surveillance. Unless there is urgent action at international and local levels to combat the problem of N. gonorrhoeae antimicrobial resistance, we are in for gloomy times ahead in terms of gonorrhoea disease and control.

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

OBJECTIVES: 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. METHODS: 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 (n = 167 strains) including the N. gonorrhoeae H041 strain and clinical specimens (n = 252) submitted for sexual health screening. The detection limits of the assays were compared with a standard N. gonorrhoeae real-time PCR method. RESULTS: 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. CONCLUSIONS: The H041-PCR2 assay is suitable for direct detection of the N. gonorrhoeae H041 ceftriaxone-resistant strain in cultured and non-cultured samples.

Evaluation of six commercial nucleic acid amplification tests for detection of Neisseria gonorrhoeae and other Neisseria species.

Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites.