Mismatch Amplification Mutation Assay (MAMA)-Based Real-Time PCR for Rapid Detection of Neisseria gonorrhoeae and Antimicrobial Resistance Determinants in Clinical Specimens

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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

Donà, Valentina (author): Institute for Infectious Diseases, University of Bern, Bern, Switzerland

Smid, Joost H. (author): Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland

Kasraian, Sara (author): Institute for Infectious Diseases, University of Bern, Bern, Switzerland

Egli-Gany, Dianne (author): Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland

Dost, Ferah (author): Ambulatorium Kanonengasse, Zurich, Switzerland

Imeri, Fatime (author): Laborgemeinschaft 1, Zurich, Switzerland

Unemo, Magnus, 1970- (author): Örebro universitet,Institutionen för medicinska vetenskaper,Region Örebro län,WHO Collaborating Centre for Gonorrhoea and other STIs, Örebro University Hospital, Örebro, Sweden

Low, Nicola (author): Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland

Endimiani, Andrea (author): Institute for Infectious Diseases, University of Bern, Bern, Switzerland

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American society for microbiology, 2018
2018
English.
In: Journal of Clinical Microbiology. - : American society for microbiology. - 0095-1137 .- 1098-660X. ; 56:9

Related links:: https://doi.org/10.1...; show more...; https://urn.kb.se/re...; https://doi.org/10.1...; show less...

Journal article (peer-reviewed)

Abstract Subject headings

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.

Subject headings

MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)
MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Microbiology in the medical area (hsv//eng)

Keyword

gonococcus
antimicrobial resistance
NAAT
real-time PCR
clinical samples
AMR
Neisseria gonorrhoeae
antibiotic resistance
clinical methods
diagnostics
rapid tests
sexually transmitted diseases

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ref (subject category)
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By the author/editor: Donà, Valentina; Smid, Joost H.; Kasraian, Sara; Egli-Gany, Diann ...; Dost, Ferah; Imeri, Fatime; show more...; Unemo, Magnus, 1 ...; Low, Nicola; Endimiani, Andre ...; show less...

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MEDICAL AND HEALTH SCIENCES: MEDICAL AND HEAL ...; and Basic Medicine; and Microbiology in ...

Articles in the publication: Journal of Clini ...

By the university: Örebro University

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