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

Donà, Valentina; Smid, Joost H; Kasraian, Sara; Egli-Gany, Dianne; Dost, Ferah; Imeri, Fatime; Unemo, Magnus; Low, Nicola; Endimiani, Andrea (2018). Mismatch Amplification Mutation Assay (MAMA)-Based Real-Time PCR for Rapid Detection of and Antimicrobial Resistance Determinants in Clinical Specimens. Journal of clinical microbiology, 56(9), e00365-18. American Society for Microbiology 10.1128/JCM.00365-18

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Molecular methods are often used for (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 (); mosaic 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 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 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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Research
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Social and Preventive Medicine

UniBE Contributor:

Donà, Valentina; Smid, Joost Hubert; Kasraian Fard, Sara; Egli, Dianne; Low, Nicola and Endimiani, Andrea

Subjects:

300 Social sciences, sociology & anthropology > 360 Social problems & social services
500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0095-1137

Publisher:

American Society for Microbiology

Language:

English

Submitter:

Tanya Karrer

Date Deposited:

05 Jul 2018 14:29

Last Modified:

18 Feb 2019 08:19

Publisher DOI:

10.1128/JCM.00365-18

PubMed ID:

29950339

BORIS DOI:

10.7892/boris.118327

URI:

https://boris.unibe.ch/id/eprint/118327

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