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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
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland.
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland.
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2018 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 56, no 9, article id e00365-18Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American society for microbiology , 2018. Vol. 56, no 9, article id e00365-18
Keywords [en]
gonococcus, antimicrobial resistance, NAAT, real-time PCR, clinical samples, AMR, Neisseria gonorrhoeae, antibiotic resistance, clinical methods, diagnostics, rapid tests, sexually transmitted diseases
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:oru:diva-67621DOI: 10.1128/JCM.00365-18ISI: 000442773300013PubMedID: 29950339Scopus ID: 2-s2.0-85052492530OAI: oai:DiVA.org:oru-67621DiVA, id: diva2:1229158
Note

Funding Agency:

SwissTransMed Initiative from the Rectors' Conference of the Swiss Universities (CRUS)  25/2013

Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2018-09-06Bibliographically approved

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Unemo, Magnus

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