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- Donà, Valentina, et al.
(författare)
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Mismatch Amplification Mutation Assay (MAMA)-Based Real-Time PCR for Rapid Detection of Neisseria gonorrhoeae and Antimicrobial Resistance Determinants in Clinical Specimens
- 2018
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Ingår i: Journal of Clinical Microbiology. - : American society for microbiology. - 0095-1137 .- 1098-660X. ; 56:9
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Tidskriftsartikel (refereegranskat)abstract
- Molecular methods are often used for Neisseria gonorrhoeae (NG) detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies NG and detects AMR determinants in clinical specimens.We designed a mismatch amplification mutation assay (MAMA)-based SYBR Green real-time PCR targeting: one NG-specific region (opa); mosaic penA alleles (Asp345 deletion, Gly545Ser) associated with decreased susceptibility to cephalosporins; alterations conferring resistance to ciprofloxacin (GyrA: Ser91Phe), azithromycin (23S rRNA: A2059G and C2611T) and spectinomycin (16S rRNA: C1192T). We applied the real-time PCR to 489 clinical specimens, of which 94 had paired culture isolates, and evaluated its performance by comparison with commercial diagnostic molecular and phenotypic tests.Our assay exhibited a sensitivity/specificity of 93%/100%, 96%/85%, 90%/91%, 100%/100% and 100%/90% for the detection of NG directly from urethral, rectal, pharyngeal, cervical and vaginal samples, respectively. The MAMA strategy allowed the detection of AMR mutations by comparing cycle threshold values with the reference opa reaction. The method accurately predicted the phenotype to four antibiotic classes when compared with the MIC values obtained from 94 paired cultures (sensitivity/specificity for cephalosporins, azithromycin, ciprofloxacin and spectinomycin resistance: 100%/95%, 100%/100%, 100%/100% and not applicable (NA)/100%, respectively, in genital specimens; NA/72%, NA/98%, 100%/97%, and NA/96%, respectively, in extra-genital specimens). False-positive results, particularly for the penA Asp345del reaction were observed predominantly in pharyngeal specimens.Our real-time PCR assay is a promising rapid method to identify NG and predict AMR directly in genital specimens, but further optimization for extra-genital specimens is needed.
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| 2. |
- Seth-Smith, Helena, et al.
(författare)
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NEISSERIA GONORRHOEAE GENOMIC DIVERSITY IN HIGH RISK GROUPS IN SWITZERLAND
- 2019
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Ingår i: Sexually Transmitted Infections. - : BMJ Publishing Group Ltd. - 1368-4973 .- 1472-3263. ; 95:Supl. 1, s. A281-A281
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background: Neisseria gonorrhoeae cases are increasing in Europe, with decreasing susceptibility to first line antibiotics. Whole genome sequencing (WGS) provides detailed information about gonococcal molecular epidemiology and prediction of antimicrobial resistance (AMR), especially if linked to epidemiological data. The aim of this study was to examine molecular, clinical and social epidemiological aspects of gonorrhoea infections in Switzerland.Methods: In 2015–2016, we cultured urethral, cervical, vaginal, rectal, and pharyngeal specimens from patients in three clinics predominantly attended by men who have sex with men (MSM) and female sex workers (FSW). MSM also completed a sexual behaviour questionnaire. Minimal inhibitory concentrations (MIC) were assessed by Etest, interpreted using EUCAST breakpoints except azithromycin (2 mg/L); WGS used an Illumina Miseq.Results: We sequenced 140 isolates from 116 participants, MSM (107, 92%, mean age 35.8 years) and FSW (6, 5%, mean age 25.3 years). Amongst MSM, 48/105 respondents (45.7%) reported recent sex abroad. Three patients (two MSM and one FSW) carried different strains at different body sites. The isolates show large genomic diversity, with 69 NG-MAST types and 37 MLST sequence types, largely embedded within characterised European Union clusters. NG-MAST 1407 was identified in n=4 isolates from two patients (FSW, not travel-associated and MSM, sex elsewhere in Europe). Mosaic penAXXXIV was seen in these isolates, and also in an NG-MAST 13488 from an MSM, which was also not travel associated. One isolate (heterosexual male, not travel-associated) with elevated cefixime MIC (0.19mg/ml) carried a mosaic penAX in an NG-MAST 10557 background. Ciprofloxacin resistance was seen in these six isolates, and overall in 59/140 (42%), all containing GyrA mutations S91F and D95A/G/N.Conclusion: Switzerland has a high diversity of circulating gonorrhoea, generally related to European clusters. Multidrug resistant isolates were not identified in this study, but NG-MAST 1407 and penA mosaics, associated with elevated cephalosporin MICs, are circulating.
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