Carolien M. Wind

Determination of in vitro synergy for dual antimicrobial therapy against resistant Neisseria gonorrhoeae using Etest and agar dilution

In response to antimicrobial resistance of Neisseria gonorrhoeae to last-resort extended-spectrum cephalosporins, combination therapy of azithromycin+ceftriaxone is now recommended. Dual therapy can be effective to treat monoresistant strains as well as multidrug-resistant strains, preferably employing the effect of in vitro synergy. As reports on in vitro synergy of azithromycin+ceftriaxone in N. gonorrhoeae are conflicting, in this study an evaluation of this combination was performed using a cross-wise Etest method and agar dilution. Synergy was defined as a fractional inhibitory concentration index (FICI) of ≤0.5. To identify other dual treatment options for gonorrhoea, in vitro synergy was evaluated for 65 dual antimicrobial combinations using Etest. Azithromycin, cefixime, ceftriaxone, colistin, ertapenem, fosfomycin, gentamicin, minocycline, moxifloxacin, rifampicin, spectinomycin and tigecycline were screened for synergy in all possible combinations. No synergy or antagonism was found for any of the 65 combinations. The geometric mean FICI ranged from 0.82 to 2.00. The mean FICI of azithromycin+ceftriaxone was 1.18 (Etest) and 0.55 (agar dilution). The difference between both methods did not result in a difference in interpretation of synergy. Ceftriaxone-resistant strain F89 was tested in all combinations and no synergy was found for any of them. Most importantly, the ceftriaxone minimum inhibitory concentration of F89 was not decreased below the breakpoint with any concentration of azithromycin.

Decreased Azithromycin Susceptibility of Neisseria gonorrhoeae Isolates in Patients Recently Treated with Azithromycin

Background Increasing azithromycin usage and resistance in Neisseria gonorrhoeae threatens current dual treatment. Because antimicrobial exposure influences resistance, we analyzed the association between azithromycin exposure and decreased susceptibility of N. gonorrhoeae. Methods We included N. gonorrhoeae isolates of patients who visited the Amsterdam STI Clinic between 1999 and 2013 (t0), with another clinic visit in the previous 60 days (t-1). Exposure was defined as the prescription of azithromycin at t-1. Using multivariable linear regression, we assessed the association between exposure and azithromycin minimum inhibitory concentration (MIC). Whole genome sequencing (WGS) was performed to produce a phylogeny and identify multilocus sequence types (MLST), N. gonorrhoeae multiantigen sequence types (NG-MAST), and molecular markers of azithromycin resistance. Results We included 323 isolates; 212 were unexposed to azithromycin, 14 were exposed ≤30 days, and 97 were exposed between 31 and 60 days before isolation. Mean azithromycin MIC was 0.28 mg/L (range, <0.016-24 mg/L). Linear regression adjusted for age, ethnicity, infection site, and calendar year showed a significant association between azithromycin exposure ≤30 days and MIC (β, 1.00; 95% confidence interval, 0.44-1.56; P = .002). WGS was performed on 31 isolates: 14 unexposed, 14 exposed to azithromycin ≤30 days before isolation, and 3 t-1 isolates. Exposure to azithromycin was significantly associated with A39T or G45D mtrR mutations (P = .046) but not with MLST or NG-MAST types. Conclusions The results suggest that frequent azithromycin use in populations at high risk of contracting N. gonorrhoeae induces an increase in MIC and may result in resistance.

Successful Combination of Nucleic Acid Amplification Test Diagnostics and Targeted Deferred Neisseria gonorrhoeae Culture

