Neisseria Gonorrhoeae Multi-antigen Sequence Typing Research Articles

P3.263 Do Men Harbour the Same Strain of Gonococcus at Multiple Anatomical Sites?

BackgroundAll N. gonorrhoeae clinical isolates in Scotland are routinely typed by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). The frequency with which individuals harboured the same N. gonorrhoeae strain at >...

P3.270 Antimicrobial Susceptibility and Molecular Characterization of Neisseria Gonorrhoeae Strains from Slovenia 2008–2012

ObjectivesAntimicrobial resistance in Neisseria gonorrhoeae is a major public health problem worldwide. Slovenia is among the countries with highest prevalence of decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) in...

P1.013 Emergence of Neisseria Gonorrhoeae Isolates with in Vitro Decreased Susceptibility to Ceftriaxone in Argentina

Background N. gonorrhoeae(Ng) has developed resistance to most antimicrobial used for treatment. The report of the first resistant isolates of Ceftriaxone (CRO) in Japan, France and Spain, highlighted the lack...

Neisseria gonorrhoeae multi-antigen sequence typing using urine samples

Objective To determine Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence types in different geographical areas of China,including Changzhou and Yangzhou cities of Jiangsu province,Wuzhou and Hezhou cities in Guangxi Zhuang Autonomous region,Sanya and Qionghai cities of Hainan province,Jiangmen and Maoming cities of Guangdong province.Methods DNA was extracted using Qiagen DX extraction kits from 88 urine samples which were collected from male patients attending sexually transmitted disease (STD) clinics and positive for nucleic acid amplification test (NAAT) for N.gonorrhoeae.Two rounds of PCR were carried out to amplify the porB and tbpB genes of N.gonorrhoeae followed by gene sequencing.Sequence alignment was performed on the NG-MAST website (http://www.ng-mast.net) to determine the genotype of N.gonorrhoeae.Results The first-round PCR yielded positive results for porB and tbpB in 13.6％ (12/88) and 14.8％ (13/88),respectively,of these urine specimens,and 12 samples were successfully genotyped with the efficiency of genotyping being 13.6％.The amplification efficiency of second-round PCR was enhanced to 71.6％ and 72.7％ for porB and tbpB,respectively,and the efficiency of genotyping increased to 70.5％ (62/88).Compared with the first-round PCR,the second-round PCR showed an increase in amplification efficiency for porB and tbpB by 58.0％ and 57.9％ respectively,as well as in genotyping efficiency by 56.9％.Forty-five genotypes were identified in the 62 samples,including 40 known genotypes and 5 novel genotypes.Of these genotypes,ST1866 was the most abundant (6/62),followed by ST1972 (4/62) and ST3356 (4/62),all of which were from Jiangsu province.The ST532 genotype was identified in 3 samples from Guangdong province,ST2221 genotype in 2 samples from Guangxi Zhuang Autonomous region.Each of the remaining genotypes was identified in only 1 sample and scattered in all of these cities.The 5 novel MAST-genotypes were as follows:porB-892 and tbpB-46 (98％ similarity),porB-130 and tbpB-504 (96％ similarity),porB-2790 and tbpB-32 (99％ similarity),porB-1053 and tbpB-856 (99％ similarity).Conclusions Urine samples can be used for NG-MAST analysis,and two rounds of PCR can enhance the efficiency of genotyping.NG-MAST genotypes appear to be diverse in different geographical areas of China. Key words: Neisseria gonorrhoeae; Genotype; Neisseria gonorrhoeae multi-antigen sequence typing

Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from Vietnam, 2011

BackgroundAntimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. In Vietnam, knowledge regarding N. gonorrhoeae prevalence and AMR is limited, and data concerning genetic characteristics of N. gonorrhoeae is totally lacking. Herein, we investigated the phenotypic AMR (previous, current and possible future treatment options), genetic resistance determinants for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolated in 2011 in Hanoi, Vietnam.MethodsN. gonorrhoeae isolates from Hanoi, Vietnam isolated in 2011 (n = 108) were examined using antibiograms (Etest for 10 antimicrobials), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and sequencing of ESC resistance determinants (penA, mtrR and penB).ResultsThe levels of in vitro resistance were as follows: ciprofloxacin 98%, tetracycline 82%, penicillin G 48%, azithromycin 11%, ceftriaxone 5%, cefixime 1%, and spectinomycin 0%. The MICs of gentamicin (0.023-6 mg/L), ertapenem (0.002-0.125 mg/L) and solithromycin (<0.016-0.25 mg/L) were relatively low. No penA mosaic alleles were found, however, 78% of the isolates contained an alteration of amino acid A501 (A501V (44%) and A501T (34%)) in the encoded penicillin-binding protein 2. A single nucleotide (A) deletion in the inverted repeat of the promoter region of the mtrR gene and amino acid alterations in MtrR was observed in 91% and 94% of the isolates, respectively. penB resistance determinants were detected in 87% of the isolates. Seventy-five different NG-MAST STs were identified, of which 59 STs have not been previously described.ConclusionsIn Vietnam, the highly diversified gonococcal population displayed high in vitro resistance to antimicrobials previously recommended for gonorrhoea treatment (with exception of spectinomycin), but resistance also to the currently recommended ESCs were found. Nevertheless, the MICs of three potential future treatment options were low. It is essential to strengthen the diagnostics, case reporting, and epidemiologic surveillance of gonorrhoea in Vietnam. Furthermore, the surveillance of gonococcal AMR and gonorrhoea treatment failures is imperative to reinforce. Research regarding novel antimicrobial treatment strategies (e.g., combination therapy) and new antimicrobials is crucial for future treatment of gonorrhoea.

Molecular epidemiological typing within the European Gonococcal Antimicrobial Resistance Surveillance Programme reveals predominance of a multidrug-resistant clone

Treatment of gonorrhoea is threatened by antimicrobial resistance, and decreased susceptibility and resistance to recommended therapies is emerging in Europe. Current associations between resistance and molecular type remain poorly understood. Gonococcal isolates (n=1,066) collected for the 2009 and 2010 European Gonococcal Antimicrobial Surveillance Programme were typed by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). A total of 406 sequence types (STs) were identified, 125 of which occurred in ≥two isolates. Seven major genogroups of closely related STs (varying by ≤1% at just one of the two target loci) were defined. Genogroup 1407 (G1407), observed in 20/21 countries and predominant in 13/21 countries, accounted for 23% of all isolates and was associated with decreased susceptibility to cefixime and resistance to ciprofloxacin and raised minimum inhibitory concentrations for ceftriaxone and azithromycin. Genogroup 225 (G225), associated with ciprofloxacin resistance, was observed in 10% of isolates from 19/21 countries. None of the other genogroups were associated with antimicrobial resistance. The predominance of a multidrug-resistant clone (G1407) in Europe is worrying given the recent reports of recommended third generation cephalosporins failing to treat infections with this clone. Identifying associations between ST and antimicrobial resistance aids the understanding of the dissemination of resistant clones within a population and could facilitate development of targeted intervention strategies.

Surveillance of gonococcal antimicrobial susceptibility resulting in early detection of emerging resistance

To undertake laboratory and clinical surveillance of gonococcal antimicrobial susceptibility to various therapeutic agents in Hong Kong, so as to monitor for emerging resistance and to inform on appropriate choice of empirical therapy. Trends in susceptibility of gonococci to ceftriaxone, spectinomycin, ceftibuten and azithromycin were monitored over time. Isolates with reduced susceptibility to oral extended-spectrum cephalosporins were further characterized by detection of the mosaic penA gene and typing by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). Correlation with clinical and epidemiological findings was undertaken on isolates positive for the mosaic penA gene. Trends in susceptibility of gonococci to ceftriaxone, spectinomycin and ceftibuten remained stable between 2005 and 2010. In 2010, 30.3% of tested strains were not susceptible to azithromycin. The percentage of gonococcal strains harbouring the mosaic penA gene increased from 1.0% during April to December 2010 to 8.2% during January to September 2011 (P < 0.0001). Review of available clinical records showed that, out of 35 patients infected by strains positive for the mosaic penA gene, 30 had laboratory-documented treatment failure. This study showed that ceftriaxone and spectinomycin remained effective against gonorrhoea in Hong Kong. There was an alarming increase in strains with reduced susceptibility to oral extended-spectrum cephalosporins associated with clinical treatment failure. One-third of gonococcal isolates were non-susceptible to azithromycin. The need to switch to agents other than oral extended-spectrum cephalosporins for empirical treatment is imminent. Continued surveillance with strain characterization is essential to monitor the effectiveness of currently recommended therapy.

Identification of Sexual Networks Through Molecular Typing of Quinolone-Resistant Neisseria gonorrhoeae in Ontario, Canada

Neisseria gonorrhoeae multi-antigen sequence typing technique demonstrated multiple sexual transmission networks in Ontario, Canada. Isolates with novel sequences had higher odds of originating in Toronto but had no association with heightened population-level quinolone exposure. Neisseria gonorrhoeae multi-antigen sequence typing technique can be a useful tool for investigation of multidrug-resistant N. gonorrhoeae emergence in North America.

Resistance to Azithromycin of Neisseria gonorrhoeae Isolates From 2 Cities in China

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is a public health concern globally, and azithromycin-resistant NG isolates have been identified in many countries. This study aimed to investigate azithromycin susceptibility in NG and to genetically characterize the azithromycin-resistant isolates identified in 2 cities in China. NG isolates (n = 318) were cultured from patients attending 2 sexually transmitted infection clinics in Nanjing and Chongqing, China, between 2008 and 2009. Minimum inhibitory concentration (MIC) of azithromycin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone was determined using the agar dilution method. NG strains ATCC 49226 and WHO G, J, L, and P were used for quality control. Azithromycin-resistant isolates were defined as having an MIC value equal to or greater than that of strain WHO P, which is internationally recognized as azithromycin-resistant (MIC = 2 mg/L), and genotyped using NG multiantigen sequence typing. The MIC values of strain WHO P for azithromycin were 2 to 4 mg/L in all runs, which showed that the method provided consistent and reliable MIC values. Seventeen isolates (5.3%) showed resistance to azithromycin. Among these isolates (n = 17), 11 sequence types (STs) were identified by NG multiantigen sequence typing, of which 5 were novel. The most common ST was ST3356, represented by 6 isolates. ST1866 was represented by 2 isolates, which were isolated from patients with an unknown relationship, and both isolates were highly resistant to azithromycin, i.e., displayed an MIC of >64 mg/L. A relatively high prevalence of azithromycin-resistant NG strains implies that azithromycin should not be recommended for the treatment of gonococcal urethritis or cervicitis in China.

P4-S1.08 Performance comparison of Neisseria gonorrhoeae (NG) sequence typing methods NG-MAST and NG-MLVA on a defined panel of isolates

BackgroundThe increase in N gonorrhoeae (NG) prevalence since the mid-1990s, increasing antibiotic resistance, and the absence of an effective vaccine, demand more efficient national and international surveillance of the NG...

Analysis of Emergence of Quinolone-Resistant Gonococci in Greece by Combined Use of Neisseria gonorrhoeae Multiantigen Sequence Typing and Multilocus Sequence Typing

The prevalence of quinolone-resistant Neisseria gonorrhoeae (QRNG) in Greece remained low from 1997 to 2003 but increased dramatically from 11% to 56% between 2004 and 2007. N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to investigate trends in quinolone resistance from 1997 to 2007 and explore the origins of the recent increase in QRNG. We characterized 295 QRNG isolates from the study period and 233 quinolone-susceptible (QS) gonococci from 2004 and 2005, when the rapid increase in QRNG occurred. From 1997 to 1999, an outbreak of QRNG was due to the dissemination of isolates of serovar Arst that belonged to two closely related genotypes. Few QRNG isolates, of diverse genotypes, were present between 2001 and 2003, whereas the sharp increase in QRNG from 2004 onwards was due to the appearance of serovar Bropyst isolates of several major NG-MAST sequence type (STs) that previously had not been identified in Greece. These isolates were shown by MLST to be variants of a single multiply antibiotic-resistant QRNG strain (ST1901) that appeared in Greece and rapidly diversified into 31 NG-MAST STs. There were no isolates of MLST ST1901 or any of the 31 NG-MAST STs among QS isolates from 2004 and 2005 or among 8 representatives of multiresistant but quinolone-susceptible serovar Bropyst isolates circulating in Greece during the 1990 s, supporting the view that the recent increase in QRNG was due to importation of a QRNG strain(s) of MLST ST1901 into Greece. Recently, multiresistant QRNG isolates of ST1901 with reduced susceptibility to the newer cephalosporins have appeared in Greece.

Molecular Surveillance of Clinical Neisseria gonorrhoeae Isolates in Russia

The choice of adequate methods for epidemiological purposes remains a challenging problem in Neisseria gonorrhoeae molecular monitoring. In this study, the collection of geographically unrelated gonococci (n = 103) isolated in Russian clinics was comparably tested by (i) a traditional serotyping scheme, (ii) por typing, (iii) Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST), and (iv) multilocus sequence typing (MLST). It is shown that, according to sequencing data, a third of the strains carried new porB1 alleles, as well as tbpB ones, and more than half of the samples had new sequence types (STs) as determined by NG-MAST or MLST. The discriminatory power for each typing method was calculated by using the Hunter-Gaston discriminatory index, D. Commonly, modern nucleic acid-based typing methods (por typing, NG-MAST, and MLST) appeared to be more efficient than the classical serotyping scheme. While the traditional serotyping gave a D value of 0.82, the por typing, NG-MAST, and MLST approaches yielded D values of 0.97, 0.98, and 0.91, respectively. Each typing technique revealed the distribution of gonococci slightly correlated with their geographical sources. However, only the MLST method STs were highly associated with certain phenotypes. Although ST1594, ST1892, and ST6720 were typical for susceptible gonococci, ST1901 and ST6716 were undoubtedly associated with a multidrug-resistant phenotype. We conclude that every tested nucleic acid-based typing method is suitable for N. gonorrhoeae molecular surveillance. However, the MLST method seems to serve large-scale epidemiological purposes, whereas the NG-MAST and por typing approaches are more appropriate for the investigation of local outbreaks.

Neisseria gonorrhoeae multi-antigen sequence typing: a new genotyping method

The typing of gonococcus involves phenotypic analysis and molecular genotyping. Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) is a sequence-based genotyping method with a broad prospect. NG-MAST system is not only suitable for cultured specimens, but also for swab samples. It allows the typing of N.gonorrhoeae strains by blasting against databases at NG-MAST Web site following amplification of variable internal regions of por and tbpB genes of gonococcal strains using PCR and sequencing of PCR products.Compared with other genotyping methods, NG-MAST appears simple and objective, and tends to produce results with satisfactory comparability between national and international laboratories. This system has been extensively used in predicting gonococcal resistance, analyzing sexual networks, identifying outbreak and epidemic strains, and so on. Key words: Neisseria gonorrhoeae ; Sequence analysis ; Evaluation studies ;

Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens

ObjectivesThe Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal...

Characteristics of patients infected with common Neisseria gonorrhoeae NG-MAST sequence type strains presenting at the Edinburgh genitourinary medicine clinic

Objective:Characterisation of population groups infected with common Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence types, presenting at the Edinburgh genitourinary medicine clinic.Methods:All patients with gonococcal infection attending over a 2-year...

To what extent does Neisseria gonorrhoeae multiantigen sequence typing of gonococcal isolates support information derived from patient interviews?

Gonococcal isolates from genitourinary (GU) medicine clinic attendees in Glasgow, Scotland were typed using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST). Correlation between named partners (contacts) and NG-MAST type was sought and associations between specific NG-MAST types, and the social, epidemiological and geographical data were explored. We found NG-MAST typing to be a supportive and confirmatory tool for contact tracing. Specific NG-MAST types were found to be associated with distinct characteristics such as sexuality or chlamydial co-infection. An increased number of gonococcal infections were reported from those resident in deprived areas of Glasgow than from those resident in more affluent areas. However, there was no clear geographic clustering of specific NG-MAST types found within the city. Routinely observing the spread of common strains of gonorrhoea is likely best done from a larger geographical perspective unless a specific outbreak occurs.

Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) of ciprofloxacin resistant isolates of Pretoria, South Africa

Gonococcal isolates resistant to ciprofloxacin collected from 2004 to 2005, in the Pretoria region, were characterised using Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). The isolates were obtained from men presenting...

Opa-typing can identify epidemiologically distinct subgroups within Neisseria gonorrhoeae multi-antigen sequence type (NG-MAST) clusters

A collection of 106 Neisseria gonorrhoeae ciprofloxacin-resistant isolates were typed using Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). Opa-typing was performed on 74 isolates which had non-unique sequence types to determine if further discrimination could be achieved and if so whether this had any epidemiological basis. The 74 isolates were separated into 12 sequence types and 20 opa-types (OT). Seven opa-type clusters were congruent with the sequence types and five sequence types could be subdivided by opa-typing. These results demonstrate that opa-typing can add a further level of discrimination compared with NG-MAST. The surveillance data for isolates in the largest sequence type cluster (ST 147) indicated that two major subdivisions OT 1 and OT 2 differed epidemiologically by patients' sexual preference and geographical location. ST 147 is a common strain that has been isolated in several countries since 1999; our results suggest that it has diverged into at least two epidemiologically discrete forms.

Dramatic increase in a single genotype of TRNG ciprofloxacin-resistant Neisseria gonorrhoeae isolates in men who have sex with men

In 2003, episodes of gonorrhoea caused by ciprofloxacin-resistant strains increased to 15.3% from 11% in 2002. This was coincident with a marked increase in strains characterized as serogroup WI, ciprofloxacin-resistant bearing the tetracycline resistance plasmid. Molecular typing of these strains, using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) revealed 71% (34/48) were of the same sequence type, ST338, accounting for 4.1% (34/824) of all strains in 2003. Epidemiological data demonstrated that transmission of ST338 was associated with men who have sex with men (MSM; 23/27), acquisition within the UK (22/26) and having two or more partners in the previous three-month period (18/27). The combined use of highly discriminatory typing and epidemiological surveillance helps to identify successful transmission networks.