A P van Dam

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

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.

P5.077 Nucleic Acid Amplification Test (NAAT) Diagnostics Combined with Delayed Neisseria Gonorrhoeae Cultivation of NAAT Positive Samples Using the ESwab™ System - the Solution For Future Gonococcal Antimicrobial Susceptibility Surveillance?

Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (Ng) is a major public health problem worldwide. Nucleic acid amplification tests (NAATs) have rapidly replaced cultivation for detection of Ng. We have evaluated the ESwab system for NAAT diagnostics combined with delayed Ng cultivation of NAAT positive samples for gonococcal AMR surveillance. Methods Based on clinical indications, a urethral, cervical or anal swab was collected from patients with purulent discharge. Gold standard for diagnosis was the APTIMA Combo 2 assay (Gen-Probe). In another study, swabs from urine (UR) and urine sediment (US) were collected if Gram-negative diplococci were observed in direct smears. Flocked swabs were stored in ESwab Liquid Amies (Copan) at room temperature (RT) and 4°C and cultured after 1, 24 and 48 hours. Results From 35 patients with Ng positive NAAT, we obtained 34 (97%) Ng cultures from ESwab samples stored for 1 hour at RT. Storage for 24 hours at 4°C and RT resulted in 32 (91%) cultures. Storage for 48 hours at 4°C yielded 25 (71%), and at RT only 13 (37%, p = 0.007) cultures. Fourteen urine samples resulted in 13 (UR) respectively 14 (US) cultures after storage for 1 hour at RT. Storage for 24 hours at 4°C and RT resulted in 11 and 7 (UR), respectively 12 and 10 (US) cultures. Storage for 48 hours at 4°C and RT gave 3 and 1 (UR), respectively 5 and 2 (US) cultures. Conclusion Delayed Ng cultivation from the ESwab system was successful after storage at 4°C for 24 hours in 91% and for 48 hours in 71% of cases. The ESwab system for NAAT diagnostics combined with delayed Ng cultivation of positive NAAT samples appears highly effective for future sustainable and essential gonococcal AMR surveillance. This approach is now being validated in routine practise.

P3.18 Monitoringchlamydia trachomatisinfections after treatment for test of cure purposes

Introduction Performing a test of cure (TOC) could demonstrate success or failure of antimicrobial treatment of C. trachomatis (CT) infection, but the value of using a nuclear acid amplification test (NAAT) based TOC after treatment is subject to discussion, as the presence of CT nucleic acids after treatment may be prolonged and intermittent without the presence of infectious bacteria. We used cell culture to assess if a NAAT positive TOC indicates the presence of viable CT. Methods We analysed follow up (FU) data from women with a CT infection who visited the STI clinic of Amsterdam, the Netherlands, from September 2015 through June 2016. After giving informed consent, participants underwent baseline and three FU speculum examinations to obtain cervical swabs for both CT culture and NAAT testing. Speculum examinations were scheduled at 7, 21 and 49 days after treatment (single dose 1000 mg azithromycin). Collected samples were analysed using a RNA and DNA-based NAAT. CT cell culture was performed on all samples at baseline, and in FU samples that were NAAT-positive. Clearance was defined as conversion to negative NAAT results at any FU visit. Results We included 78 women with NAAT proven CT infection prior to receiving treatment of whom 58 (74%) were also culture positive. At the first visit after treatment (median 7 days; IQR 7–8) 44 (47%) women were NAAT positive, of whom three tested also positive by culture. CT infection was cleared in 73 women (94%), of whom 61 (78%) at their second FU visit (median 21 days; IQR 21–25). Of the five women who did not clear their infection, three were also culture positive indicating a viable infection. All five reported unprotected sexual contact after inclusion prior to their last FU visit, indicating potentially new infections. Conclusion We observed prolonged and intermittent positive results over time for both NAAT tests. For three participants (4%) viable CT infections were detected 49 days after treatment. All three cases reported new sexual contacts. In conclusion, persisting infections or treatment failure were rare. Support: Hologic provided Aptima test materials and kits in-kind. Roche provided Cobas test materials and kits in- kind. Copan provided Universal Transport Medium in-kind

001.4 Recent rise in reduced susceptibility to ceftriaxone in neisseria gonorrhoeae is not caused by strains with a pena mosaic gene

