Molecular Antimicrobial Resistance Surveillance for Neisseria gonorrhoeae, Northern Territory, Australia

Emerging Infectious Disease's an open access journal in the public domain. All content is freely available without charge to the user or his/her institution.Neisseria gonorrhoeae antimicrobial resistance (AMR) is a globally recognized health threat; new strategies are needed to enhance AMR surveillance. The Northern Territory of Australia is unique in that 2 different first-line therapies, based primarily on geographic location, are used for gonorrhea treatment. We tested 1,629 N. gonorrhoeae nucleic acid amplification test-positive clinical samples, collected from regions where ceftriaxone plus azithromycin or amoxicillin plus azithromycin are recommended first-line treatments, by using 8 N. gonorrhoeae AMR PCR assays. We compared results with those from routine culture-based surveillance data. PCR data confirmed an absence of ceftriaxone resistance and a low level of azithromycin resistance (0.2%), and that penicillin resistance was <5% in amoxicillin plus azithromycin regions. Rates of ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae were lower when molecular methods were used. Molecular methods to detect N. gonorrhoeae AMR can increase the evidence base for treatment guidelines, particularly in settings where culture-based surveillance is limited

Diversity of penA Alterations and Subtypes in Neisseria gonorrhoeae Strains from Sydney, Australia, That Are Less Susceptible to Ceftriaxone▿

Increasing numbers of Neisseria gonorrhoeae strains with decreased susceptibilities to ceftriaxone and other oral cephalosporins widely used for the treatment of gonorrhea have been isolated in Sydney, Australia, over several years. In this study, we examined the complete penicillin-binding protein 2 (PBP 2) amino acid sequences of 109 gonococci, selected on the basis of their diverse temporal and geographic origins and because they exhibited a range of ceftriaxone MICs: ≤0.03 μg/ml (n = 59), 0.06 μg/ml (n = 43), and 0.125 μg/ml (n = 7). Auxotyping, serotyping, and genotyping by N. gonorrhoeae multiantigen sequence typing sequence-based analysis was also performed. In total, 20 different amino acid sequence patterns were identified, indicating considerable variation in the PBP 2 sequences in this study sample. Only some of the N. gonorrhoeae isolates with significantly higher ceftriaxone MICs contained a mosaic PBP 2 pattern, while more isolates exhibited a nonmosaic PBP 2 pattern containing an A501V substitution. Although particular N. gonorrhoeae genotypes in our sample were shown to be less susceptible to ceftriaxone, the reduced susceptibility to ceftriaxone was not specific to any particular genotype and was observed in a broad range of auxotypes, serotypes, and genotypes. Overall, the results of our study show that N. gonorrhoeae strains exhibiting reduced sensitivity to ceftriaxone are not of a particular subtype and that a number of different mutations in PBP 2 may contribute to this phenomenon

Dynamics of Appearance and Expansion of a Prolyliminopeptidase- Negative Subtype among Neisseria gonorrhoeae Isolates Collected in Sydney, Australia, from 2002 to 2005

Recent studies have demonstrated a wide geographic circulation of isolates of Neisseria gonorrhoeae that fail to produce prolyliminopeptidase (PIP). Tests based on the production of this enzyme are important elements of a number of identification systems for gonococci. We documented the appearance, expansion, and contraction of subtypes of 165 PIP-negative gonococci detected in an extended and systematic sample of the 3,926 N. gonorrhoeae isolates collected in Sydney, Australia, from July 2002 to September 2005. Their appearance, peak, and decline followed an “epidemic” curve. At the peak of their prevalence in 2003, PIP-negative gonococci comprised 22% of all isolates. Closely related phenotypes accounted for 162/165 of the PIP-negative gonococci. Algorithms for confirmation of N. gonorrhoeae should take account of the temporal and geographic variability of gonococci by utilizing two or more distinct confirmatory methods

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

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

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target

Aims: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR