| 1. |
- 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. |
- Donà, Valentina, et al.
(författare)
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Multiplex real-time PCR assay with high-resolution melting analysis for characterization of antimicrobial resistance in neisseria gonorrhoeae
- 2016
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Ingår i: Journal of Clinical Microbiology. - Washington, USA : American Society for Microbiology. - 0095-1137 .- 1098-660X. ; 54:8, s. 2074-2081
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.
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| 3. |
- Golparian, Daniel, 1984-, et al.
(författare)
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Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.
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| 4. |
- Hauser, Christoph, et al.
(författare)
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In Vitro Activity of Fosfomycin Alone and in Combination with Ceftriaxone or Azithromycin against Clinical Neisseria gonorrhoeae Isolates
- 2015
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 59:3, s. 1610-1616
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Tidskriftsartikel (refereegranskat)abstract
- New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae. In this study, fosfomycin (FOS) was tested against 89 Neisseria gonorrhoeae isolates using the Etest method, showing MIC50/MIC(90)s of only 8/16 mu g/ml (range, <= 1 to 32 mu g/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including Neisseria gonorrhoeae F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations that included FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values, 1.2 to 2.3 for FOS plus CRO, 1.8 to 3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT, and their combinations (at 0.5x, 1x, 2x, and 4x the MIC) were performed against Neisseria gonorrhoeae strain ATCC 49226, one Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence type 1407 (ST1407) strain, F89, and AZT-HLR. For all strains, at 24 h, the results indicated that (i) FOS was bactericidal at 2X the MIC, but after >24 h, there was regrowth of bacteria; (ii) CRO was bactericidal at 0.5x the MIC; (iii) AZT was bactericidal at 4x the MIC; (iv) CRO plus AZT was less bactericidal than was CRO alone; (v) FOS plus AZT was bactericidal at 2x the MIC; and (vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5x the MIC, but FOS plus CRO had more rapid effects. FOS is appealing for use in the management of Neisseria gonorrhoeae infections because of its single and oral formulation. However, our results suggest it be used in combination with CRO. After the appropriate clinical trials are conducted, this strategy could be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT.
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| 5. |
- 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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