Background Resistance of Neisseria gonorrhoeae against third generation cephalosporins is a threat to public health. A known determinant is the presence of a mosaic penA gene in N.gonorrhoeae , partially derived from commensal Neisseria spp. We report resistance figures of N.gonorrhoeae against ceftriaxone from 2010 to 2013 and looked at penA characteristics of specific strains. Methods MICs for ceftriaxone were assesed from 2010–13 (4191 strains). A specific PCR identifying strains with a mosaic penA gene and partial sequence analysis (aa 180 – 550) of the penA gene were used for further characterisation of specific strains. Results Strains resistant to ceftriaxone were not found during the study period. The frequency of strains with an increased MIC (>0.032) to ceftriaxone was 5.2% in 2010, this rate dropped to 2.0 and 3.1% in 2011 and 2012 respectively, but increased to 7.8% in 2013. In 2010, 46/48 (96%) strains with an increased MIC against ceftriaxone contained a mosaic penA gene; in 2013, only 15/68 (22%) of such strains contained this gene. Sequence analysis of 16 of the strains isolated in 2013 with reduced susceptibility to ceftriaxone and lacking a mosaic penA gene showed that they all had an identical penA gene which was similar to type XVIII, including a 502 A-T mutation, but lacking the 543 G-S mutation. 1 Conclusion The recent increase of the frequency of strains with reduced susceptibility to ceftriaxone in 2013 is due to strains with a penA sequence not yet found in the Netherlands in 2010 among strains with reduced susceptibility to ceftriaxone. Disclosure of interest statement Nothing to declare Reference Whiley DM, Limnios EA, Ray S, et al . Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Austrlia, that are less susceptible to ceftriaxone. Antimicrob Agents Chemother . 2007; 51 :3111–6

P2.097 PCR For Direct Detection of the Mosaic Neisseria Gonorrhoeae PenA Gene in Urines and Cervical, Rectal and Tonsillar Swabs

Introduction The mosaic penA gene, partly derived from commensal Neisseria strains, is strongly associated with diminished susceptibility of Neisseria gonorrhoeae (Ng) against cephalosporins. We developed a direct PCR test for Ng-positive clinical specimens to detect the mosaic penA gene. Methods Swabs and urines from patients with gonorrhoea were in medium for NAAT testing (Aptima Combo 2). Corresponding Ng strains were obtained by culture on selective GC agar plates and stored at –80°C. Presence of a mosaic penA gene in these strains was demonstrated by PCR. Results Using one conserved forward primer and two reverse primers, specific for mosaic- and wild type PenA genes, and SYBR green as a fluorescing agent, two real-time PCRs were developed. Testing diluted DNA samples showed that the mosaic penA gene PCR was 10–100 fold more sensitive than the wild type gene PCR. Both PCRs were negative with strains belonging to N.meningitidis (n = 3), N.lactamica (n = 4), N.subflava (n = 2), N.cinerea (n = 1) and N.elongata (n = 1). Ten urine (U), 10 cervical (C), 10 rectal (R) and 10 tonsillar (T) samples, all negative in the NAAT for Ng, were negative in both PCRs. Testing paired samples from patients, who had a positive culture and NAAT (10 R, 9 U, 8 C, 9 T) showed concordant results in 35/36 samples: 4 pairs tested positive in the mosaic PCR and 31 in the wild type PCR. From one patient a wild type strain had been cultured from the throat, but both PenA PCRs on the swab were negative, possibly due to a low amount of DNA. Conclusion We successfully developed discriminating PCRs with which the Ng mosaic penA gene can be detected without culture of Ng. This test can be used to estimate the prevalence of diminished susceptibility of Ng against cephalosporins in regions where culture is no longer performed.

P3.271 Identical Multilocus Sequence Typing (MLST) Analysis in Sequential Samples from Patients with Pharyngeal Chlamydia Infections

Introduction Pharyngeal Chlamydia trachomatis (PCt) must persist to contribute to ongoing transmission. In a retrospective study, we examined MLST-types of PCt in patients who had a positive pharyngeal swab on two visits, and had not been treated for this infection at first visit. Methods From 1/1/2008 to 14/7/2010, pharyngeal swabs from patients at risk for pharyngeal gonorrhoea were tested with the AC2 (Hologic-GenProbe) test. Since at that time PCt detection was not considered to represent an infection, PCt results were not reported and patients were not treated, unless they had a chlamydial infection at another anatomic site. We looked for patients who had a positive PCt test on two different occasions with an interval of at least 3 weeks. For inclusion in the study, patients were required to have no Chlamydia infections at other anatomic locations at first visit and therefore received no treatment. PCt typing was done by MLST on stored specimens. Results Sixteen patients could be included and paired pharyngeal samples from four of those patients contained enough DNA for MLST analysis. The intervals between the two visits were 112, 168, 207 and 268 days, respectively. In all four patients MLST types of both pharyngeal samples were completely identical. Patients were two women and two men who had sex with men (MSM). At second visit one woman and one MSM reported commercial sex work and had 30 and 150 sexual partners in the last 6 months, respectively. The second woman reported sex with two known persons and the second MSM reported sex with 15 known persons. None reported sex with a steady partner. Conclusion Our findings of identical MLST types are consistent with persistent PCt infection for a period of 3–9 months, although repetitive exposure to untreated partners with identical C. trachomatis strains can not be excluded.

