Ella Trembizki

High-throughput informative single nucleotide polymorphism-based typing of Neisseria gonorrhoeae using the Sequenom MassARRAY iPLEX platform

OBJECTIVES Neisseria gonorrhoeae antimicrobial resistance (AMR) is a global problem heightened by emerging resistance to ceftriaxone. Appropriate molecular typing methods are important for understanding the emergence and spread of N. gonorrhoeae AMR. We report on the development, validation and testing of a Sequenom MassARRAY iPLEX method for multilocus sequence typing (MLST)-style genotyping of N. gonorrhoeae isolates. METHODS An iPLEX MassARRAY method (iPLEX14SNP) was developed targeting 14 informative gonococcal single nucleotide polymorphisms (SNPs) previously shown to predict MLST types. The method was initially validated using 24 N. gonorrhoeae control isolates and was then applied to 397 test isolates collected throughout Queensland, Australia in the first half of 2012. RESULTS The iPLEX14SNP method provided 100% accuracy for the control isolates, correctly identifying all 14 SNPs for all 24 isolates (336/336). For the 397 test isolates, the iPLEX14SNP assigned results for 5461 of the possible 5558 SNPs (SNP call rate 98.25%), with complete 14 SNP profiles obtained for 364 isolates. Based on the complete SNP profile data, there were 49 different sequence types identified in Queensland, with 11 of the 49 SNP profiles accounting for the majority (n = 280; 77%) of isolates. AMR was dominated by several geographically clustered sequence types. Using the iPLEX14SNP method, up to 384 isolates could be tested within 1 working day for less than Aus

Characterization of a Novel Neisseria gonorrhoeae Penicillinase-Producing Plasmid Isolated in Australia in 2012

10 per isolate. CONCLUSIONS The iPLEX14SNP offers an accurate and high-throughput method for the MLST-style genotyping of N. gonorrhoeae and may prove particularly useful for large-scale studies investigating the emergence and spread of gonococcal AMR.

Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance : a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains

Neisseria gonorrhoeae plasmids harboring a penicillinase gene are responsible for dissemination of high-level penicillin resistance among gonococci worldwide. Penicillinase-producing N. gonorrhoeae (PPNG) strains are believed to have originally acquired such plasmids via horizontal genetic transfer

A national quality assurance survey of Neisseria gonorrhoeae testing

ABSTRACT A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca ). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n = 109) were identified as unrelated singletons. NG-STAR had a high Simpsons diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n = 100; 13.0%), ST-42 and ST-91 (n = 45; 5.9%), ST-64 (n = 44; 5.72%), and ST-139 (n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

A Neisseria gonorrhoeae strain with a meningococcal mtrR sequence.

The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility

Genetic exchange between species within the Neisseria genus is well recognized (Linz et al., 2000) and can have important implications for the management of gonorrhoea, both in terms of detecting gonorrhoea by nucleic acid amplification tests and through the development of gonococcal antimicrobial resistance. For example, cross-reaction of commercial and in-house Neisseria gonorrhoeae nucleic acid amplification tests with commensal Neisseria species has been well documented (Farrell, 1999; Linz et al., 2000; Tabrizi et al., 2011). Furthermore, it is becoming increasingly clear that the acquisition of resistance determinants from commensal Neisseria species is one of the prime pathways leading to N. gonorrhoeae antimicrobial resistance; such determinants include the well-documented N. gonorrhoeae ‘mosaic’ penA sequences, which are thought to have been acquired from commensal Neisseria and are implicated in N. gonorrhoeae resistance to extended-spectrum cephalosporins (Ameyama et al., 2002; Unemo et al., 2012). Here, we report a novel ‘Neisseria meningitidis-like’ mtrR sequence in N. gonorrhoeae isolates with reduced susceptibility to azithromycin.

Opportunities and pitfalls of molecular testing for detecting sexually transmitted pathogens

OBJECTIVES The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. METHODS The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. RESULTS The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. CONCLUSIONS The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

Further evidence to support the individualised treatment of gonorrhoea with ciprofloxacin

Summary In the last 20 years, nucleic acid amplification tests (NAATs) have gradually replaced traditional methods for the detection of sexually transmitted infections. NAAT technology comes with some considerable benefits for diagnosis, including increased sensitivity, rapid result turnaround and suitability for high throughput screening of asymptomatic individuals using more-readily available specimens. However, the transition to NAAT has not come without its problems. False-negative and false-positive results have been reported owing to various technical issues. Furthermore, increased reliance on NAATs for diagnosis have created the need to develop NAAT-based methods to inform treatment, being an area that presents its own set of challenges. In this review article, we explore NAAT-based detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. In doing so, we consider the benefits and limitations of NAAT-based technology and highlight areas where further research and development is in need.

Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis

www.thelancet.com/infection Vol 16 September 2016 1005 over several months, if not years, not truly representing force of infection or disease incidence. Third, to highlight urbanisation as a major contributor to the disparity in the incidence of cutaneous leishmaniasis between countries in the past two decades is misleading. Urbanisation is not what has been driving the disparity, it is the paucity of prevention, control programming, and diagnostic and treatment services, particularly in times of war. Consequently, and contrary to the Karimkhani and colleagues’ assertions, their findings are quite different from previous studies. Although the findings confirm those of Alvar and colleagues that Afghanistan, Sudan, Syria, and Peru are among the top ten highest burden cutaneous leishmaniasis countries, Yemen, Iraq, Bolivia, Venezuela, Burkina Faso, and Haiti are not among the top ten; on the basis of published scientific literature and existing country data, Venezuela, Burkina Faso, and Haiti are particularly not deemed to be highburden countries and they exemplify why Karimkhani and colleagues’ fi ndings are not completely accurate. Compared with other endemic countries, Venezuela and Burkina Faso had few cutaneous leishmaniasis cases reported in 2011 (1551 and 1389, respectively) and Haiti is not even listed by WHO as a country with autochthonous cutaneous leishmaniasis transmission.

Multitarget PCR assay for direct detection of penicillinase-producing neisseria gonorrhoeae for enhanced surveillance of gonococcal antimicrobial resistance

In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001–2002 and 2007–2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the ‘M3L7’ subtype at this centre during 2007–2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007–2009 than 2001–2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1–13.5 and LasR: OR = 2.5; 95%CI: 1.3–5.0). Surveillance at the adult centre in 2007–2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2–39.2) and non-AUST-02 strains (adjusted HR = 4.8; 95%CI: 1.4–16.2). This suggests ongoing microevolution of the shared CF strain, AUST-02, was associated with an emerging multi-drug resistant subtype and possibly poorer clinical outcomes.