ABSTRACT Nucleic acid amplification tests (NAATs) are recommended for the diagnosis of N. gonorrhoeae infections because of their superior sensitivity. Increasing NAAT use causes a decline in crucial antimicrobial resistance (AMR) surveillance data, which rely on culture. We analyzed the suitability of the ESwab system for NAAT diagnostics and deferred targeted N. gonorrhoeae culture to allow selective and efficient culture based on NAAT results. We included patients visiting the STI Clinic Amsterdam, The Netherlands, in 2013. Patient characteristics and urogenital and rectal samples for direct N. gonorrhoeae culture, standard NAAT, and ESwab were collected. Standard NAAT and NAAT on ESwab samples were performed using the Aptima Combo 2 assay for N. gonorrhoeae and C. trachomatis. Two deferred N. gonorrhoeae cultures were performed on NAAT-positive ESwab samples after storage at 4°C for 1 to 3 days. We included 2,452 samples from 1,893 patients. In the standard NAAT, 107 samples were N. gonorrhoeae positive and 284 were C. trachomatis positive. The sensitivities of NAAT on ESwab samples were 83% (95% confidence interval [CI], 75 to 90%) and 87% (95% CI, 82 to 90%), respectively. ESwab samples were available for 98 of the gonorrhea-positive samples. Of these, 82% were positive in direct culture and 69% and 56% were positive in the 1st and 2nd deferred cultures, respectively (median storage times, 27 and 48 h, respectively). Deferred culture was more often successful in urogenital samples or when the patient had symptoms at the sampling site. Deferred N. gonorrhoeae culture of stored ESwab samples is feasible and enables AMR surveillance. To limit the loss in NAAT sensitivity, we recommend obtaining separate samples for NAAT and deferred culture.

Trends in antimicrobial susceptibility for azithromycin and ceftriaxone in Neisseria gonorrhoeae isolates in Amsterdam, the Netherlands, between 2012 and 2015

Resistance of Neisseria gonorrhoeae to azithromycin and ceftriaxone has been increasing in the past years. This is of concern since the combination of these antimicrobials is recommended as the first-line treatment option in most guidelines. To analyse trends in antimicrobial resistance, we retrospectively selected all consultations with a positive N. gonorrhoeae culture at the sexually transmitted infection clinic, Amsterdam, the Netherlands, from January 2012 through September 2015. Minimum inhibitory concentrations (MICs) for azithromycin and ceftriaxone were analysed per year, and determinants associated with decreased susceptibility to azithromycin (MIC > 0.25 mg/L) or ceftriaxone (MIC > 0.032 mg/L) were assessed. Between 2012 and 2015 azithromycin resistance (MIC > 0.5 mg/L) was around 1.2%, the percentage of isolates with intermediate MICs (> 0.25 and ≤ 0.5 mg/L) increased from 3.7% in 2012, to 8.6% in 2015. Determinants associated with decreased azithromycin susceptibility were, for men who have sex with men (MSM), infections diagnosed in the year 2014, two infected sites, and HIV status (HIV; associated with less decreased susceptibility); for heterosexuals this was having ≥ 10 sex partners (in previous six months). Although no ceftriaxone resistance (MIC > 0.125 mg/L) was observed during the study period, the proportion of isolates with decreased ceftriaxone susceptibility increased from 3.6% in 2012, to 8.4% in 2015. Determinants associated with decreased ceftriaxone susceptibility were, for MSM, infections diagnosed in 2014, and pharyngeal infections; and for heterosexuals, infections diagnosed in 2014 or 2015, being of female sex, and having ≥ 10 sex partners. Continued decrease of azithromycin and ceftriaxone susceptibility will threaten future treatment of gonorrhoea. Therefore, new treatment strategies are warranted.

Test of cure for anogenital gonorrhoea using modern RNA-based and DNA-based nucleic acid amplification tests - a prospective cohort study

BACKGROUND The use of nucleic acid amplification tests (NAATs) to diagnose Neisseria gonorrhoeae infections complicates the performance of a test of cure (TOC) to monitor treatment failure, if this is indicated. As evidence for the timing of TOC using modern NAATs is limited, we performed a prospective cohort study to assess time to clearance when using modern RNA- and DNA-based NAATs. METHODS We included patients with anogenital gonorrhoea visiting the Sexually Transmitted Infection Clinic Amsterdam from March through October 2014. After treatment with ceftriaxone mono- or dual therapy (with azithromycin or doxycycline), anal, vaginal, or urine samples were self-collected during 28 consecutive days, and analyzed using an RNA-based NAAT (Aptima Combo 2) and a DNA-based NAAT (Cobas 4800). Clearance was defined as 3 consecutive negative results, and blips as isolated positive results following clearance. RESULTS We included 77 patients; 5 self-cleared gonorrhoea before treatment and 10 were lost to follow-up. Clearance rate of the remaining 62 patients was 100%. Median time to clearance was 2 days, with a range of 1-7 days for RNA-based NAAT and 1-15 days for DNA-based NAAT. The risk of finding a blip after clearance was 0.8% and 1.5%, respectively. One patient had a reinfection. CONCLUSIONS If indicated, we recommend that TOC be performed for anogenital gonorrhoea at least 7 or 14 days after administering therapy, when using modern RNA- or DNA-based NAATs, respectively. When interpreting TOC results for possible treatment failure, both the occurrence of blips and a possible reinfection need to be taken into account.

A Case-Control Study of Molecular Epidemiology in Relation to Azithromycin Resistance in Neisseria gonorrhoeae Isolates Collected in Amsterdam, the Netherlands, between 2008 and 2015

ABSTRACT Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin is increasing, which threatens the recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n = 143) were selected from patients visiting the Amsterdam STI Outpatient Clinic from January 2008 through September 2015. We included all 69 azithromycin-resistant isolates (MIC ≥ 2.0 mg/liter) and 74 frequency-matched susceptible controls (MIC ≤ 0.25 mg/liter). The methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae multilocus variable-number tandem-repeat analysis (NG-MLVA), and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin-resistant isolates had C2611T mutations in 23S rRNA (n = 62, 89.9%, P < 0.001) and were NG-MAST genogroup G2992 (P < 0.001), G5108 (P < 0.001), or G359 (P = 0.02) significantly more often than susceptible isolates and were more often part of NG-MLVA clusters (P < 0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) had wild-type 23S rRNA. No association between mtrR mutations and azithromycin resistance was found. Twenty-four isolates, including 10 azithromycin-resistant isolates, showed reduced susceptibility to extended-spectrum cephalosporins. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates included strains from men who have sex with men and from heterosexual males and females. The co-occurrence of resistant and susceptible strains in NG-MLVA clusters and the frequent occurrence of resistant strains outside of clusters suggest that azithromycin resistance develops independently from the background genome.

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.

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) techniques

BackgroundControl of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions.MethodsIn 2014, urogenital Ng strains were isolated from clients of sexually transmitted infection clinics in three Indonesian cities. Strains were typed using Multiple-Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) and Ng Multi-Antigen Sequence Typing (NG-MAST) at the Public Health Service, Amsterdam, the Netherlands, and compared to Dutch strains collected from 2012 to 2015. Minimum spanning trees (MSTs) were constructed using MLVA profiles incorporating demographics and NG-MAST genogroups. A cluster was defined as ≥5 strains differing in ≤1 VNTR locus. The concordance between MLVA and NG-MAST was examined with the adjusted Wallace coefficients (AW).ResultsWe collected a total of 78 Indonesian strains from Yogyakarta (n = 44), Jakarta (n = 25), and Denpasar (n = 9). Seven MLVA clusters and 16 non-clustered strains were identified. No cluster was specific for any geographic area, risk group or age group.Combined with 119 contemporary Dutch strains, 8 MLVA clusters were identified, being four clusters of Indonesian strains, two of Dutch strains, and two of both Indonesian and Dutch strains. Indonesian strains (79.5%) were more often clustered compared to Dutch strains (24.3%).The most prevalent NG-MAST genogroups among Indonesian strains was G1407 (51.3%) and among Dutch strains was G2992 (19.3%). In Indonesian strains, the AW [95% confidence interval] for MLVA to NG-MAST was 0.07 [0.00–0.27] and for NG-MAST to MLVA was 0.03 [0.00–0.12].ConclusionIndonesian Ng strains are more often clustered than Dutch strains, but show no relation with geographical area, risk group, or age group, suggesting a more clonal Ng epidemic in Indonesia. Some similar Ng strains circulate in both Indonesia and the Netherlands.

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.