P5.078 False-Positive Neisseria Gonorrhoeae Results in Urine Samples Using a Highly Sensitive NAAT Tests: The Sampling Site as a Source of Contamination?

Introduction False-positive results due to contamination of NAATs have been described. Apart from the laboratory, also the area where samples from patients are collected can be the source of the contamination. Methods and results: In a 46 days period, 62 (7.3%) of male patients visiting the STI outpatient clinic with a low risk for gonorrhoea showed a positive NAAT (AC 2, Hologic-GenProbe) for Neisseria gonorrhoeae (NG) in urine. This was only 0.8% in the previous 6 months. The prevalence of positive NAAT results for Chlamydia trachomatis (CT) remained unchanged. Culture was positive in only 2/24 NG-NAAT-positive patients whose cultures were available. The prevalence of NG among high-risk patients as determined by culturing, and the positive NG-NAAT results from vaginal, rectal and pharyngeal swabs from the STI clinic and from urines received from other practises remained unchanged. All 5 environmental swabs from the male bathroom and all 4 swabs from transport trays were positive in NG-NAAT, but only 1 of these 9 was positive for CT. Swabs from trays from the laboratory, routinely cleaned with chlorine, were negative. An audit showed that some clients do not deliver their urine samples in a hygienic way and employees who transferred urine into Aptima tubes might have touched the seal of these tubes. The pseudo-outbreak ended after daily cleaning of bathrooms and trays with chlorine and strictly following anti-contamination guidelines. Afterwards only 0.2% of low-risk male patients had a positive NG-NAAT in urine. Thirty-seven patients who had been treated for gonorrhoea were informed about the possible incorrect diagnosis. Conclusion This pseudo-outbreak was most likely a consequence of external contamination of trays and test tubes with nucleic acids from the sampling site, in combination with inadequate handling of tubes during pipetting.

O21.4 In Vitro Synergy Determination For Dual Antibiotic Therapy Against Resistant Neisseria Gonorrhoeae Using Etest® and Agar Dilution

Background Antimicrobial resistance (AMR) of Neisseria gonorrhoeae (Ng) is increasing. With recent resistance to last resort extended-spectrum cephalosporins, combination therapy of azithromycin (AZ) and ceftriaxone (TX) is now widely recommended. We used 2 methods to study in vitro synergy of recommended and new dual antibiotic combinations. Methods A panel of 15 Ng strains with a minimal inhibitory concentration (MIC) of 0.064–8 for AZ and 0.012–2 for TX was tested for in vitro synergy, using both Etest and agar dilution checkerboard methods. Combinations of cefixime with AZ, colistin, ertapenem, gentamicin and moxifloxacin were also tested using the Etest method on 10 stains of the panel. Etests were placed crosswise at the MIC of each antibiotic in a 90° angle. All tests were performed in duplicate. MIC’s were red after 16–18 hours (Etest) or 24–48 hours (checkerboard) incubation. Synergy was defined as a fractional inhibitory concentration index (FICI) ≤ 0.5. Results Using the Etest method no synergy was found in any strain for any of the used combinations. Mean FICI for each combination was between 0.77–1.27. Individual FICI’s varied between 0.49–2.00. Values ≤ 0.5 could not be confirmed in repeat testing. No antagonism was found. Mean FICI for AZ+TX was 1.27 (0.58–2.00). The results of the checkerboard for AZ+TX indicated synergy for only 2 of the 15 strains (FICI: 0.16 and 0.5). The mean FICI of all strains was 0.64 (0.16–1.01). Adding AZ to TX could not lower the TX MIC below 0.25 for one TX resistant strain (MIC for TX alone: 2). Conclusion The recommended combination therapy against Ng (AZ+TX) showed no in vitro synergy using either the Etest or the agar dilution method. Other combinations of antibiotics from various groups showed no indication of in vitro synergy using the Etest method.